ORIGINAL PAPER

Female fitness, sperm traits and patterns of paternityin an Australian polyandrous mouse

Renée C. Firman

Received: 26 August 2013 /Revised: 9 October 2013 /Accepted: 10 October 2013 /Published online: 5 November 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Multiple mating is a common reproductive strategyamong mammals, and rodents living in communal, mixed sexsocial groups are predisposed to a polygamous existence. Thesandy inland mouse is a naturally polyandrous species thatoccurs across most of Australia’s arid region. Femalestypically have greater reproductive restrictions compared withmales and are therefore expected to acquire substantial fitnessbenefits from copulating with more than one male. Here, Ishow that the reproductive output of female sandy inlandmicedid not differ between females mated monandrously (singlemale) or polyandrously (two males). Paternity data obtainedfrom the polyandrous litters revealed that in most cases, therewas a first male-to-mate advantage. I discuss this result inrelation to the chastity enforcement hypothesis for theevolution of the copulatory plug. Finally, I compared ejaculatetraits of competing males and found that the paternity loss ofmales that mated first was attributable to their own spermdensity and sperm quality, and not to that of their rivals. Thesperm data also revealed that second males gained greaterpaternity representation when sperm velocities and motilitieswere higher in first-mated males. This investigation indicatesthat mating position is a critical determinant of male fitness inmammalian sperm competition.

Keywords Sperm competition .Multiple paternity .

Mammals . Copulatory plug . Genetic benefits .

Ejaculate quality

Introduction

Mating systems evolve as individuals strive to maximise theirreproductive output (Thornhill and Alcock 1983; Clutton-Brock 1989). Copulation can be costly for both males andfemales (Daly 1978). However, due to restrictions on eggproduction and/or offspring support during development,females typically have greater fitness constraints than males.Certainly, a single mating event that results in the successfultransfer of sperm should be more than adequate to ensure thatall the eggs are fertilised (Bateman 1948; Trivers 1972).Consequently, accounting for the evolution of multiple matingby females (polyandry) has presented to be a complexproblem in evolutionary biology.

Females may solicit multiple partners to facilitate ejaculateoverlap within the reproductive tract and thus force the spermof different males to compete for fertilizations (Parker 1970).Females may accrue genetic benefits for their offspring ifmales that are of greater genetic quality are successful in spermcompetition and achieve high fertilisation rates (Yasui 1997).Direct support for the ‘good sperm’ hypothesis is currentlylimited to only a few studies across a diverse range of species(Hosken et al. 2003; Fisher et al. 2006; García-González andSimmons 2007; Firman and Simmons 2012). Alternatively,although not mutually exclusive, polyandry may allowpaternity to be biased toward genetically compatible malegenotypes and save females from failed reproductive attemptsthat would occur when monogamous matings are destined tobe incompatible (Zeh and Zeh 1997). As an extension of thishypothesis, a competitive fertilisation bias has also beenobserved to be skewed toward males whose genotypes aremore disparate to that of the female, which may assistindividuals in avoiding costs associated with inbreeding(Bretman et al. 2004; Simmons et al. 2006; Firman andSimmons 2008a). Of course, the outcome of spermcompetition is also conditional to female influences over spermuse (Eberhard 1996), and genotypic interactions among malesand between males and females (García-González 2008).

Communicated by C. Soulsbury

Electronic supplementary material The online version of this article(doi:10.1007/s00265-013-1643-1) contains supplementary material,which is available to authorized users.

R. C. Firman (*)Centre for Evolutionary Biology, School of Animal Biology (M092),University of Western Australia, 25 Stirling Hwy, Crawley,WA 6009, Australiae-mail: renee.firman@uwa.edu.au

Behav Ecol Sociobiol (2014) 68:283–290DOI 10.1007/s00265-013-1643-1

In competitive situations, paternity outcomes can beinfluenced by male genetic quality and make-up, as well asmale sperm competitive ability. The density and quality ofsperm that rival males contribute to the competition will be asignificant determinant of each male’s success (Parker 1990).Males have been found to invest differentially in theirejaculate expenditure and more specifically spermexpenditure, under different sperm competition situations orwhen they have been reared in different sperm competitionenvironments (Gage 1991; Pizzari et al. 2003; delBarco-Trilloand Ferkin 2006; Ramm and Stockley 2009; Firman et al.2013b). Indeed, the high cost that males incur in producingejaculates ensures such prudency in expenditure and generatesthe need for males to protect their investment (Dewsbury1982). In some species, males deposit a copulatory plugfollowing sperm transfer (Devine 1975; Barker 1994;Dixson and Anderson 2002; Ramm et al. 2005). Althoughthe precise function of the copulatory plug is unknown, it hasbeen suggested that plugs might reduce the risk of spermcompetition by preventing subsequent males frominseminating females (Voss 1979). Therefore, in somepolyandrous species, mating position may be criticallyimportant in determining the outcome of male paternitysuccess. Specifically among mammals, the timing of matingsrelative to the time of ovulation will also influence theoutcome of sperm competition (Gomendio et al. 1998).

The sandy inland mouse (Pseudomys hermannsburgensis)is an arid-adapted rodent that occurs in semi-arid and aridareas of Australia (Breed and Ford 2007). Like many desertrodents, the sandy inland mouse inhabits burrows in large,intersexual groups (Breed and Ford 2007). A grouped socialorganisation may facilitate reproduction in the harsh,unpredictable arid environment by ensuring access to matesand/or generating synchronised breeding. A recent studyrevealed that sandy inland mouse litters are usually sired bymore than one male and provided evidence that females matepolyandrously and males are forced to engage in spermcompetition (Firman et al. 2013a). In this investigation, Idocumented patterns of paternity among litters of femalesandy inland mice that had mated with two males in a singlereceptive period and compared the reproductive output offemales mated multiply to females mated with a single male.I also extracted sperm from males, documented relationshipsamong a range of different sperm traits and determined whichsperm traits contributed to competitive paternity success.

Methods

Source population and experimental animals

Sandy inland mice (Pseudomys hermannsburgensis ) weresourced from a wild population located approximately

25 km south of Laverton, Western Australia (28 °37′S,122 °24′E). Male (n=30) and female (n=30) mice were trappedin Elliot small mammal traps baited with peanut butter and rolledoats and transported to the University of Western Australia. Theanimals were held in a constant temperature room (CTR; 26 °C)on a reversed light–dark cycle, housed in large mouse boxes(28×74×41 cm) and provided with native Australian rodentpellets and water ad libitum . To supplement their diet, theanimals received ∼5 g of large parrot seed three times a week.The wild-caught animals were used to establish a small, captive-bred colony. Male–female pairs were housed together until thefemale was noted as being pregnant, or for a maximum of3 weeks. Pregnant females were separated from the males andprovided with shredded paper for nesting. Litters were weanedfrom their mother at 28 days old. The mice were outbred forthree generations before being used in this experiment.

Experimental matings

The housing and maintenance regime for the experimentalanimals was the same as described above (i.e. held in a CTRon a reversed light–dark cycle inside large mouse boxes andfed native Australian rodent pellets and large parrot seed). At3 months of age, females (n =20) and males (n =30) that hadreproduced once previously and thus were known to be fertile,were used in the experimental matings. Ten females and tenmales were randomly assigned to the monandrous treatment,and 10 females and 20 males were randomly assigned to thepolyandrous treatment. Each animal was used once. Thematings were conducted during the dark phase of the lightcycle, under a red light. The females were placed in the males’box and then inspected every 2 h for oestrus condition (Byerset al. 2012). When females were in estrus, they were inspectedhalf-hourly for the presence of a mating plug. In most rodentspecies, the copulatory plug is evidence of a completeejaculatory series and therefore assumed to be a successfulmating that resulted in sperm transfer (Rugh 1968; Breed andAdams 1992). In the polyandrous treatment, once a plug wasdetected, the female was placed in the box of a second male.The mating pairs were then observed continuously until themale mounted and successfully copulated with the female.Thus, I assumed that successful sperm transfer occurred inboth the first-male and the second-male matings. To ensureequal mating opportunities for competing males, females inthe polyandrous treatment spent the same amount of time witheach of their mates (mean=16.2±0.4 h). In the monandroustreatment, the time at which a mating plug was first observedwas noted; females then remained with their partner for twicethat duration of time (mean=34.2±0.8 h). In both treatments,females that had mated were placed in a box with paper fornesting. Nests were checked for pups daily from 28 days aftermating. Length of gestation was recorded as the number ofdays from mating until the day the litter was born.

284 Behav Ecol Sociobiol (2014) 68:283–290

Genotyping and paternity analysis

To examine patterns of paternity in the ten litters produced bythe polyandrous females, the mice were genotyped at fourmicrosatellite loci. DNA was extracted from 1 mm2 of eartissue of females (n =10), males (n =20) and the offspring (36)using the EDNA HISPEX extraction kit (Fisher Biotec,Subiaco, WA, Australia). Microsatellite markers developedfor the western pebble-mound mouse (Pseudomys chapmani)had previously been shown to cross-amplify in the sandyinland mouse (Moro and Spencer 2003). I selected four ofthese markers (pPc1A7, pPc9A8, pPc6E8 and pPc10E12) andobtained labelled forward primers from Geneworks(Hindmarsh, SA, Australia) (FAM) and Life Technologies(Foster City, CA, USA) (PET, VIC) and unlabelled primersfrom Geneworks. For each sample, two polymerase chainreactions (PCRs) were performed in combinations ofpPc1A7+pPc10E12 and pPc9A8+pPc6E8. PCRs wereperformed in 10 μL reactions containing 10 mM Tris (LifeTechnologies), 50 mM KCL (Life Technologies), 1.5 mMMgCl2 (Life Technologies), 200 mM of each dNTP (LifeTechnologies), 250 nM of each labelled forward primer,250 nM of each unlabelled reverse primer, 0.5 units ofPlatinum Taq polymerase (Life Technologies) and ∼100 ngof template DNA. The thermocycling profile for each PCRwas 5 min denature at 94 °C, 35 cycles of 90 °C for 30 s, 62 °Cfor 20 s and 72 °C for 45 s, followed by 72 °C for 10 min [38].PCR products (1.5 μL) were run on an ABI3730 Sequencer,sized using Genescan-500 LIZ size standard and genotypedusing Genemapper software (v3.0; Life Technologies).Paternity was unambiguously assigned by manual exclusion.

Male reproductive traits and sperm analysis

Approximately 2 weeks after mating, the males from thepolyandrous treatment (n =20) were sacrificed, weighed andused for sperm analysis. The testes were removed andweighed. I then used a CEROS computer-assisted spermanalyser (CASA; Hamilton and Thorne, version 10) toquantify epididymal sperm number and sperm motilityparameters. Upon leaving the testes, sperm cells maturethrough their migration along the epididymis and are storedin the caudal epididymis prior to ejaculation (Jones 1999).Thus, epididymal sperm number is representative of thenumber of sperm within an ejaculate. The protocol forin vitro activation of epididymal sperm has been describedpreviously (Firman et al. 2013b). Briefly, the caudal tissuewasremoved and repeatedly cut in a 500 μL drop of human tubalfluid that was immersed in mineral oil (Firman et al. 2013b).After an initial 10-min incubation (37 °C) period, whichallowed the sperm to swim into the medium, the epididymaltissue was removed. The sperm suspension was thenincubated for a further 50 min. Finally, an aliquot of the sperm

suspension was loaded into a Neubauer haemocytometer(∼10 μL) and scanned at 40× magnification with the CASA.

Each sperm sample was scanned ten times, and measuresof (1) sperm number (×106 sperm), (2) % motile sperm, (3) %sperm with rapid motility, (4) % sperm with progressivemotility, (5) average path velocity (VAP; μm/s), (6) straightline velocity (VSL; μm/s) and (7) curvilinear velocity (VCL;μm/s) were retrieved. The replicate measures were found to behighly repeatable for each trait (analyses not provided). Foreach trait, the mean value of the ten replicate CASA scans wasused in the statistical analyses.

Statistical analyses

The litter size data were normally distributed (Shapiro–Wilktest: W =0.910; P=0.063; dispersion estimate: σ=1.27) andthus analysed with analysis of variance (ANOVA). ANOVAswere applied to assess whether the fixed effects of (1)treatment and (2) total amount of time spent with the maleinfluenced litter size and gestation length. I performed a poweranalysis for a two-sample t -test in R and report the effect size(Cohen’s d ) and the 95 % confidence intervals (CIs)(Nakagawa 2004) to assess the power that I had to detectdifferences in litter sizes between the two treatments.

To determine if therewas amating order effect evident amongthe paternity data obtained from the litters of the polyandroustreatment, I used a Wilcoxon signed-rank test and compared thenumber of pups sired by themalemating in the first positionwiththe number of pups sired by the male mating second.

Pairwise correlations revealed strong covariation among themale reproductive traits (Table 1). Consequently, a principalcomponent analysis (PCA) was applied to reduce the datasetand summarise the variation among the seven sperm traits. ThePCA produced two principal components with eigenvalues>0.9 that collectively accounted for 95 % of the variation(Table 2). Variables that were 0.7 times as large as the largesteigenvector were considered to have contributed significantlyto that principal component (PC) (Mardia et al. 1979). PC1accounted for 82 % of the variation and was weighted by thedescriptors of spermmotility (%motile, % rapid, % progressivesperm) and sperm velocity (VAP, VSL, VCL) (Table 2). PC2,which explained 13 % of the variation, was weighted by spermnumber (Table 2). PC1 and PC2 were modelled as fixed effectsin ANOVAs to determine whether variation among sperm traitsaccounted for competitive paternity success.

Results

Female reproductive output

In nature, the litter size of sandy inland mice is typically 3 or 4(Breed 1990; Firman et al. 2013a). Here, litter sizes ranged

Behav Ecol Sociobiol (2014) 68:283–290 285

from one to five, with an average of 3.4±0.3. ANOVArevealed that the total amount of time spent with male/s(F1, 17=1.036,P=0.323) and treatment did not influence littersize (F1, 17=0.652, P=0.431) (whole model: F2, 17=0.903,P=0.424). In the case of treatment on litter size, a poweranalysis revealed low power (0.213) to detect a significanteffect based on this sample size and size difference (n =10/group, sig=0.05, delta=0.5, sd=1.27, alternative= ‘onesided’). However, the effect size was moderate (d =0.392),and the 95 % CIs (−0.264, 0.592) provided confidence inaccepting the null hypothesis of no effect. The total amountof time spent with males (F 1, 17=0.680, P =0.421) andtreatment (F 1, 17=0.649, P =0.432) did not influencegestation length (whole model: F 2, 17=0.340, P =0.717).Thus, litter sizes (M =3.1±0.5, P=3.6±0.3) and gestationlengths (M =31.1±0.2, P=31.1±0.3) did not differ amongfemales from the two treatments (also see Table S1 of the

electronic supplementary material). In addition, pup survivaluntil weaning was comparable between treatments (number ofpup deaths: M =1, P=2).

Sperm traits and competitive paternity success

The Wilcoxon signed-rank test revealed that males mating inthe first position on average sired more pups than malesmating second (Z =2.446, P=0.014). The average proportionof pups sired by males mating second (P2) was 0.24±0.1(Fig. 1).

I assessed whether variation in sperm traits of males matingin the different positions influenced paternity success. Thus,the PC values of males that mated both first and second, andtheir interaction, weremodelled in anANOVAonP2 (Table 3).The analyses revealed that the sperm PC1 and PC2 values of

Table 1 Correlation matrix of male reproductive traits of sandy inland mice

Bodyweight (g)

Testesweight (mg)

Sperm no.(×106)

% motile % rapid % progressive VAP (μm/s) VSL (μm/s) VCL (μm/s)

Range 12.6–21.7 162.9–268.1 2.3–16.2 25.5–95.3 15.6–78.8 9.9–61.4 77.3–149.1 58.9–133.0 128.9–233.5

Mean±s.e. 15.4±0.5 220.5±6.9 8.8±0.9 72.2±4.7 51.2±4.5 40.2±3.6 119.7±5.5 102.8±5.4 185.9±7.1

Body weight —

Testes weight 0.460 —

Sperm no. 0.385 0.461 —

% motile 0.206 0.373 0.650 —

% rapid 0.117 0.258 0.554 0.927 —

% progressive 0.104 0.293 0.502 0.887 0.932 —

VAP 0.054 0.207 0.340 0.844 0.904 0.902 —

VSL 0.058 0.254 0.289 0.832 0.881 0.911 0.991 —

VCL −0.039 0.189 0.351 0.790 0.889 0.857 0.968 0.942 —

The Table is divided into two parts: descriptive statistics for body weight, testes weight and seven sperm variables as measured by the computer-assistedsperm analyser (top), and a matrix showing correlations between the variables (significant correlations are presented in bold; bottom).

Table 2 Principal component analyses of sperm motility and velocitytraits in sandy inland mice

Trait PC1 PC2

Sperm number 0.225 0.863

% motile sperm 0.391 0.223

% progressive sperm 0.405 0.063

% rapid sperm 0.400 −0.002Average path velocity (μm/s) 0.402 −0.246Straight line velocity (μm/s) 0.396 −0.293Curvilinear velocity (μm/s) 0.391 −0.234Eigenvalue 5.74 0.92

Proportion of total 0.82 0.13

Eigenvectors in bold contribute significantly to the PC (Mardia et al. 1979)

Fig. 1 Paternity success of male sandy inland mice mating in the first(blue) and second (green) male-to-mate positions. The size of each litteris provided in the parentheses on the x-axis

286 Behav Ecol Sociobiol (2014) 68:283–290

males mating first had a significant effect on P2, whilst thePC1 and PC2 values of males mating second did not (Table 3).Surprisingly, when males that mated first had faster sperm andgreater proportions of motile sperm, the paternity success ofmales that mated second was greater (Fig. 2a). In addition, P2correlated with sperm densities of males that mated first, withlower sperm numbers contributing to greater success amongmales that mated second (Fig. 2b).

Discussion

Studies of different vertebrate taxa have shown that multiplepaternity is a common characteristic of litters/clutches in thewild and thus provided evidence that polyandry is a commonmating strategy adopted by many species (e.g. Baker et al.1999; Kraaijeveld-Smit et al. 2002; Bretman and Tregenza2005; Dean et al. 2006; Simmons et al. 2007; Firman andSimmons 2008c). As mating is costly, a number of theorieshave been developed to explain why female multiple-matingbehaviour exists (Yasui 1997). It has been shown thatpolyandrous mating does indeed result in enhanced femalefitness in some species (Simmons 2005; Fisher et al. 2006;Firman and Simmons 2008b; Klemme et al. 2008). However,I found no evidence that female sandy inland mice benefitfrom mating with more than one male. Given that the femalescould not escape the males, increased maternal stressassociated with increased sexual harassment and coercioncould have counteracted the fitness benefits of polyandry.However, considering that the average litter size of femalesfrom both treatments was comparable to those of wild-caughtanimals, this seems to be an unlikely explanation. Further, iffemales did benefit from multiple mating, for example byselecting sperm from fit or compatible males, a strong matingorder effect would not be expected, which is certainly not thecase here. Although I cannot eliminate fitness benefits that

might become apparent during later life-history stages,measures of early reproductive success did not differ betweenfemales mated to a single male and females mated to twomales. Fitness benefits from polyandrous mating have beenobserved in a number of mammal species when individualswere forced to compete for access to resources in a semi-natural environment (Fisher et al. 2006; Klemme et al. 2008;Firman 2011). Therefore, it is possible that the benignlaboratory conditions of the current experiment may havemasked fitness benefits that influence reproduction andsurvival in nature.

The number of mates that female sandy inlandmice chooseto mate with is currently not known. A maximum of two sireshas been observed among sandy inland mouse littersconceived in the wild (Firman et al. 2013a). However, giventhat offspring number in this species is so small, sire number islikely to be quite a conservative estimate of the number ofmales with which a female copulates. Indeed, in thepolyandrous treatment, all the females mated with two males,

Table 3 ANOVAs between the principal component values and P2 insandy inland mice

Test S df F P

(a) PC1—sperm motility/velocity

First male to mate 0.288 1 6.044 0.049

Second male to mate 0.054 1 1.133 0.328

First male×second male 0.244 1 5.121 0.064

Error 0.286 6

(a) PC2—sperm number

First male to mate 0.735 1 14.704 0.009

Second male to mate 0.157 1 3.138 0.127

First male×second male 0.274 1 5.489 0.058

Error 0.300 64-4 -3 -2 -1 0 1 2 3

1.0

0

0.2

0.4

0.6

0.8

Sperm motility (PC1)

P2

3-1 0 1 2

1.0

0

0.2

0.4

0.6

0.8

Sperm number (PC2)

P2

a

b

Fig. 2 The proportion of the litters sired by the second male to mate wasinfluenced by the a sperm motility (PC1) and b sperm number (PC2) ofthe first male to mate

Behav Ecol Sociobiol (2014) 68:283–290 287

and yet more than half of the litters were sired entirely by thefirst male to mate. The first male-to-mate advantage is furtherevident with first males obtaining some degree of paternityrepresentation in 90% of litters. Male paternity success can beinfluenced by a range of different factors, including time ofmating in relation to the time of ovulation, cryptic femalechoice and maternal effects, interactions between male andfemale genotypes, and male competitive ability (in relation tothat of rival males). However, a first male-to-mate advantagehas been commonly observed in rodent species (Huck et al.1985; Firman and Simmons 2008b), suggesting that matingposition is a critical factor influencing male fitness in spermcompetition.

The first male to mate is in the best position to secure andprotect his ejaculate with the deposition of a copulatory plug.The mammalian copulatory plug is typically formed byproteins secreted by the seminal vesicles that coagulate withthe contribution of enzymes from the seminal vesicles andcoagulating gland (Williams-Ashman 1984). The specific roleof the copulatory plug is currently unclear. A recentinvestigation on mice revealed that the copulatory plug isnecessary for normal fertility (Dean 2013). Interspecificcomparisons have provided evidence to suggest thatcopulatory plugs are an evolutionary product of spermcompetition (Dixson and Anderson 2002; Ramm et al. 2005)and function to prevent inseminations by subsequent mates(Voss 1979). The first male advantage observed here amongthe litters of doubly-mated female sandy inland mice suggestthat the copulatory plug does play a part in preventingsubsequent inseminations by rival males. However, thecopulatory plug is not 100 % effective, as evidenced withmixed paternity occurring in almost half of the litters.

The loss of paternity of first-matedmales could be attributedto sperm and/or behavioural traits that are important in spermcompetition. Among house mouse populations, testes size andejaculate quality have been found to vary with the level ofsperm competition (Firman and Simmons 2008c; Firman et al.2013b), and increased sperm numbers within ejaculates willenhance the ability of males to gain paternity undercompetitive conditions (Firman and Simmons 2011; Klemmeand Firman 2013). Here in sandy inland mice, I found thatsecond-male success was dependent upon the number of spermthe first male contributed; as first-male sperm numbersdecreased, rivals gained greater paternity representation, whichaligns with the theoretical prediction that sperm competitionconforms to the raffle principal (Parker 1990).

Plasticity in sperm expenditure under a risk of spermcompetition could also account for this result and explainwhy second-male sperm number did not influence second-male paternity success. In a number of different species, maleshave been shown to exhibit strategic adjustments in spermexpenditure according to the risk and intensity of spermcompetition (Gage 1991; Pizzari et al. 2003; delBarco-Trillo

and Ferkin 2006; Ramm and Stockley 2009; Firman et al.2013b). Male house mice (Mus musculus domesticus) willcopulate repeatedly but invest fewer sperm per ejaculationwhen mating in the presence of a rival male (Ramm andStockley 2007). In the current investigation, the experimentalmatings were not observed, and so I am unable to comment onthe copulation/ejaculation frequency of sandy inland mice.However, male rodents have been reported to be able todiscriminate between mated and unmated females (Hucket al. 1984). Therefore, males mating in the disfavoured roleof second male to mate, with the knowledge that they areengaging in a sperm competition, might adjust theircopulatory behaviour accordingly and become more‘competitive’. Certainly, males are likely to be more effectivein dislodging rivals’ plugs by increasing their intromissionfrequency. Future research is required to properly elucidate thespecific tactics that male rodents use to circumvent thecopulatory plugs of rival males.

The paternity share of the second males to mate seemed tobe influenced by the quality of the sperm of the first males thatmated. In some vertebrate species, it has been shown thatmales that produce ejaculates containing high quality sperm(e.g. increased motility/velocity) have greater competitivesuccess (Birkhead et al. 1999; Gasparini et al. 2010; Firmanand Simmons 2011). Here, analyses revealed a negativecorrelation between male sperm quality and paternity share,and that second males gained greater paternity representationwhen sperm velocities and motilities were higher in first-mated males. This pattern may be representative of a trade-off between sperm motility and sperm longevity. Mouseejaculates containing faster swimming sperm exhibit reducedlongevities (Firman and Simmons 2010). Therefore, secondmales would have greater opportunity to gain fertilizationsand sire offspring when first-mated males have fast swimmingsperm that die quickly. A trade-off between sperm velocityand sperm longevity might be particularly pertinent ininfluencing the outcome of sperm competition when the delaybetween matings is quite considerable, as was the case in thecurrent study.

In conclusion, I found no evidence that female sandy inlandmice accrue fitness benefits from mating with more than onemale. Microsatellite data revealed that paternity was skewedtoward males that mated first, suggesting that the copulatoryplug contributes to restricting subsequent males from gainingfertilizations. Analyses of sperm traits revealed that paternityloss to first-mated males was influenced by their own spermdensity and sperm quality, but not that of their rivals. Thisstudy highlights the importance that mating position has onmale fitness in mammalian sperm competition.

Acknowledgments I thank Shresta Lobind for assistance with animalhusbandry. This research was funded by the Fortescue Metals Group Ltd.and the Australian Research Council (Australian Postdoctoral Fellowship).

288 Behav Ecol Sociobiol (2014) 68:283–290

Ethical standards The author declares that this research complies withthe current laws of the country in which it was performed (Australia). Theresearch was approved by the University of Western Australia animalethics committee (approval number: 07/100/607).

References

Baker RJ, Makova KD, Chesser RK (1999) Microsatellites indicate ahigh frequency of multiple paternity in Apodemus (Rodentia). MolEcol 8:107–111

Barker DM (1994) Copulatory plugs and paternity assurance in thenematode Caenorhabditis elegans. Anim Behav 48:147–156

Bateman AJ (1948) Intra-sexual selection in Drosophila. Heredity 2:349–368

Birkhead TR, Martinez JG, Burke T, Froman DP (1999) Sperm mobilitydetermines the outcome of sperm competition in the domestic fowl.Proc R Soc Lond B 266:1759–1764

BreedWG (1990) Comparative studies on the timing of reporduction andfoetal number in six species of Australian conilurine rodents(Muridae: Hydromyinae). J Zool 221:1–10

Breed WG, Adams M (1992) Breeding systems of spinifix hopping mice(Notomys alexis) and plains rats (Pseudomys australis): a test formultiple paternity in the laboratory. Aust J Zool 40:13–20

Breed B, Ford F (2007) Native mice and rats. CSIRO Publishing,Collingwood, Victoria

BretmanA, Tregenza T (2005)Measuring polyandry in wild populations:a case study using promiscuous crickets. Mol Ecol 14:2169–2179

Bretman A, Wedell N, Tregenza T (2004) Molecular evidence of post-copulatory inbreeding avoidance in the field cricket Gryllusbimaculatus. Proc R Soc Lond B 271:159–164

Byers SL, Wiles MV, Dunn SL, Taft RA (2012) Mouse estrous cycleidentification tool and images. PLoS ONE 7:e35538

Clutton-Brock TH (1989) Mammalian mating systems. Proc R Soc LondB 236:339–372

Daly M (1978) The cost of mating. Am Nat 112:771–774Dean MD (2013) Genetic disruption of the copulatory plug in mice leads

to severely reduced fertility. PLoS Genet 9:e1003185Dean MD, Ardlie KG, Nachman MW (2006) The frequency of multiple

paternity suggests that sperm competition is common in house mice(Mus domesticus). Mol Ecol 15:4141–4151

delBarco-Trillo J, Ferkin MH (2006) Male meadow voles responddifferently to risk and intensity of sperm competition. Behav Ecol17:581–585

Devine MC (1975) Copulatory plugs in snakes: enforced chastity.Science 187:844–845

Dewsbury DA (1982) Ejaculate cost and male choice. Am Nat 119:601–610

Dixson AF, Anderson MJ (2002) Sexual selection, seminal coagulationand copulatory plug formation in primates. Folia Primatol 73:63–69

Eberhard WG (1996) Female control: sexual selection by cryptic femalechoice. Princeton University Press, Princeton

Firman RC (2011) Polyandrous females benefit by producing sons thatachieve high reproductive success in a competitive environment.Proc R Soc Lond B 278:2823–2831

Firman RC, Simmons LW (2008a) Polyandry facilitates postcopulatoryinbreeding avoidance in house mice. Evolution 62:601–611

Firman RC, Simmons LW (2008b) Polyandry, sperm competition, andreproductive success in mice. Behav Ecol 19:695–702

Firman RC, Simmons LW (2008c) The frequency of multiple paternitypredicts variation in testes size among island populations of housemice. J Evol Biol 21:1524–1533

Firman RC, Simmons LW (2010) Sperm midpiece length predicts spermswimming velocity in house mice. Biol Lett 6:513–516

Firman RC, Simmons LW (2011) Experimental evolution of spermcompetitiveness in a mammal. BMC Evol Biol 11:19

Firman RC, Simmons LW (2012) Male house mice evolving with post-copulatory sexual selection sire embryos with increased viability.Ecol Lett 15:42–46

Firman RC, Bentley B, Bowman F, Garcia-SolisMarchant F, Parthenay J,Sawyer J, Stewart T, O’Shea JE (2013a) No evidence of spermconjugate formation in sn Australian mouse bearing sperm withthree hooks. Ecol Evol 3:1856–1863

Firman RC, Klemme I, Simmons LW (2013b) Strategic adjustments insperm production within and between two island populations ofhouse mice. Evolution 67:3061–3070

Fisher DO, DoubleMC, Blomberg SP, JennionsMD, Cockburn A (2006)Post-mating sexual selection increase lifetime fitness of polyandrousfemales in the wild. Nature 444:89–92

Gage MJG (1991) Risk of sperm competition directly affects ejaculatesize in the Mediterranean fruit-fly. Anim Behav 42:1036–1037

García-González F (2008) The relative nature of fertilization success:Implications for the study of post-copulatory sexual selection. BMCEvol Biol 8:140

García-González F, Simmons LW (2007) Shorter sperm confer highercompetitive fertilization success. Evolution 61:816–824

Gasparini C, Simmons LW, Beveridge M, Evans JP (2010) Spermswimming velocity predicts competitive fertilization success in thegreen swordtail Xiphophorus helleri . PLoS ONE 5:1–5

Gomendio M, Harcourt AH, Roldan ERS (1998) Sperm competition inmammals. In: Sperm competition and sexual selection. AcademicPress, London, pp 667–781

Hosken DJ, Garner TWJ, Tregenza T,Wedell N,Ward PI (2003) Superiorsperm competitors sire higher-quality young. Proc R Soc Lond B270:1933–1938

Huck UW, Banks EM, Coopersmith CB (1984) Social olfaction in malebrown lemmings (Lemmus sibiricus=trimucronatus) and collaredlemmings (Dicrostonyx groenlandicus): II. Discrimination of matedand unmated females. J Comp Psychol 98:60–65

Huck UW, Quinn RP, Lisk RD (1985) Determinants of mating success inthe golden hamster (Mesocricetus auratus) iv. Sperm competition.Behav Ecol Sociobiol 17:239–252

Jones R (1999) To store or mature sperm? The primary role of theepididymis. Int J Androl 22:57–67

Klemme I, Firman RC (2013) Male house mice that have evolved withsperm competition have increased mating duration and paternitysuccess. Anim Behav 85:751–758

Klemme I, Ylonen H, Eccard JA (2008) Long-term fitness benefits ofpolyandry in a small mammal, the bank vole Clethrionomysglareolus . Proc R Soc Lond B 275:1095–1100

Kraaijeveld-Smit FJL, Ward SJ, Temple-Smith PD (2002) Multiplepaternity in a field population of a small carnivorous marsupial, theagile antechinus, Antechinus agilis . Behav Ecol Sociobiol 52:84–91

Mardia KV, Kent JT, Bibby JM (1979) Multivariate analysis. AcademicPress, London

Moro D, Spencer PBS (2003) Microsatellite primers for the WesternPebble-mound Mouse (Pseudomys chapmani ) that show crossamplification for other species of Australian rodent. Mol EcolNotes 3:259–261

Nakagawa S (2004) A farewell to Bonferroni: the problems of lowstatistical power and publication bias. Behav Ecol 15:1044–1045

Parker GA (1970) Sperm competition and its evolutionary consequencesin the insects. Biol Rev 45:525–567

Parker GA (1990) Sperm competition games: raffles and roles. Proc RSoc Lond B 242:120–126

Pizzari T, Cornwallis CK, Løvlie H, Jakobsson S, Birkhead TR (2003)Sophisticated sperm allocation in male fowl. Nature 426:70–74

Ramm SA, Stockley P (2007) Ejaculate allocation under varying spermcompetition risk in the house mouse, Mus musculus domesticus .Behav Ecol 18:491–495

Behav Ecol Sociobiol (2014) 68:283–290 289

Ramm SA, Stockley P (2009) Adaptive plasticity of mammalian spermproduction in response to social experience. Proc R Soc LondB 276:745–751

Ramm SA, Parker GA, Stockley P (2005) Sperm competition and theevolution of male reproductive anatomy in rodents. Proc R SocLond B 272:949–955

Rugh R (1968) The mouse: its reproduction and development. BurgessPublishing Company, Minneapolis

Simmons LW (2005) The evolution of polyandry: sperm competition,sperm selection, and offspring viability. Annu Rev Ecol Evol Syst36:125–146

Simmons LW, Beveridge M, Wedell N, Tregenza T (2006)Postcopulatory inbreeding avoidance by female crickets onlyrevealed by molecular markers. Mol Ecol 15:3817–3824

Simmons LW, Beveridge M, Kennington J (2007) Polyandry in the wild:temporal changes in female mating frequency and sperm

competition intensity in natural populations of the tettigoniidRequena verticalis. Mol Ecol 16:4613–4623

Thornhill R, Alcock J (1983) The evolution of insect mating systems.Harvard University Press, Cambridge

Trivers RL (1972) Parental investment and sexual selection. In: CampbellB (ed) Sexual Selection and the Descent of Man. Aldine-Atherton,Chicago, pp 136–179

Voss R (1979) Male accessory glands and the evolution of copulatoryplugs in rodents. Occ Pap Mus Zool Univ Mich 689:1–17

Williams-Ashman HG (1984) Transglutaminases and the clotting ofmammalian seminal fluids. Mol Cell Biochem 58:51–61

Yasui Y (1997) A ‘good sperm’model can explain the evolution of costlymultiple mating by females. Am Nat 149:573–584

Zeh JA, Zeh DW (1997) The evolution of polyandry II: post-copulatorydefences against genetic incompatibility. Proc R Soc Lond B 264:69–75

290 Behav Ecol Sociobiol (2014) 68:283–290