BACKGROUND
mating systems in nature range from strict monogamy across a lifespan to mating with many mating partners  <cit> .
striking natural variation in the number of mates is often observed between closely related species and even within species .
this variation has important implications for a wide array of evolutionary and ecological processes, including sexual selection, sexual conflict, social organization and evolution, life history evolution, sexually-transmitted pathogen dynamics, speciation, sperm competition and cryptic female choice .
as a result, there is great potential for variation in the number of mates to drive phenotypic evolution in both sexes.
because the number of mates and mating frequency are the outcome of interacting male and female mating strategies, understanding how mating systems evolve requires characterizing optimal mating strategies in both sexes and how they co-evolve between the sexes.
a powerful tool for investigating the evolutionary consequences of variation in mate numbers is experimental evolution.
several studies have adopted this approach by evolving populations at natural or restricted numbers of partners.
these studies have revealed that variation in mating frequency can drive the evolution of a wide range of phenotypes, including mating behaviour , sperm competitiveness  <cit> , genital and gonad size , cuticular hydrocarbons  <cit> , mate harming and resistance to harm  <cit> , cognitive function  <cit> , and sex-specific transcription  <cit> .
however, we know little about the evolutionary consequences of increased mating frequency.
this is because restricting mating is experimentally tractable, but increasing mating frequency is challenging because high mating frequencies are constrained by limited sexual receptivity in one or both sexes.
to overcome this limitation, several studies have experimentally manipulated ecological settings to create conditions favouring higher mating frequencies.
for example, mating frequency often increases when the sex with the highest sexual receptivity  is rarer in a population .
these studies demonstrate that when the ecological setting favours increased mating, evolutionary change in mating and life history traits can occur, including increased investment in male reproductive organs  <cit> , increased courtship frequency  <cit> , decreased male ejaculate depletion  <cit> , increased male stimulation of female oviposition after mating  <cit> , increased suppression of female receptivity  <cit> , and decreased male but increased female lifespan  <cit> .
yet, the evolutionary consequences of increases in mating frequency that are driven by changes in mating behaviour, independent of environmental variation, remain unknown.
here, we use a genetic manipulation of the fruit fly drosophila melanogaster to experimentally increase mating frequencies in experimental populations.
we allowed the populations to evolve under these hyper-promiscuous mating conditions to explore the evolutionary consequences for both sexes.
to manipulate mating frequencies, we used flies deficient in the sex peptide receptor  gene  <cit> , which encodes a female receptor for the male seminal protein, sex peptide   <cit> .
sex peptide elicits several post-mating responses in females via the spr, including inhibiting female sexual receptivity  <cit> .
thus, females that lack the spr  have a greatly reduced refractory period after mating   <cit> .
when spr is removed from populations, males are less able to prevent females from re-mating, creating a novel, highly promiscuous mating system.
the increase in mating frequency in spr- populations is within the range of naturally occurring mating frequencies found in closely related, more promiscuous drosophilid species.
we allowed spr- and control populations to evolve for  <dig> generations.
we first confirmed that mating frequencies were elevated in spr- populations and tracked changes in mating frequency over evolutionary time.
we then assayed behavioural, morphological and transcriptional phenotypes expected to evolve in response to increased population mating frequencies, at several time points during experimental evolution.
theory suggests that, under increased promiscuity, selection on males should shift from pre-copulatory  to post-copulatory , and that males should decrease per-mating ejaculate investment because the returns from each mating are likely to be lower .
we therefore tested for experimentally evolved differences in both pre- and post-copulatory phenotypes in males, including courtship behaviour, copulation duration, effects on female post-mating fecundity and re-mating behaviour, testes and accessory gland size, and success in sperm competition.
because males in spr- populations cannot influence female re-mating through sp, male investment in sp should decrease, as should male investment in the related seminal protein ductus ejaculatorius peptide 99b , which also binds to spr  <cit>  and suppresses female post-mating receptivity .
to this end, we investigated the expression of both sp and dup99b genes using rt-qpcr.
we also assayed female mating strategies – including female latency to mating, post-mating fecundity and re-mating behaviour – to determine how the reproductive behaviour of the sexes co-evolves under the novel mating regime.
RESULTS
we experimentally evolved  <dig> replicate spr- populations  and  <dig> genetically matched control populations, from a dahomey stock into which the w <dig> allele for white eye colour had been backcrossed to facilitate tracking the spr deficiency.
we introduced the spr deficiency to the stock population by backcrossing in the deletion dfexel <dig> , which carries a white + transgene that partially rescues the w <dig> mutation.
thus, the  <dig> spr- populations bore the spr deficiency and the w + transgene and had red eyes, whereas the  <dig> control populations bore wild-type spr and had white eyes.
white-eyed flies have reduced vision relative to wild-type  eyes  <cit> ; therefore, the red-eyed spr- flies likely had partially improved visual performance compared with controls.
for each replicate population, each generation began with  <dig> adult males and  <dig> adult females, which were permitted to interact in chambers for 9 days before progeny were collected for the next generation.
initial tests of male courtship behaviour revealed that spr- and white-eyed control males had comparable courtship rates, whereas wild-type males courted females significantly more  than both spr- and white-eyed males .
due to this substantial difference in courtship behaviour we therefore did not include wild-type populations in the subsequent experimental evolution.
for most phenotypes, we first used unselected spr- and control flies to establish baseline differences caused by the spr deficiency itself or by the difference in eye colour between control and spr- flies.
thus, ‘unselected spr-’ and ‘unselected control’ refer to flies that have not undergone experimental evolution, and for which any differences are a result of the genetic manipulation rather than evolution under varying levels of promiscuity.
we then tested for evolved differences between the replicate spr- and control populations.
behavioural evolution
for most behavioural phenotypes, we first tested for effects of the spr deficiency itself in both sexes.
we then tested for evolved differences in pairings between  experimentally-evolved males and wild-type females,  experimentally-evolved females and wild-type males, and  experimentally-evolved males and females within treatments .table  <dig> summary of tests for phenotypic differences related to experimental evolution under high promiscuity
phenotypes tested for differences between sex peptide receptor deficient  flies and controls that expressed spr .
we tested experimental males with wild-type females, experimental females with wild-type males, or within-treatment tests of spr- or control males and females paired within replicate line
mating frequency
as expected, we observed mating ~15-fold more frequently in populations of spr- males and spr- females, compared with control populations .
this difference was maintained during experimental evolution despite overall declining mating frequency .fig.
<dig> mating frequency over the course of experimental evolution.
the percentage of time in which flies were observed mating in replicate populations  in which flies did  or did not express sex peptide receptor .
the proportion of time in copula for spr- populations relative to control populations
courtship frequency
unselected spr- males  courted wild-type females more frequently than did unselected control males , likely due to improved vision in spr- populations relative to controls.
at generation  <dig>  experimentally-evolved spr- males similarly courted females more frequently than control males, although the difference was not statistically significant .
however, at generation  <dig> we detected no difference nor any trend for experimentally-evolved spr- males to court more frequently , suggesting an evolved loss of courtship activity in the hyper-promiscuous spr- lines relative to control lines.table  <dig> male courtship frequency
effects of spr- deficiencya
acourtship frequency varied between blocks
effects of the spr- deficiency itself in unselected flies, or experimental evolution in spr- and control populations, on the number of courtship events observed during male courtship of wild-type females
latency until mating
unselected spr- males were faster to mate with wild-type females, at both a male’s first mating and fifth consecutive mating , as expected from the relatively better vision of spr- males compared with controls.
although experimentally-evolved spr- males were also faster to achieve first matings at generation  <dig>  we detected no difference at generation  <dig> nor any trend for spr- males to mate faster.
at generation  <dig>  the pattern had reversed, with experimentally-evolved control males being faster than spr- males to achieve a fifth mating .
this suggests that spr- males lost their mating speed advantage  over evolutionary time.table  <dig> latency until mating
p
experimental evolution of malesb
amales and females were spr- or genetically matched controls , or wild-type
bfull model details for generation  <dig> are given in additional file  <dig>
effects of the spr- deficiency itself in unselected flies, or experimental evolution in spr- and control populations, on latency until a first mating by  experimental males  paired with wild-type females,  experimental females paired with wild-type males, or  experimental males and females paired within treatment and replicate population.
risk ratios >  <dig> indicate that spr- males were faster to mate; values <  <dig> indicate control males were faster
we found no evidence for evolutionary change in latency to mating in either spr- females or in within-treatment pairings.
spr- females were faster to mate with wild-type males than control females in both unselected  and experimentally-evolved flies, with a similar magnitude of difference .
likewise, spr- males and females paired together were faster to mate than controls in both unselected and experimentally evolved flies .
copulation duration
the spr deficiency itself did not alter male copulation duration in unselected flies, but experimentally-evolved spr- males had shorter copulations than controls .
in contrast, the spr deficiency itself caused a shorter female copulation duration, but experimentally-evolved spr- and control females did not differ .
in within-treatment pairings, we found only weak evidence that the spr deficiency itself affected copulation duration .
however, experimentally-evolved spr- males and females had significantly shorter matings .
the above results are for matings between virgin flies; we found no differences in copulation duration in second matings .fig.
<dig> copulation duration in unselected and experimentally evolved spr- and control flies.
copulation duration measured in flies that either did  or did not express sex peptide receptor , for unselected flies  and experimentally-evolved selected flies.
dotted lines demarcate different comparisons where we tested for experimentally evolved differences in males by pairing spr- or c males with wild-type females , in females by pairing spr- or c females with wild-type males , or in within-treatment pairings of spr-  males with spr-  females .
asterisks indicate significant differences between spr- and c flies.
least squares means are presented with 95 % confidence intervals
p
effects of spr- deficiency in malesb
amales and females were spr- or genetically matched controls , or wild-type
bcopulation duration did not vary between blocks
effects of the spr- deficiency itself in unselected flies, or experimental evolution in spr- and control populations, on copulation duration in virgin flies for  experimental males paired with wild-type females,  experimental females paired with wild-type males, or  experimental males and females paired within treatment and replicate population
post-mating fecundity
neither unselected nor experimentally-evolved spr- and control males differed in their ability to stimulate female oviposition .
when expressed in females, the spr deficiency itself reduced fecundity in unselected flies, as expected given sp’s role in fecundity stimulation  <cit>  .
although we hypothesized that experimentally evolved spr- females would evolve relatively increased post-mating fecundity, through selection to decrease reliance on sp, the difference in egg-laying at generation  <dig> was similar to that caused by the spr deficiency in unselected flies .
the spr- deficiency did not alter male fertility , suggesting that fertility differences did not confound the sperm competition experiments .
re-mating behaviour
as expected given that spr is not known to impact male ejaculate parameters, the spr deficiency did not affect male ability to inhibit re-mating by their wild-type mates, whether measured as latency to re-mating  or  as the number of females re-mating .
although experimentally-evolved males likewise did not differ in ability to inhibit re-mating at generation  <dig>  spr- males were significantly worse at inhibiting re-mating at generation  <dig> .table  <dig> re-mating behaviour
p
effects of spr- deficiency in malesb
experimental evolution of males, generation 36b
amales and females were spr- or genetically matched controls , or wild-type
bfull model details are given in additional file 1: table s4
effects of the spr- deficiency itself in unselected flies, or experimental evolution in spr- and control populations, on  experimental male ability to inhibit re-mating by wild-type females, or  experimental female propensity to re-mate with wild-type males following a mating with a  wild-type male or  experimental male of the same treatment group
surprisingly – given that sp inhibits female re-mating – when the spr- deficiency was expressed in unselected females, we did not detect significant changes in latency until re-mating ; nonetheless, the confidence interval for the difference between groups is  <dig>  –  <dig>  , indicating no strong support for rejecting the null hypothesis of no difference.
however, experimentally-evolved spr- females re-mated more quickly after mating with wild-type males .
in within-treatment pairings, we found the expected faster re-mating following matings between spr- males and spr- females, compared with control males and control females, with a similar magnitude of difference for both unselected and experimentally-evolved flies .
morphological evolution
evolving in populations that lacked spr had no significant effect on testes or accessory gland size – neither on absolute organ size nor on body mass-corrected size – or on body mass, nor did the spr deficiency itself influence these traits .
we tested whether the spr deficiency influenced female mass as a test for pleiotropic effects of the deficiency, but found no difference .
ejaculate evolution
sperm precedence as first male to mate
neither the spr deficiency itself nor evolution in spr- populations affected either male success in sperm competition as the first male to mate  or male ability to maintain p <dig> over multiple matings .table  <dig> sperm precedence
p
p1a
p2b
afull model results in additional file 1: table s6
bfull model results in additional file 1: table s7
effects of the spr- deficiency itself, or experimental evolution in spr- and control populations, on the proportion of offspring sired by a focal male as the first or second male to mate with a wild-type female .
p <dig> tests include a male’s first and fifth mating to test for differences in male ejaculate depletion across successive matings.
effects of the spr deficiency itself were evaluated in unselected flies or in experimentally evolved backcrossed or outcrossed controls
sperm precedence as second male to mate
we found mixed evidence that the spr deficiency itself affected male success in sperm competition as the second male to mate , but experimentally evolved spr- and control males showed little difference .
sex peptide and dup99b gene expression evolution
surprisingly, we found a trend towards higher sp expression in experimentally evolved spr- males, compared with controls .
in contrast, there was no significant difference in dup99b expression .
characteristics of the rt-qpcr calibration curves are given in additional file  <dig> .fig.
<dig> experimental evolution of sex peptide  and dup99b gene expression.
mean levels of gene expression of  sp and  dup99b in four experimentally evolved replicate populations each of the sex peptide receptor-deficient  treatment  and the control treatment.
gene expression was measured at generation  <dig>  error bars represent ± 2 s.e
discussion
our study investigated the evolutionary consequences of increased mating frequencies above naturally occurring levels.
our main findings are that, after evolution at high mating frequency in spr- populations, males from these populations evolved shorter copulation duration and decreased ability to inhibit female re-mating.
these changes are consistent with decreased male post-copulatory investment at each mating.
surprisingly, males from spr- populations also tended to show an evolved increase in sp gene expression, and we discuss hypotheses that might explain this pattern below.
furthermore, although spr- males initially courted females more frequently and mated faster than controls, we found that after experimental evolution these differences were no longer detectable  or had reversed , results consistent with decreased pre-copulatory investment.
reduced ejaculate investment
sperm competition theory predicts that males should decrease their investment in ejaculate at each mating as the intensity of sperm competition  increases because the returns from investing in a given mating decrease .
we confirmed that mating frequency was increased in spr- populations, such that sperm competition would have been intensified if increased mating frequency resulted in greater temporal overlap of ejaculates from multiple males  .
this is an especially likely scenario here because spr regulates female sperm storage, such that spr- females retain more sperm in the days after mating  <cit> .
two lines of evidence support the prediction that per-mating ejaculate investment decreased in males from spr- populations.
first, spr- males evolved decreased copulation duration compared with control males.
copulation duration in matings with virgin females is positively correlated with ejaculate transfer in drosophila spp.
, suggesting that spr- males might transfer less ejaculate per mating.
second, spr- males were less able to inhibit female re-mating, consistent with the hypothesis that they transfer fewer receptivity-inhibiting seminal proteins.
below, we discuss hypotheses to reconcile this result with our finding of increased sp gene expression for these males.
although these results are consistent with decreased per-mating ejaculate investment, we did not find differences in either male stimulation of female fecundity or success in sperm competition.
low statistical power is a potential concern, but our sample sizes were sufficient to allow detection of some effects of the spr deficiency itself on these traits.
taken together, these changes in ejaculate-related traits are consistent with decreased investment in some ejaculate components more than others and a shift in ejaculate composition  <cit> .
expression of sex peptide
if expressing sp is costly, then males co-evolving with spr- females should evolve lower sp gene expression because sp is functionless with these females.
there are several potential explanations for our finding that sp expression was weakly increased, in addition to the possibility that the marginally non-significant result  arose by chance.
first, expressing sp may not be costly for males; however, this alone does not explain increased sp expression in spr- males.
second, sp expression measured in males might not reflect sp protein transfer to females at mating.
indeed, a recent study found no correlation between the two  <cit> .
sp expression in males could instead reflect male sp stores, with larger stores evolving to increase male capacity to mate many times in spr- populations.
however, we detected no change in accessory gland size, nor evidence that spr- males could better sustain ejaculate transfer over multiple matings, as spr- and control males had similar success in sperm competition in first and fifth matings.
third, if sp expression is in fact positively correlated with sp protein transfer, then increased sp expression in spr- males appears inconsistent with our finding of decreased ability of spr- males to inhibit female re-mating.
however, if functionality of the sp protein degraded through genetic drift, a likely scenario given that sp is effectively neutral in spr- lines, this inconsistency is reconciled.
under this scenario, there are two potential explanations for increased sp gene expression.
first, sp expression might be genetically correlated with other ejaculate components under positive selection .
a second mechanism relates to sp protein structure, which consists of a carboxy-terminal end that binds to spr and mediates female post-mating oviposition and re-mating responses, and an amino-terminal end that does not bind to spr but to unknown female receptors  <cit> .
positive selection on the amino-terminal end might occur in spr- populations to compensate for the lack of oviposition stimulation from the carboxy-terminal end, because the amino-terminal end induces juvenile hormone synthesis in adult females and stimulates oogenesis .
we did not find that spr- males increased the post-mating fecundity of wild-type females, although the wild-type female fecundity response might be dominated by carboxy-terminal end effects.
this hypothesis is consistent with our finding of no increase in dup99b expression, as dup99b is homologous with sp only at the carboxy-terminal end.
finally, sp might have other as yet unknown functions that are selected positively under high promiscuity.
evidence for decreased pre-copulatory performance
we found two effects on pre-copulatory performance that were most likely caused by the improved vision of red-eyed spr- males relative to white-eyed controls: unselected spr- males courted females more frequently and were faster to mate.
both effects remained detectable at generation  <dig>  but by generation  <dig>  both were either not statistically significant or significant and reversed .
although type ii error is a possibility such that we simply failed to detect the same differences, the trends were no longer in the previous direction: at generation  <dig>  mean courtship frequency was higher in control males and the risk ratio for latency to mating was  <dig> .
the results are consistent with decreased pre-copulatory investment in spr- males, which might result from weaker selection to overcome re-mating resistance with spr- females.
declining courtship frequency might be accompanied by decreased courtship intensity or quality of courtship signals.
furthermore, if the increased mating frequency in spr- populations caused sexual selection to shift from pre-copulatory to post-copulatory, we would expect increased displays of post-mating guarding behaviour  in spr- males.
we are currently investigating these hypotheses.
CONCLUSIONS
our study shows that spr- males evolving at high promiscuity evolve shorter copulations and decreased inhibition of female re-mating, consistent with decreased post-copulatory investment per mating, and less frequent courtship and longer latency until mating, consistent with decreased pre-copulatory investment.
curiously, males that evolved with spr- females also showed a tendency for increased sp expression.
previous experimental evolution studies have demonstrated that reducing a population’s mating frequency by imposing monogamy can result in evolutionary change in reproductive traits .
other studies have increased or decreased mating frequency by altering the sex ratio , but changes in sex ratio tend to alter mating frequency and mate competition in opposite directions .
the effects of increased promiscuity per se, as observed in our study, seem to be different from simultaneously changing mating frequency and mate competition.
our study highlights the utility of genetic manipulations of the socio-sexual environment, and the sensitivity of experimental evolution studies of mating system to the method by which mating frequency is manipulated.

 mating systems in nature range from strict monogamy across a lifespan to mating with many mating partners. striking natural variation in the number of mates is often observed between closely related species and even within species. this variation has important implications for a wide array of evolutionary and ecological processes, including sexual selection, sexual conflict, social organization and evolution, life history evolution, sexually-transmitted pathogen dynamics, speciation, sperm competition and cryptic female choice. as a result, there is great potential for variation in the number of mates to drive phenotypic evolution in both sexes. because the number of mates and mating frequency are the outcome of interacting male and female mating strategies, understanding how mating systems evolve requires characterizing optimal mating strategies in both sexes and how they co-evolve between the sexes. a powerful tool for investigating the evolutionary consequences of variation in mate numbers is experimental evolution. several studies have adopted this approach by evolving populations at natural or restricted numbers of partners. these studies have revealed that variation in mating frequency can drive the evolution of a wide range of phenotypes, including mating behaviour, sperm competitiveness, genital and gonad size, cuticular hydrocarbons, mate harming and resistance to harm, cognitive function, and sex-specific transcription. however, we know little about the evolutionary consequences of increased mating frequency. this is because restricting mating is experimentally tractable, but increasing mating frequency is challenging because high mating frequencies are constrained by limited sexual receptivity in one or both sexes. to overcome this limitation, several studies have experimentally manipulated ecological settings to create conditions favouring higher mating frequencies. for example, mating frequency often increases when the sex with the highest sexual receptivity is rarer in a population. these studies demonstrate that when the ecological setting favours increased mating, evolutionary change in mating and life history traits can occur, including increased investment in male reproductive organs, increased courtship frequency, decreased male ejaculate depletion, increased male stimulation of female oviposition after mating, increased suppression of female receptivity, and decreased male but increased female lifespan. yet, the evolutionary consequences of increases in mating frequency that are driven by changes in mating behaviour, independent of environmental variation, remain unknown. here, we use a genetic manipulation of the fruit fly drosophila melanogaster to experimentally increase mating frequencies in experimental populations. we allowed the populations to evolve under these hyper-promiscuous mating conditions to explore the evolutionary consequences for both sexes. to manipulate mating frequencies, we used flies deficient in the sex peptide receptor gene, which encodes a female receptor for the male seminal protein, sex peptide. sex peptide elicits several post-mating responses in females via the spr, including inhibiting female sexual receptivity. thus, females that lack the spr have a greatly reduced refractory period after mating. when spr is removed from populations, males are less able to prevent females from re-mating, creating a novel, highly promiscuous mating system. the increase in mating frequency in spr- populations is within the range of naturally occurring mating frequencies found in closely related, more promiscuous drosophilid species. we allowed spr- and control populations to evolve for generations. we first confirmed that mating frequencies were elevated in spr- populations and tracked changes in mating frequency over evolutionary time. we then assayed behavioural, morphological and transcriptional phenotypes expected to evolve in response to increased population mating frequencies, at several time points during experimental evolution. theory suggests that, under increased promiscuity, selection on males should shift from pre-copulatory to post-copulatory, and that males should decrease per-mating ejaculate investment because the returns from each mating are likely to be lower. we therefore tested for experimentally evolved differences in both pre- and post-copulatory phenotypes in males, including courtship behaviour, copulation duration, effects on female post-mating fecundity and re-mating behaviour, testes and accessory gland size, and success in sperm competition. because males in spr- populations cannot influence female re-mating through sp, male investment in sp should decrease, as should male investment in the related seminal protein ductus ejaculatorius peptide 99b, which also binds to spr and suppresses female post-mating receptivity. to this end, we investigated the expression of both sp and dup99b genes using rt-qpcr. we also assayed female mating strategies – including female latency to mating, post-mating fecundity and re-mating behaviour – to determine how the reproductive behaviour of the sexes co-evolves under the novel mating regime.  we experimentally evolved replicate spr- populations and genetically matched control populations, from a dahomey stock into which the w allele for white eye colour had been backcrossed to facilitate tracking the spr deficiency. we introduced the spr deficiency to the stock population by backcrossing in the deletion dfexel, which carries a white + transgene that partially rescues the w mutation. thus, the spr- populations bore the spr deficiency and the w + transgene and had red eyes, whereas the control populations bore wild-type spr and had white eyes. white-eyed flies have reduced vision relative to wild-type eyes; therefore, the red-eyed spr- flies likely had partially improved visual performance compared with controls. for each replicate population, each generation began with adult males and adult females, which were permitted to interact in chambers for 9 days before progeny were collected for the next generation. initial tests of male courtship behaviour revealed that spr- and white-eyed control males had comparable courtship rates, whereas wild-type males courted females significantly more than both spr- and white-eyed males. due to this substantial difference in courtship behaviour we therefore did not include wild-type populations in the subsequent experimental evolution. for most phenotypes, we first used unselected spr- and control flies to establish baseline differences caused by the spr deficiency itself or by the difference in eye colour between control and spr- flies. thus, ‘unselected spr-’ and ‘unselected control’ refer to flies that have not undergone experimental evolution, and for which any differences are a result of the genetic manipulation rather than evolution under varying levels of promiscuity. we then tested for evolved differences between the replicate spr- and control populations. behavioural evolution for most behavioural phenotypes, we first tested for effects of the spr deficiency itself in both sexes. we then tested for evolved differences in pairings between experimentally-evolved males and wild-type females, experimentally-evolved females and wild-type males, and experimentally-evolved males and females within treatments.table summary of tests for phenotypic differences related to experimental evolution under high promiscuity     phenotypes tested for differences between sex peptide receptor deficient flies and controls that expressed spr. we tested experimental males with wild-type females, experimental females with wild-type males, or within-treatment tests of spr- or control males and females paired within replicate line mating frequency as expected, we observed mating ~15-fold more frequently in populations of spr- males and spr- females, compared with control populations. this difference was maintained during experimental evolution despite overall declining mating frequency.fig. mating frequency over the course of experimental evolution. the percentage of time in which flies were observed mating in replicate populations in which flies did or did not express sex peptide receptor. the proportion of time in copula for spr- populations relative to control populations courtship frequency unselected spr- males courted wild-type females more frequently than did unselected control males, likely due to improved vision in spr- populations relative to controls. at generation experimentally-evolved spr- males similarly courted females more frequently than control males, although the difference was not statistically significant. however, at generation we detected no difference nor any trend for experimentally-evolved spr- males to court more frequently, suggesting an evolved loss of courtship activity in the hyper-promiscuous spr- lines relative to control lines.table male courtship frequency effects of spr- deficiencya acourtship frequency varied between blocks effects of the spr- deficiency itself in unselected flies, or experimental evolution in spr- and control populations, on the number of courtship events observed during male courtship of wild-type females latency until mating unselected spr- males were faster to mate with wild-type females, at both a male’s first mating and fifth consecutive mating, as expected from the relatively better vision of spr- males compared with controls. although experimentally-evolved spr- males were also faster to achieve first matings at generation we detected no difference at generation nor any trend for spr- males to mate faster. at generation the pattern had reversed, with experimentally-evolved control males being faster than spr- males to achieve a fifth mating. this suggests that spr- males lost their mating speed advantage over evolutionary time.table latency until mating p experimental evolution of malesb amales and females were spr- or genetically matched controls, or wild-type bfull model details for generation are given in additional file effects of the spr- deficiency itself in unselected flies, or experimental evolution in spr- and control populations, on latency until a first mating by experimental males paired with wild-type females, experimental females paired with wild-type males, or experimental males and females paired within treatment and replicate population. risk ratios >  indicate that spr- males were faster to mate; values <  indicate control males were faster we found no evidence for evolutionary change in latency to mating in either spr- females or in within-treatment pairings. spr- females were faster to mate with wild-type males than control females in both unselected and experimentally-evolved flies, with a similar magnitude of difference. likewise, spr- males and females paired together were faster to mate than controls in both unselected and experimentally evolved flies. copulation duration the spr deficiency itself did not alter male copulation duration in unselected flies, but experimentally-evolved spr- males had shorter copulations than controls. in contrast, the spr deficiency itself caused a shorter female copulation duration, but experimentally-evolved spr- and control females did not differ. in within-treatment pairings, we found only weak evidence that the spr deficiency itself affected copulation duration. however, experimentally-evolved spr- males and females had significantly shorter matings. the above results are for matings between virgin flies; we found no differences in copulation duration in second matings.fig. copulation duration in unselected and experimentally evolved spr- and control flies. copulation duration measured in flies that either did or did not express sex peptide receptor, for unselected flies and experimentally-evolved selected flies. dotted lines demarcate different comparisons where we tested for experimentally evolved differences in males by pairing spr- or c males with wild-type females, in females by pairing spr- or c females with wild-type males, or in within-treatment pairings of spr- males with spr- females. asterisks indicate significant differences between spr- and c flies. least squares means are presented with 95 % confidence intervals p effects of spr- deficiency in malesb amales and females were spr- or genetically matched controls, or wild-type bcopulation duration did not vary between blocks effects of the spr- deficiency itself in unselected flies, or experimental evolution in spr- and control populations, on copulation duration in virgin flies for experimental males paired with wild-type females, experimental females paired with wild-type males, or experimental males and females paired within treatment and replicate population post-mating fecundity neither unselected nor experimentally-evolved spr- and control males differed in their ability to stimulate female oviposition. when expressed in females, the spr deficiency itself reduced fecundity in unselected flies, as expected given sp’s role in fecundity stimulation. although we hypothesized that experimentally evolved spr- females would evolve relatively increased post-mating fecundity, through selection to decrease reliance on sp, the difference in egg-laying at generation was similar to that caused by the spr deficiency in unselected flies. the spr- deficiency did not alter male fertility, suggesting that fertility differences did not confound the sperm competition experiments. re-mating behaviour as expected given that spr is not known to impact male ejaculate parameters, the spr deficiency did not affect male ability to inhibit re-mating by their wild-type mates, whether measured as latency to re-mating or as the number of females re-mating. although experimentally-evolved males likewise did not differ in ability to inhibit re-mating at generation spr- males were significantly worse at inhibiting re-mating at generation.table re-mating behaviour p effects of spr- deficiency in malesb experimental evolution of males, generation 36b amales and females were spr- or genetically matched controls, or wild-type bfull model details are given in additional file 1: table s4 effects of the spr- deficiency itself in unselected flies, or experimental evolution in spr- and control populations, on experimental male ability to inhibit re-mating by wild-type females, or experimental female propensity to re-mate with wild-type males following a mating with a wild-type male or experimental male of the same treatment group surprisingly – given that sp inhibits female re-mating – when the spr- deficiency was expressed in unselected females, we did not detect significant changes in latency until re-mating; nonetheless, the confidence interval for the difference between groups is –, indicating no strong support for rejecting the null hypothesis of no difference. however, experimentally-evolved spr- females re-mated more quickly after mating with wild-type males. in within-treatment pairings, we found the expected faster re-mating following matings between spr- males and spr- females, compared with control males and control females, with a similar magnitude of difference for both unselected and experimentally-evolved flies. morphological evolution evolving in populations that lacked spr had no significant effect on testes or accessory gland size – neither on absolute organ size nor on body mass-corrected size – or on body mass, nor did the spr deficiency itself influence these traits. we tested whether the spr deficiency influenced female mass as a test for pleiotropic effects of the deficiency, but found no difference. ejaculate evolution sperm precedence as first male to mate neither the spr deficiency itself nor evolution in spr- populations affected either male success in sperm competition as the first male to mate or male ability to maintain p over multiple matings.table sperm precedence p p1a p2b afull model results in additional file 1: table s6 bfull model results in additional file 1: table s7 effects of the spr- deficiency itself, or experimental evolution in spr- and control populations, on the proportion of offspring sired by a focal male as the first or second male to mate with a wild-type female. p tests include a male’s first and fifth mating to test for differences in male ejaculate depletion across successive matings. effects of the spr deficiency itself were evaluated in unselected flies or in experimentally evolved backcrossed or outcrossed controls sperm precedence as second male to mate we found mixed evidence that the spr deficiency itself affected male success in sperm competition as the second male to mate, but experimentally evolved spr- and control males showed little difference. sex peptide and dup99b gene expression evolution surprisingly, we found a trend towards higher sp expression in experimentally evolved spr- males, compared with controls. in contrast, there was no significant difference in dup99b expression. characteristics of the rt-qpcr calibration curves are given in additional file.fig. experimental evolution of sex peptide and dup99b gene expression. mean levels of gene expression of sp and dup99b in four experimentally evolved replicate populations each of the sex peptide receptor-deficient treatment and the control treatment. gene expression was measured at generation error bars represent ± 2 s.e discussion our study investigated the evolutionary consequences of increased mating frequencies above naturally occurring levels. our main findings are that, after evolution at high mating frequency in spr- populations, males from these populations evolved shorter copulation duration and decreased ability to inhibit female re-mating. these changes are consistent with decreased male post-copulatory investment at each mating. surprisingly, males from spr- populations also tended to show an evolved increase in sp gene expression, and we discuss hypotheses that might explain this pattern below. furthermore, although spr- males initially courted females more frequently and mated faster than controls, we found that after experimental evolution these differences were no longer detectable or had reversed, results consistent with decreased pre-copulatory investment. reduced ejaculate investment sperm competition theory predicts that males should decrease their investment in ejaculate at each mating as the intensity of sperm competition increases because the returns from investing in a given mating decrease. we confirmed that mating frequency was increased in spr- populations, such that sperm competition would have been intensified if increased mating frequency resulted in greater temporal overlap of ejaculates from multiple males. this is an especially likely scenario here because spr regulates female sperm storage, such that spr- females retain more sperm in the days after mating. two lines of evidence support the prediction that per-mating ejaculate investment decreased in males from spr- populations. first, spr- males evolved decreased copulation duration compared with control males. copulation duration in matings with virgin females is positively correlated with ejaculate transfer in drosophila spp., suggesting that spr- males might transfer less ejaculate per mating. second, spr- males were less able to inhibit female re-mating, consistent with the hypothesis that they transfer fewer receptivity-inhibiting seminal proteins. below, we discuss hypotheses to reconcile this result with our finding of increased sp gene expression for these males. although these results are consistent with decreased per-mating ejaculate investment, we did not find differences in either male stimulation of female fecundity or success in sperm competition. low statistical power is a potential concern, but our sample sizes were sufficient to allow detection of some effects of the spr deficiency itself on these traits. taken together, these changes in ejaculate-related traits are consistent with decreased investment in some ejaculate components more than others and a shift in ejaculate composition. expression of sex peptide if expressing sp is costly, then males co-evolving with spr- females should evolve lower sp gene expression because sp is functionless with these females. there are several potential explanations for our finding that sp expression was weakly increased, in addition to the possibility that the marginally non-significant result arose by chance. first, expressing sp may not be costly for males; however, this alone does not explain increased sp expression in spr- males. second, sp expression measured in males might not reflect sp protein transfer to females at mating. indeed, a recent study found no correlation between the two. sp expression in males could instead reflect male sp stores, with larger stores evolving to increase male capacity to mate many times in spr- populations. however, we detected no change in accessory gland size, nor evidence that spr- males could better sustain ejaculate transfer over multiple matings, as spr- and control males had similar success in sperm competition in first and fifth matings. third, if sp expression is in fact positively correlated with sp protein transfer, then increased sp expression in spr- males appears inconsistent with our finding of decreased ability of spr- males to inhibit female re-mating. however, if functionality of the sp protein degraded through genetic drift, a likely scenario given that sp is effectively neutral in spr- lines, this inconsistency is reconciled. under this scenario, there are two potential explanations for increased sp gene expression. first, sp expression might be genetically correlated with other ejaculate components under positive selection. a second mechanism relates to sp protein structure, which consists of a carboxy-terminal end that binds to spr and mediates female post-mating oviposition and re-mating responses, and an amino-terminal end that does not bind to spr but to unknown female receptors. positive selection on the amino-terminal end might occur in spr- populations to compensate for the lack of oviposition stimulation from the carboxy-terminal end, because the amino-terminal end induces juvenile hormone synthesis in adult females and stimulates oogenesis. we did not find that spr- males increased the post-mating fecundity of wild-type females, although the wild-type female fecundity response might be dominated by carboxy-terminal end effects. this hypothesis is consistent with our finding of no increase in dup99b expression, as dup99b is homologous with sp only at the carboxy-terminal end. finally, sp might have other as yet unknown functions that are selected positively under high promiscuity. evidence for decreased pre-copulatory performance we found two effects on pre-copulatory performance that were most likely caused by the improved vision of red-eyed spr- males relative to white-eyed controls: unselected spr- males courted females more frequently and were faster to mate. both effects remained detectable at generation but by generation both were either not statistically significant or significant and reversed. although type ii error is a possibility such that we simply failed to detect the same differences, the trends were no longer in the previous direction: at generation mean courtship frequency was higher in control males and the risk ratio for latency to mating was. the results are consistent with decreased pre-copulatory investment in spr- males, which might result from weaker selection to overcome re-mating resistance with spr- females. declining courtship frequency might be accompanied by decreased courtship intensity or quality of courtship signals. furthermore, if the increased mating frequency in spr- populations caused sexual selection to shift from pre-copulatory to post-copulatory, we would expect increased displays of post-mating guarding behaviour in spr- males. we are currently investigating these hypotheses.  our study shows that spr- males evolving at high promiscuity evolve shorter copulations and decreased inhibition of female re-mating, consistent with decreased post-copulatory investment per mating, and less frequent courtship and longer latency until mating, consistent with decreased pre-copulatory investment. curiously, males that evolved with spr- females also showed a tendency for increased sp expression. previous experimental evolution studies have demonstrated that reducing a population’s mating frequency by imposing monogamy can result in evolutionary change in reproductive traits. other studies have increased or decreased mating frequency by altering the sex ratio, but changes in sex ratio tend to alter mating frequency and mate competition in opposite directions. the effects of increased promiscuity per se, as observed in our study, seem to be different from simultaneously changing mating frequency and mate competition. our study highlights the utility of genetic manipulations of the socio-sexual environment, and the sensitivity of experimental evolution studies of mating system to the method by which mating frequency is manipulated. 
here, we use a genetic manipulation of mating frequency in drosophila melanogaster to create a novel, highly promiscuous mating system. our study highlights the utility of genetic manipulations of animal social and sexual environments coupled with experimental evolution. we generated d. melanogaster populations in which flies were deficient for the sex peptide receptor gene – resulting in spr- females that mated more frequently – and genetically-matched control populations, and allowed them to evolve for generations. in response to experimentally increased mating frequency, spr- males evolved behavioural responses consistent with decreased male post-copulatory investment at each mating and decreased overall pre-copulatory performance. at several time-points during this experimental evolution, we assayed behavioural, morphological and transcriptional reproductive phenotypes expected to evolve in response to increased population mating frequencies. copulationcourtshipdrosophila melanogasterejaculateexperimental evolutionmatingsex peptidesexual selectionsperm competitionfoundation for polish sciencempd/2009-3/5radwan jacek institute of environmental sciences, jagiellonian universityds/mnd/wbinoz/inos/15/2013radwan jacek http://dx.doi.org/ /501100000530association for the study of animal behaviourhttp://dx.doi.org/ /501100000270natural environment research councilne/j018937/1wigby stuart http://dx.doi.org/ /501100000268biotechnology and biological sciences research councilbb/k014544/1wigby stuart http://dx.doi.org/ /100004440wellcome trusthttp://dx.doi.org/ /501100000038natural sciences and engineering research council of canadahttp://dx.doi.org/ /501100000645jesus college, university of oxfordhttp://dx.doi.org/ /501100001287lloyd's tercentenary research foundationissue-copyright-statement© the author 2016 the trend towards increased sp gene expression might plausibly relate to functional differences in the two domains of the sp protein. males from spr- populations initially exhibited more frequent courtship and faster time until mating relative to controls, but over evolutionary time these differences diminished or reversed. we found that males from the high mating frequency spr- populations evolved decreased ability to inhibit the receptivity of their mates and decreased copulation duration, in line with predictions of decreased per-mating investment with increased sperm competition. although previous research has investigated the evolutionary consequences of reductions in the number of mates, we know little about the costs and benefits of increased numbers of mates.
 the number of partners that individuals mate with over their lifetime is a defining feature of mating systems, and variation in mate number is thought to be a major driver of sexual evolution. although previous research has investigated the evolutionary consequences of reductions in the number of mates, we know little about the costs and benefits of increased numbers of mates. here, we use a genetic manipulation of mating frequency in drosophila melanogaster to create a novel, highly promiscuous mating system. we generated d. melanogaster populations in which flies were deficient for the sex peptide receptor gene – resulting in spr- females that mated more frequently – and genetically-matched control populations, and allowed them to evolve for generations. at several time-points during this experimental evolution, we assayed behavioural, morphological and transcriptional reproductive phenotypes expected to evolve in response to increased population mating frequencies.  we found that males from the high mating frequency spr- populations evolved decreased ability to inhibit the receptivity of their mates and decreased copulation duration, in line with predictions of decreased per-mating investment with increased sperm competition. unexpectedly, spr- population males also evolved weakly increased sex peptide gene expression. males from spr- populations initially exhibited more frequent courtship and faster time until mating relative to controls, but over evolutionary time these differences diminished or reversed.  in response to experimentally increased mating frequency, spr- males evolved behavioural responses consistent with decreased male post-copulatory investment at each mating and decreased overall pre-copulatory performance. the trend towards increased sp gene expression might plausibly relate to functional differences in the two domains of the sp protein. our study highlights the utility of genetic manipulations of animal social and sexual environments coupled with experimental evolution. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords copulationcourtshipdrosophila melanogasterejaculateexperimental evolutionmatingsex peptidesexual selectionsperm competitionfoundation for polish sciencempd/2009-3/5radwan jacek institute of environmental sciences, jagiellonian universityds/mnd/wbinoz/inos/15/2013radwan jacek http://dx.doi.org/ /501100000530association for the study of animal behaviourhttp://dx.doi.org/ /501100000270natural environment research councilne/j018937/1wigby stuart http://dx.doi.org/ /501100000268biotechnology and biological sciences research councilbb/k014544/1wigby stuart http://dx.doi.org/ /100004440wellcome trusthttp://dx.doi.org/ /501100000038natural sciences and engineering research council of canadahttp://dx.doi.org/ /501100000645jesus college, university of oxfordhttp://dx.doi.org/ /501100001287lloyd's tercentenary research foundationissue-copyright-statement© the author 2016 