Pre- and post-copulatory sexual selection increase offspring quality but impose survival costs to female field crickets.

Whether sexual selection increases or decreases fitness is under ongoing debate. Sexual selection operates before and after mating. Yet, the effects of each episode of selection on individual reproductive success remain largely unexplored. We ask how disentangled pre- and post-copulatory sexual selection contribute to fitness of field crickets Gryllus bimaculatus. Treatments allowed exclusively for (i) pre-copulatory selection, with males fighting and courting one female, and the resulting pair breeding monogamously, (ii) post-copulatory selection, with females mating consecutively to multiple males and (iii) relaxed selection, with enforced pair monogamy. While standardizing the number of matings, we estimated a number of fitness traits across treatments and show that females experiencing sexual selection were more likely to reproduce, their offspring hatched sooner, developed faster and had higher body mass at adulthood, but females suffered survival costs. Interestingly, we found no differences in fitness of females or their offspring from pre- and post-copulatory sexual selection treatments. Our findings highlight the potential for sexual selection in enhancing indirect female fitness while concurrently imposing direct survival costs. By potentially outweighing these costs, increased offspring quality could lead to beneficial population-level consequences of sexual selection.

Experimental extra-pair copulations provide proof of concept for fertility insurance in a socially monogamous bird.

Extra-pair paternity is common among socially monogamous birds, but whether females benefit from having extra-pair copulations remains unresolved. In this study, I staged single extra-pair copulations in captive pairs of socially monogamous Japanese quail (Coturnix japonica) and used extra-pair males of a different colour to identify extra-pair fertilizations. This eliminated among-female variation in extra-pair copulations as a source of variation in extra-pair fertilizations and tested whether a single extra-pair copulation would insure against infertility of the social male. Overall, the probability of extra-pair fertilization was 0.46 for the first egg that was fertilized after extra-pair copulation, but this rapidly declined over consecutive eggs in the laying sequence. However, a single extra-pair copulation was effective in ensuring fertilization of the majority of a typical clutch in the few cases where the social male was completely infertile. Hence, my results show that variation in extra-pair paternity can be independent from variation in extra-pair copulation behaviour and that a single, strategically timed, extra-pair copulation can largely insure against social male infertility. Among-female variation in extra-pair fertilizations, and their declining probability over the laying sequence, as typical for many bird species, can thus, in principle, be parsimoniously explained by a uniform female fertility insurance strategy.

Differential oxidative costs of locomotory and genital damage in an orb-weaving spider.

In animals that regularly experience tissue loss, physiological responses may have evolved to overcome the related costs. Changes in oxidative status may reflect such self-maintenance mechanisms. Here, we investigated how markers of oxidative status vary in female orb-weaving spiders (Larinia jeskovi) by mimicking two distinct types of tissue loss they may naturally encounter: damage to their locomotory system and damage to their external genital structure (scapus), as inflicted by males during copulation (external female genital mutilation). Damage to the locomotory system resulted in a significant shift in oxidative status, reflecting investment in self-maintenance. In contrast, the loss of the scapus did not result in quantitative changes of oxidative markers. This lack of a physiological response suggests negligible physiological costs of genital mutilation for female spiders. However, not being able to remate with other males might be costly for females.

Female sperm storage mediates post-copulatory costs and benefits of ejaculate anticipatory plasticity in the guppy.

Males of many species evolved the capability of adjusting their ejaculate phenotype in response to social cues to match the expected mating conditions. When females store sperm for a prolonged time, the expected fitness return of plastic adjustments of ejaculate phenotype may depend on the interval between mating and fertilization. Although prolonged female sperm storage (FSS) increases the opportunity for sperm competition, as a consequence of the longer temporal overlap of ejaculates from several males, it may also create variable selective forces on ejaculate phenotype, for example by exposing trade-offs between sperm velocity and sperm survival. We evaluated the relationship between the plasticity of ejaculate quality and FSS in the guppy, Poecilia reticulata, a polyandrous live-bearing fish in which females store sperm for several months and where stored sperm contribute significantly to a male's lifelong reproductive success. In this species, males respond to the perception of future mating opportunities by increasing the quantity (number) and quality (swimming velocity) of ready-to-use sperm (an anticipatory response called 'sperm priming'). Here we investigated (a) the effect of sperm priming on in vitro sperm viability at stripping and its temporal decline (as an estimate of sperm survival), and (b) the in vivo competitive fertilization success in relation to female sperm storage using artificial insemination. As expected, sperm-primed males produced more numerous and faster sperm, but with a reduced in vitro sperm viability at stripping and after 4 hr, compared with their counterparts. Artificial insemination revealed that the small (nonsignificant) advantage of primed sperm when fertilization immediately follows insemination is reversed when eggs are fertilized by female-stored sperm, weeks after insemination. By suggesting a plastic trade-off between sperm velocity and viability, these results demonstrate that prolonged female sperm storage generates divergent selection pressures on ejaculate phenotype.

Female mating tactics in lekking fallow deer (Dama dama): experience explains inter-individual variability more than costs.

Most studies on ungulate reproduction have focused on the covariates of male reproductive success, while there is much less information on female tactics of mate choice. The aim of this work is to fill this gap and to assess condition-dependent variations in female tactics in a lekking fallow deer (Dama dama) population. In particular, we investigated three indirect selection mechanisms: i) aggregation: when females join an already formed female group; ii) copying: when females copy the mate choice of other females and iii) territory choice: when females select a territory where many copulations had previously occurred. Our results show that female fallow deer, which are less experienced (younger) and/or incur higher travel costs (home range far from the lek), adopt indirect forms of mate selection more often than older females or females residing near the lek, respectively. Compared to adults, younger females remained longer in the lek (almost three times) and in male territories, returning to the lek after copulation. However, despite the time spent at the lek, younger females were not able to select the highest-rank males, and relied on territory choice more often than older females. Farther does visited the lek less frequently (farthest females only once) and arrived on average 5 days later than closer females (which performed up to 7 visits), but they were seen more often within female groups (aggregation). We did not find a different amount of copying in younger or in farther females. Our results contribute to advance our understanding of female behaviours in ungulate leks.

Individual Quality and Extra-Pair Paternity in the Blue Tit: Sexy Males Bear the Costs.

Adaptive explanations for the evolution of extra-pair paternity (EPP) suggest that females seek extra-pair copulations with high quality males. Still, the link between ornamentation, individual quality, and paternity remains unclear. Moreover, honest signaling is essential when explaining EPP because it is needed for sexual selection to occur; yet, it is understudied in multiple ornaments. Because blue tits (Cyanistes caeruleus) show variable color expression in several plumage patches, we tested: (i) over two seasons, whether males in better condition, more ornamented and less infected by blood parasites gain EPP and have higher reproductive success, and (ii) over three seasons, whether mating patterns affect color change. Males with more saturated yellow feathers, brighter tails, and in better condition had higher reproductive success in one of the seasons. Contrary to expectation, in another season, males that gained EPP were parasitized by blood parasites, suggesting increased vector exposure during extra-pair copulations. Our results for two seasons show that males siring more extra-pair young were older and grew brighter cheek or tail feathers for the following season. Despite the increased mating costs, in socially monogamous avian systems, high quality males incur in EPP without compromising traits that may be under sexual selection.

Costs of weaponry: Unarmed males sire more offspring than armed males in a male-dimorphic mite.

Morphological structures used as weapons in male-male competition are not only costly to develop but are also probably costly to maintain during adulthood. Therefore, having weapons could reduce the energy available for other fitness-enhancing actions, such as post-copulatory investment. We tested the hypothesis that armed males make lower post-copulatory investments than unarmed males, and that this difference will be most pronounced under food-limited conditions. We performed two experiments using the male-dimorphic bulb mite Rhizoglyphus robini, in which males are either armed "fighters" or unarmed "scramblers." Firstly, we tested whether fighters and scramblers differed in their reproductive output after being starved or fed for 1 or 2 weeks. Secondly, we measured the reproductive output of scramblers and fighters (starved or fed) after one, two or three consecutive matings. Scramblers sired more offspring than fighters after 1 week, but scramblers and fighters only sired a few offspring after 2 weeks. Scramblers also sired more offspring than fighters at the first mating, and males rarely sired offspring after consecutive matings. Contrary to our hypothesis, the fecundity of starved and fed males did not differ. The higher reproductive output of scramblers suggests that, regardless of nutritional state, scramblers make larger post-copulatory investments than fighters. Alternatively, (cryptic) female choice generally favours scramblers. Why the morphs differed in their reproductive output is unclear. Neither morph performed well relatively late in life or after multiple matings. It remains to be investigated to what extent the apparent scrambler advantage contributes to the maintenance and evolution of male morph expression.

Prenatal and Postnatal Assessment in Rabbits with Evacetrapib: A Cholesteryl Ester Transfer Protein Inhibitor.

BACKGROUND: Evacetrapib, a potent and selective inhibitor of cholesteryl ester transfer protein (CETP), was under development for the treatment of cardiovascular (CV) disease. The purpose of this pre-postnatal study in rabbits was to evaluate the effects of evacetrapib on pregnancy, parturition, and lactation of the maternal animals and on the growth, viability, development, and reproductive performance of the first filial (F1) offspring. The rabbit is considered a relevant species for toxicity testing with evacetrapib as it demonstrates significant CETP expression, whereas mice and rats do not express significant levels of CETP. METHODS: Evacetrapib was administered daily by oral gavage from gestation day (GD) 7 through lactation day (LD) 41 at dose levels of 0, 10, 30, and 100 mg/kg/day. RESULTS: There were no adverse effects on maternal survival, clinical signs, gestation length, parturition, and litter size. There were no effects on F1 clinical observations, body weight, sexual maturation, conditioned eye blink, functional observational battery, or pathology findings. Treatment-related decreases in F1 postnatal survival and equivocal reductions in F1 mating, fertility, and copulation/conception indices without changes in sperm parameters or pathology of reproductive organs were noted in F1 animals. CONCLUSIONS: The maternal no observed adverse effect level (NOAEL) after evacetrapib administration in female rabbits was 100 mg/kg/day. Based on the decreased F1 postnatal survival and equivocal changes in F1 fertility, the NOAEL for F1 neonatal developmental was 30 mg/kg/day. Birth Defects Research 109:486-496, 2017.© 2017 Wiley Periodicals, Inc.

How sexual selection can drive the evolution of costly sperm ornamentation.

Post-copulatory sexual selection (PSS), fuelled by female promiscuity, is credited with the rapid evolution of sperm quality traits across diverse taxa. Yet, our understanding of the adaptive significance of sperm ornaments and the cryptic female preferences driving their evolution is extremely limited. Here we review the evolutionary allometry of exaggerated sexual traits (for example, antlers, horns, tail feathers, mandibles and dewlaps), show that the giant sperm of some Drosophila species are possibly the most extreme ornaments in all of nature and demonstrate how their existence challenges theories explaining the intensity of sexual selection, mating-system evolution and the fundamental nature of sex differences. We also combine quantitative genetic analyses of interacting sex-specific traits in D. melanogaster with comparative analyses of the condition dependence of male and female reproductive potential across species with varying ornament size to reveal complex dynamics that may underlie sperm-length evolution. Our results suggest that producing few gigantic sperm evolved by (1) Fisherian runaway selection mediated by genetic correlations between sperm length, the female preference for long sperm and female mating frequency, and (2) longer sperm increasing the indirect benefits to females. Our results also suggest that the developmental integration of sperm quality and quantity renders post-copulatory sexual selection on ejaculates unlikely to treat male-male competition and female choice as discrete processes.

Impaired sperm quality, delayed mating but no costs for offspring fitness in crickets winning a fight.

The outcome of male-male contest competition is known to affect male mating success and is believed to confer fitness benefits to females through preference for dominant males. However, by mating with contest winners, females can incur significant costs spanning from decreased fecundity to negative effects on offspring. Hence, identifying costs and benefits of male dominance on female fitness is crucial to unravel the potential for a conflict of interests between the sexes. Here, we investigated males' pre- and post-copulatory reproductive investment and its effect on female fitness after a single contest a using the field cricket Gryllus bimaculatus. We allowed males to fight and immediately measured their mating behaviour, sperm quality and offspring viability. We found that males experiencing a fight, independently of the outcome, delayed matings, but their courtship effort was not affected. However, winners produced sperm of lower quality (viability) compared to losers and to males that did not experience fighting. Results suggest a trade-off in resource allocation between pre- and post-mating episodes of sexual selection. Despite lower ejaculate quality, we found no fitness costs (fecundity and viability of offspring) for females mated to winners. Overall, our findings highlight the importance of considering fighting ability when assessing male reproductive success, as winners may be impaired in their competitiveness at a post-mating level.

Assessment of rival males through the use of multiple sensory cues in the fruitfly Drosophila pseudoobscura.

Environments vary stochastically, and animals need to behave in ways that best fit the conditions in which they find themselves. The social environment is particularly variable, and responding appropriately to it can be vital for an animal's success. However, cues of social environment are not always reliable, and animals may need to balance accuracy against the risk of failing to respond if local conditions or interfering signals prevent them detecting a cue. Recent work has shown that many male Drosophila fruit flies respond to the presence of rival males, and that these responses increase their success in acquiring mates and fathering offspring. In Drosophila melanogaster males detect rivals using auditory, tactile and olfactory cues. However, males fail to respond to rivals if any two of these senses are not functioning: a single cue is not enough to produce a response. Here we examined cue use in the detection of rival males in a distantly related Drosophila species, D. pseudoobscura, where auditory, olfactory, tactile and visual cues were manipulated to assess the importance of each sensory cue singly and in combination. In contrast to D. melanogaster, male D. pseudoobscura require intact olfactory and tactile cues to respond to rivals. Visual cues were not important for detecting rival D. pseudoobscura, while results on auditory cues appeared puzzling. This difference in cue use in two species in the same genus suggests that cue use is evolutionarily labile, and may evolve in response to ecological or life history differences between species.

Optimal numbers of matings: the conditional balance between benefits and costs of mating for females of a nuptial gift-giving spider.

In species where females gain a nutritious nuptial gift during mating, the balance between benefits and costs of mating may depend on access to food. This means that there is not one optimal number of matings for the female but a range of optimal mating numbers. With increasing food availability, the optimal number of matings for a female should vary from the number necessary only for fertilization of her eggs to the number needed also for producing these eggs. In three experimental series, the average number of matings for females of the nuptial gift-giving spider Pisaura mirabilis before egg sac construction varied from 2 to 16 with food-limited females generally accepting more matings than well-fed females. Minimal level of optimal mating number for females at satiation feeding conditions was predicted to be 2-3; in an experimental test, the median number was 2 (range 0-4). Multiple mating gave benefits in terms of increased fecundity and increased egg hatching success up to the third mating, and it had costs in terms of reduced fecundity, reduced egg hatching success after the third mating, and lower offspring size. The level of polyandry seems to vary with the female optimum, regulated by a satiation-dependent resistance to mating, potentially leaving satiated females in lifelong virginity.

Mating opportunities in Sangalopsis veliterna females: costs and benefits.

In nature, females of several animal taxa exhibit considerable variation in their mating system, and this variation involves different balances of costs (e.g., energetic, reproductive) and benefits (e.g., increased net reproductive rate of the female, increased longevity). Many studies have focused on discovering the potential advantages and disadvantages that females could have when increasing their mating rate and the possible evolutionary consequences that may result. Butterflies and moths are an ideal study system because it is easy to determine and to manipulate experimentally their mating frequency. In this study, the effect of continuous availability of different numbers of males (1, 2, 4, 8) on female mating rate and fitness components was estimated by comparing the number of spermatophores in the corpus bursa (an estimate of the number of copulations, but not of the number males involved in these copulations), female longevity, lifetime number of laid eggs (fecundity), and proportion of hatching eggs (fertility) in the moth Sangalopsis veliterna Druce (Lepidoptera: Geometridae). The results showed that there were no significant differences in either fertility or fecundity when treatments were compared, but longevity and in some cases fecundity increased when females had several matings.

Social variables affecting mate preferences, copulation and reproductive outcome in a pack of free-ranging dogs.

Mating and reproductive outcome is often determined by the simultaneous operation of different mechanisms like intra-sexual competition, mating preferences and sexual coercion. The present study investigated how social variables affected mating outcome in a pack of free-ranging dogs, a species supposed to have lost most features of the social system of wolves during domestication. We found that, although the pack comprised multiple breeding individuals, both male copulation success and female reproductive success were positively influenced by a linear combination of dominance rank, age and leadership. Our results also suggest that mate preferences affect mating outcome by reinforcing the success of most dominant individuals. In particular, during their oestrous period bitches clearly searched for the proximity of high-ranking males who displayed affiliative behaviour towards them, while they were more likely to reject the males who intimidated them. At the same time, male courting effort and male-male competition for receptive females appeared to be stronger in the presence of higher-ranking females, suggesting a male preference for dominant females. To our knowledge, these results provide the first clear evidence of social regulation of reproductive activities in domestic dogs, and suggest that some common organizing mechanisms may contribute to shape the social organization of both dogs and wolves.

Extreme cost of rivalry in a monandrous species: male-male interactions result in failure to acquire mates and reduced longevity.

Mating system variation is profound in animals. In insects, female willingness to remate varies from mating with hundreds of males (extreme polyandry) to never remating (monandry). This variation in female behaviour is predicted to affect the pattern of selection on males, with intense pre-copulatory sexual selection under monandry compared to a mix of pre- and post-copulatory forces affecting fitness under polyandry. We tested the hypothesis that differences in female mating biology would be reflected in different costs of pre-copulatory competition between males. We observed that exposure to rival males early in life was highly costly for males of a monandrous species, but had lower costs in the polyandrous species. Males from the monandrous species housed with competitors showed reduced ability to obtain a mate and decreased longevity. These effects were specific to exposure to rivals compared with other types of social interactions (heterospecific male and mated female) and were either absent or weaker in males of the polyandrous species. We conclude that males in monandrous species suffer severe physiological costs from interactions with rivals and note the significance of male-male interactions as a source of stress in laboratory culture.

Observation of intra-group and extra-group copulation and reproductive characters in free ranging groups of western black crested gibbon (Nomascus concolor jingdongensis).

Black crested gibbons (Nomascus concolor) are 1 of only 3 gibbon species that have been observed in long-term polygynous groups, but their mating behavior and reproductive characters have never been reported. Based on population monitoring over 7 years and direct observation for 26 months of the study groups in Wuliang Mountain, central Yunnan, we describe for the first time the copulation behavior and breeding pattern of free-ranging western black crested gibbons. The gestation period of black crested gibbons is estimated to be between 6 and 7 months. The average breeding interval is 3.5 years, with infant independence at approximately 2.5 years. We observed 2 intra-group copulations and 5 extra-group copulations. Copulations were initiated when a female gave a 'solicitation' gesture. When a male made any mating attempt, the female might refuse it. These results demonstrate direct female mate choice. Both male and female gibbons dispersed from their natal groups and sometimes replaced paired adults in other groups. We observed no evidence of infanticide during inter-group conflicts or after replacement of adults. Together with extra-group copulations, these phenomena indicate a flexible social organization and complex mating system. We also observed a male-biased sex ratio among offspring. More genetic work is necessary to describe the effects of inter-group copulation and the genetic diversity of this population.

Extreme costs of mating for male two-spot ladybird beetles.

Male costs of mating are now thought to be widespread. The two-spot ladybird beetle (Adalia bipunctata) has been the focus of many studies of mating and sexual selection, yet the costs of mating for males are unknown. The mating system of A. bipunctata involves a spermatophore nuptial gift ingested by females after copulation. In this study, we investigate the cost to males of mating and of transferring spermatophores in terms of lifespan, ejaculate production and depletion of nutritional reserves. We found that males faced a strong trade-off between mating and survival, with males that were randomly assigned to mate a single time experiencing a 53% reduction in post-mating lifespan compared to non-mating males. This is among the most severe survival costs of a single mating yet reported. However, spermatophore transfer did not impact male survival. Instead, the costs associated with spermatophores appeared as a reduced ability to transfer spermatophores in successive matings. Furthermore, males ingested more food following spermatophore transfer than after matings without spermatophores, suggesting that spermatophore transfer depletes male nutritional reserves. This is to our knowledge the first report of an effect of variation in copulatory behaviour on male foraging behaviour. Overall, our study highlights the advantages of assessing mating costs using multiple currencies, and suggests that male A. bipunctata should exhibit mate choice.

The energetic cost of mating in a promiscuous cephalopod.

Costs that individuals incur through mating can play an important role in understanding the evolution of life histories and senescence, particularly in promiscuous species. Copulation costs, ranging from energy expenditure to reduced longevity, are widely studied in insects but have received substantially less attention in other taxa. One cost of mating, the energetic cost, is poorly studied across all taxa despite its potential importance for the many species where copulation is physically demanding and/or frequent. Here, we investigated the energetic cost of mating in both male and female dumpling squid (Euprymna tasmanica). In this species, copulation can last up to 3 h and requires that the male physically restrains the female. We report that the act of copulation halves the swimming endurance of both sexes, and that they take up to 30 min to recover. Such a reduction in post-copulatory performance may have important implications for predator avoidance, foraging ability and energy allocation. Therefore, quantifying this cost is essential to understand the evolution of reproductive strategies and behaviours such as female receptivity and male and female mating frequency.

Avaliação de dois métodos para condicionamento e coleta de sêmen em quatro espécies do gênero Mazama / Assessment of two methods for conditioning and semen collection in four species of Mazama genus

O desenvolvimento de técnicas não invasivas para a obtenção de sêmen de cervídeos facilita a criação de bancos genômicos, que são importantes instrumentos para a conservação ex situ e in situ. Este trabalho teve como objetivo criar uma metodologia não-invasiva de coleta de sêmen e comparar duas técnicas de coleta em quatro espécies do gênero Mazama: M. americana, M. gouazoubira, M. nana e M. nemorivaga. Para tanto, foram utilizados seis machos (M) e duas fêmeas (F) da espécie M. americana, 3M e 2F de M. gouazoubira, 1M e 1F de M. nana e 2M e 1F de M. nemorivaga. Para cada técnica testada, foi realizado um período de habituação dos animais ao manejo. Em seguida, duas técnicas de condicionamento e coleta foram avaliadas. Na primeira delas foi utilizada uma fêmea em estro com desvio lateral do pênis para vagina artificial (FEDL), obtendo-se a coleta de 50% dos indivíduos (100% dos machos de M. gouazoubira e 50% dos machos de M. americana), não obtendo ejaculados das demais espécies. Na segunda técnica, utilizando um manequim taxidermizado com urina de fêmea em estro (MUFE) não foi possível a coleta de nenhum ejaculado. Em todas as fases foi observado o comportamento do macho quanto ao tempo de interesse e aproximação, reflexo de "Flehmen", ato de cheirar ou lamber, exposição do pênis, ereção, número de falsas montas, tentativas de cópula e ocorrência de agressividade entre os animais.

The development of noninvasive techniques for obtaining semen from deer facilitates the creation of genome banks, which are important tools for ex situ and in situ conservation. This study aimed to establish a noninvasive method of semen collection and compare two techniques of collection in four species of the genus Mazama: M. americana, M. gouazoubira, M. nana and M. nemorivaga. To achieve this, 6 males (M) and 2 females (F) of the species M. Americana, 3M and 2F of M. gouazoubira, 1M and 1F of M. nana and 2M and 1F of M. nemorivaga were used. For each technique tested, a period of habituation to animal handling was conducted; then, the two conditioning techniques and collection were evaluated. In the first, a female in estrus was used with lateral deviation of the penis to an artificial vagina (FEDL), yielding collection from 50% of the males (100% from M. gouazoubira and 50% from M. americana), with no ejaculate from the remaining species. In the second technique, using a taxidermized dummy with urine from females in estrus (MUFE), no semen collection was possible. During all stages, male behavior was observed regarding the time of interest and approximation, the "Flehmen" response, the act of sniffing or licking, exposure of the penis, erection, number of false mounts, attempts at copulation and the occurrence of aggression between the deer.

The evolution of multiple mating: Costs and benefits of polyandry to females and of polygyny to males.

Polyandry is a paradox: why do females mate multiple times when a single ejaculate often provides enough sperm for lifetime egg production? Gowaty et al. addressed explanations for polyandry in Drosophila pseudoobscura from the perspective of hypotheses based on sex differences in costs of reproduction (CoR). Contrary to CoR, Gowaty et al. showed that (1) a single ejaculate was inadequate for lifetime egg production; (2) polyandry provided fitness benefits to females beyond provision of adequate sperm and (3) fitness benefits of polyandry were not offset by costs. Here, I discuss predictions of the ad hoc hypotheses of CoR and three alternative hypotheses to CoR to facilitate a discussion and further development of a strong inference approach to experiments on the adaptive significance of polyandry for females. Each of the hypotheses makes testable predictions; simultaneous tests of the predictions will provide a strong inference approach to understanding the adaptive significance of multiple mating. I describe a sex-symmetric experiment meant to evaluate variation in fitness among lifelong virgins (V); monogamous females and males with one copulation (MOC); monogamous females and males with multiple copulations (MMC); PAND, polyandrous females; and PGYN, polygynous males. Last, I recommend the study of many different species, while taking care in choice of study species and attention to the assumptions of specific hypotheses. I particularly urge the study of many more Drosophila species both in laboratory and the wild to understand the "nature of flies in nature," where opportunities and constraints mold evolutionary responses.