Hypodermic self-insemination as a reproductive assurance strategy

Self-fertilization occurs in a broad range of hermaphroditic plants and animals, and is often thought to evolve as a reproductive assurance strategy under ecological conditions that disfavour or prevent outcrossing. Nevertheless, selfing ability is far from ubiquitous among hermaphrodites, and may be constrained in taxa where the male and female gametes of the same individual cannot easily meet. Here, we report an extraordinary selfing mechanism in one such species, the free-living flatworm Macrostomum hystrix. To test the hypothesis that adaptations to hypodermic insemination of the mating partner under outcrossing also facilitate selfing, we experimentally manipulated the social environment of these transparent flatworms and then observed the spatial distribution of received sperm in vivo. We find that this distribution differs radically between conditions allowing or preventing outcrossing, implying that isolated individuals use their needle-like stylet (male copulatory organ) to inject own sperm into their anterior body region, including into their own head, from where they then apparently migrate to the site of (self-)fertilization. Conferring the ability to self could thus be an additional consequence of hypodermic insemination, a widespread fertilization mode that is especially prevalent among simultaneously hermaphroditic animals and probably evolves due to sexual conflict over the transfer and subsequent fate of sperm.     

Genomic Signatures of Sexual Selection on Pollen-Expressed Genes in Arabis alpina

Fertilization in angiosperms involves the germination of pollen on the stigma, followed by the extrusion of a pollen tube that elongates through the style and delivers two sperm cells to the embryo sac. Sexual selection could occur throughout this process when male gametophytes compete for fertilization. The strength of sexual selection during pollen competition should be affected by the number of genotypes deposited on the stigma. As increased self-fertilization reduces the number of mating partners, and the genetic diversity and heterozygosity of populations, it should thereby reduce the intensity of sexual selection during pollen competition. Despite the prevalence of mating system shifts, few studies have directly compared the molecular signatures of sexual selection during pollen competition in populations with different mating systems. Here we analyzed whole-genome sequences from natural populations of Arabis alpina, a species showing mating system variation across its distribution, to test whether shifts from cross- to self-fertilization result in molecular signatures consistent with sexual selection on genes involved in pollen competition. We found evidence for efficient purifying selection on genes expressed in vegetative pollen, and overall weaker selection on sperm-expressed genes. This pattern was robust when controlling for gene expression level and specificity. In agreement with the expectation that sexual selection intensifies under cross-fertilization, we found that the efficacy of purifying selection on male gametophyte-expressed genes was significantly stronger in genetically more diverse and outbred populations. Our results show that intra-sexual competition shapes the evolution of pollen-expressed genes, and that its strength fades with increasing self-fertilization rates.     

TESTING THE EFFECTS OF MATING SYSTEM VARIATION ON RATES OF MOLECULAR EVOLUTION IN PRIMATES

Post‐copulatory sexual selection has been proposed to drive the rapid evolution of reproductive proteins, and, more recently, to increase genome‐wide mutation rates. Comparisons of rates of molecular evolution between lineages with different levels of female multiple mating represent a promising, but under‐utilized, approach for testing the effects of sperm competition on sequence evolution. Here, I use comparisons between primate species with divergent mating systems to examine the effects of sperm competition on reproductive protein evolution, as well as on sex‐averaged mutation rates. Rates of nonsynonymous substitution are higher for testis‐specific genes along the chimpanzee lineage in comparison to the human lineage, consistent with expectations. However, the data reported here do not allow firm conclusions concerning the effects of mating system on genome‐wide mutation rates, with different results obtained from different species pairs. Ultimately, comparative studies encompassing a range of mating systems and other life history traits will be required to make broad generalizations concerning the genomic effects of sperm competition.     

Mating systems and protein–protein interactions determine evolutionary rates of primate sperm proteins

To assess the relative impact of functional constraint and post-mating sexual selection on sequence evolution of reproductive proteins, we examined 169 primate sperm proteins. In order to recognize potential genome-wide trends, we additionally analysed a sample of altogether 318 non-reproductive (brain and postsynaptic) proteins. Based on cDNAs of eight primate species (Anthropoidea), we observed that pre-mating sperm proteins engaged in sperm composition and assembly show significantly lower incidence of site-specific positive selection and overall lower non-synonymous to synonymous substitution rates (d_N/d_S) across sites as compared with post-mating sperm proteins involved in capacitation, hyperactivation, acrosome reaction and fertilization. Moreover, database screening revealed overall more intracellular protein interaction partners in pre-mating than in post-mating sperm proteins. Finally, post-mating sperm proteins evolved at significantly higher evolutionary rates than pre-mating sperm and non-reproductive proteins on the branches to multi-male breeding species, while no such increase was observed on the branches to unimale and monogamous species. We conclude that less protein–protein interactions of post-mating sperm proteins account for lowered functional constraint, allowing for stronger impact of post-mating sexual selection, while the opposite holds true for pre-mating sperm proteins. This pattern is particularly strong in multi-male breeding species showing high female promiscuity.     

The direct cost of traumatic secretion transfer in hermaphroditic land snails: individuals stabbed with a love dart decrease lifetime fecundity

Several taxa of simultaneously hermaphroditic land snails exhibit a conspicuous mating behaviour, the so-called shooting of love darts. During mating, such land snail species transfer a specific secretion by stabbing a mating partner''s body with the love dart. It has been shown that sperm donors benefit from this traumatic secretion transfer, because the secretions manipulate the physiology of a sperm recipient and increase the donors'' fertilization success. However, it is unclear whether reception of dart shooting is costly to the recipients. Therefore, the effect of sexual conflict and antagonistic arms races on the evolution of traumatic secretion transfer in land snails is still controversial. To examine this effect, we compared lifetime fecundity and longevity between the individuals that received and did not receive dart shooting from mating partners in Bradybaena pellucida. Our experiments showed that the dart-receiving snails suffered reduction in lifetime fecundity and longevity. These results suggest that the costly mating behaviour, dart shooting, generates conflict between sperm donors and recipients and that sexually antagonistic arms races have contributed to the diversification of the morphological and behavioural traits relevant to dart shooting. Our findings also support theories suggesting a violent escalation of sexual conflict in hermaphroditic animals.     

Shell shape and mating behaviour in pulmonate gastropods (Mollusca)

Previous work suggests that low-spired hermaphroditic snails mate face-to-face and have reciprocal sperm exchange, whereas high-spired snails mate by shell mounting and have unilateral sperm exchange. This dichotomy lead others to speculate on the evolution of enigmatic mating behaviours and whole-body enantiomorphy. In the present study, we review the current literature on mating behaviour in pulmonate snails and show that: (1) several pulmonate species show considerable intraspecific variation in mating behaviour; (2) mating position does not predict reciprocity of penis intromission and sperm exchange; (3) dart-shooting may be correlated with reciprocity of sperm exchange but other factors must explain the gain or loss of darts; (4) it is unlikely that the degree of reciprocity is the most important factor in explaining the relationship of whole-body enantiomorphy and shell shape; and (5) the reciprocal intromission of penises does not necessarily involve the reciprocal transfer of sperm. Hence, our survey shows that current ideas on the evolutionary relationship between shell shape and reciprocity with sexual selection (including dart-use) and whole-body enantiomorphy in hermaphroditic snails should be refined. The results obtained demonstrate that our current knowledge on gastropod mating behaviour is too limited to detect general evolutionary trajectories in gastropod mating behaviour and genital anatomy.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 306–321.     

Experimentally evolved and phenotypically plastic responses to enforced monogamy in a hermaphroditic flatworm

Sexual selection is considered a potent evolutionary force in all sexually reproducing organisms, but direct tests in terms of experimental evolution of sexual traits are still lacking for simultaneously hermaphroditic animals. Here, we tested how evolution under enforced monogamy affected a suite of reproductive traits (including testis area, sex allocation, genital morphology, sperm morphology and mating behaviour) in the outcrossing hermaphroditic flatworm Macrostomum lignano, using an assay that also allowed the assessment of phenotypically plastic responses to group size. The experiment comprised 32 independent selection lines that evolved under either monogamy or polygamy for 20 generations. While we did not observe an evolutionary shift in sex allocation, we detected effects of the selection regime for two male morphological traits. Specifically, worms evolving under enforced monogamy had a distinct shape of the male copulatory organ and produced sperm with shorter appendages. Many traits that did not evolve under enforced monogamy showed phenotypic plasticity in response to group size. Notably, individuals that grew up in larger groups had a more male‐biased sex allocation and produced slightly longer sperm than individuals raised in pairs. We conclude that, in this flatworm, enforced monogamy induced moderate evolutionary but substantial phenotypically plastic responses.     

Molecular evolution of seminal proteins in field crickets

In sexually reproducing organisms, male ejaculates are complex traits that are potentially subject to many different selection pressures. Recent experimental evidence supports the hypothesis that postmating sexual selection, and particularly sexual conflict, may play a key role in the evolution of the proteinaceous components of ejaculates. However, this evidence is based almost entirely on the study of Drosophila, a species with a mating system characterized by a high cost of mating for females. In this paper, we broaden our understanding of the role of selection on the evolution of seminal proteins by characterizing these proteins in field crickets, a group of insects in which females appear to benefit from mating multiply. We have used an experimental protocol that can be applied to other organisms for which complete genome sequences are not yet available. By combining an evolutionary expressed sequence tag screen of the male accessory gland in 2 focal species (Gryllus firmus and Gryllus pennsylvanicus) with a bioinformatics approach, we have been able to identify as many as 30 seminal proteins. Evolutionary analyses among 5 species of the genus Gryllus suggest that seminal protein genes evolve more rapidly than genes encoding proteins that are not involved with reproduction. The rates of synonymous substitution (dS) are similar in genes encoding seminal proteins and genes encoding "housekeeping" proteins. For the same comparison, the rate of fixation of nonsynonymous substitutions (dN) is 3 times higher in genes encoding seminal proteins, suggesting that the divergence of seminal proteins in field crickets has been accelerated by positive Darwinian selection. In spite of the contrasting characteristics of the Drosophila and Gryllus mating systems, the mean selection parameter omega and the proportion of loci estimated to be affected by positive selection are very similar.     

Rates of Evolution of Hominoid Seminal Proteins are Correlated with Function and Expression, Rather than Mating System

In species where females mate with multiple males during a single ovulatory cycle, sperm competition is hypothesized to increase the rate of adaptive evolution of proteins expressed in male reproductive tissues through recurrent selective sweeps (positive selection). The hominoids, comprising apes and humans, are a group of closely related primates with extensive variation in mating behaviors and predicted levels of sperm competition. Since previous studies of individual male reproductive genes have shown evidence of positive selection, we estimated rates of evolution of a comprehensive set of proteins expressed in ejaculated semen. Our results show that these proteins in hominoids do not have elevated rates of nonsynonymous substitutions (Ka) compared with a control dataset of nonreproductive genes. Species with greater sperm competition do not have faster rates of seminal protein evolution. Although at these broad levels our hypotheses were not confirmed, further analyses indicate specific patterns of molecular evolution. Namely, the Ka of seminal genes is more strongly correlated with measures of tissue specificity than nonreproductive genes, suggesting that the former may more readily adapt to tissue-specific functions. Proteins expressed from the seminal vesicles evolve more rapidly than those from other male reproductive tissues. Also, several gene ontology categories show elevated rates of protein evolution, not seen in the control data set. While the generalization that male reproductive genes evolve rapidly in hominoids is an oversimplification, a subset of proteins can be identified that are likely targets for adaptive evolution driven by sexual selection.     

Multiple speciation events in an arthropod with divergent evolution in sexual morphology

Sexual selection can facilitate divergent evolution of traits related to mating and consequently promote speciation. Theoretically, independent operation of sexual selection in different populations can lead to divergence of sexual traits among populations and result in allopatric speciation. Here, we show that divergent evolution in sexual morphology affecting mating compatibility (body size and genital morphologies) and speciation have occurred in a lineage of millipedes, the Parafontaria tonominea species complex. In this millipede group, male and female body and genital sizes exhibit marked, correlated divergence among populations, and the diverged morphologies result in mechanical reproductive isolation between sympatric species. The morphological divergence occurred among populations independently and without any correlation with climatic variables, although matching between sexes has been maintained, suggesting that morphological divergence was not a by-product of climatic adaptation. The diverged populations underwent restricted dispersal and secondary contact without hybridization. The extent of morphological difference between sympatric species is variable, as is diversity among allopatric populations; consequently, the species complex appears to contain many species. This millipede case suggests that sexual selection does contribute to species richness via morphological diversification when a lineage of organisms consists of highly divided populations owing to limited dispersal.     

Sexual selection drives the coevolution of male and female reproductive traits in Peromyscus mice

When females mate with multiple partners within a single reproductive cycle, sperm from rival males may compete for fertilization of a limited number of ova, and females may bias the fertilization of their ova by particular sperm. Over evolutionary timescales, these two forms of selection shape both male and female reproductive physiology when females mate multiply, yet in monogamous systems, post-copulatory sexual selection is weak or absent. Here, we examine how divergent mating strategies within a genus of closely related mice, Peromyscus, have shaped the evolution of reproductive traits. We show that in promiscuous species, males exhibit traits associated with increased sperm production and sperm swimming performance, and females exhibit traits that are predicted to limit sperm access to their ova including increased oviduct length and a larger cumulus cell mass surrounding the ova, compared to monogamous species. Importantly, we found that across species, oviduct length and cumulus cell density are significantly correlated with sperm velocity, but not sperm count or relative testes size, suggesting that these female traits may have coevolved with increased sperm quality rather than quantity. Taken together, our results highlight how male and female traits evolve in concert and respond to changes in the level of post-copulatory sexual selection.     

Selection on bristle length has the ability to drive the evolution of male abdominal appendages in the sepsid fly Themira biloba

Many exaggerated and novel traits are strongly influenced by sexual selection. Although sexual selection is a powerful evolutionary force, underlying genetic interactions can constrain evolutionary outcomes. The relative strength of selection vs. constraint has been a matter of debate for the evolution of male abdominal appendages in sepsid flies. These abdominal appendages are involved in courtship and mating, but their function has not been directly tested. We performed mate choice experiments to determine whether sexual selection acts on abdominal appendages in the sepsid Themira biloba. We tested whether appendage bristle length influenced successful insemination by surgically trimming the bristles. Females paired with males that had shortened bristles laid only unfertilized eggs, indicating that long bristles are necessary for successful insemination. We also tested whether the evolution of bristle length was constrained by phenotypic correlations with other traits. Analyses of phenotypic covariation indicated that bristle length was highly correlated with other abdominal appendage traits, but was not correlated with abdominal sternite size. Thus, abdominal appendages are not exaggerated traits like many sexual ornaments, but vary independently from body size. At the same time, strong correlations between bristle length and appendage length suggest that selection on bristle length is likely to result in a correlated increase in appendage length. Bristle length is under sexual selection in T. biloba and has the potential to evolve independently from abdomen size.     

Polyandry and paternity skew in natural and experimental populations of Drosophila serrata

Many species engage in polyandry, resulting in the potential for sexual selection to continue post-copulation through sperm competition and/or cryptic female choice. The relative importance of pre- vs. post-copulatory processes remains unknown for most species despite this information being fundamental for understanding the evolutionary consequences of sexual selection. The Australian fruit fly Drosophila serrata has become a prominent model system for studying precopulatory sexual selection, such as mating preferences and their influence on the evolution of sexually selected traits. Here, we investigated polyandry and the potential for post-copulatory sexual selection in this species using indirect paternity analysis. We genotyped 21 wild-caught and 19 laboratory-reared mothers and their offspring (a total of 787 flies) at six microsatellite loci and found extensive polyandry, with all broods surveyed having at least two sires. Female remating rates were higher than in other Drosophila surveyed to date and no significant differences were found between laboratory and field populations. Additionally, we found evidence for biased sperm usage in several broods of D. serrata . Paternity skew occurred more frequently in broods from the field population than the laboratory one, suggesting differences between the two environments in the level of post-copulatory sexual selection. Our data suggest that D. serrata represents a promising system for studying the interaction between pre- and post-copulatory sexual selection in driving the evolution of sexually selected phenotypes.     

Precopulatory but not postcopulatory male reproductive traits diverge in response to mating system manipulation in Drosophila melanogaster

Competition between males creates potential for pre‐ and postcopulatory sexual selection and conflict. Theory predicts that males facing risk of sperm competition should evolve traits to secure their reproductive success. If those traits are costly to females, the evolution of such traits may also increase conflict between the sexes. Conversely, under the absence of sperm competition, one expectation is for selection on male competitive traits to relax thereby also relaxing sexual conflict. Experimental evolution studies are a powerful tool to test this expectation. Studies in multiple insect species have yielded mixed and partially conflicting results. In this study, we evaluated male competitive traits and male effects on female costs of mating in Drosophila melanogaster after replicate lines evolved for more than 50 generations either under enforced monogamy or sustained polygamy, thus manipulating the extent of intrasexual competition between males. We found that in a setting where males competed directly with a rival male for access to a female and fertilization of her ova polygamous males had superior reproductive success compared to monogamous males. When comparing reproductive success solely in double mating standard sperm competition assays, however, we found no difference in male sperm defense competitiveness between the different selection regimes. Instead, we found monogamous males to be inferior in precopulatory competition, which indicates that in our system, enforced monogamy relaxed selection on traits important in precopulatory rather than postcopulatory competition. We discuss our findings in the context of findings from previous experimental evolution studies in Drosophila ssp. and other invertebrate species.     

Evolution of female sperm-storage organs in the carrefour of stylommatophoran gastropods

The presence of specialized female sperm-storage organs has been recognized as an important factor influencing postcopulatory sexual selection via sperm competition and cryptic female choice in internally fertilizing species. We morphologically examined the complexity of sperm-storage organs in the carrefour (spermatheca and fertilization pouch) in 47 species of stylommatophoran gastropods. We used partial 28S rDNA sequences to construct a molecular phylogeny, and applied maximum likelihood (ML) and Bayesian methods to investigate the history of spermatheca diversification and to test different hypotheses of sperm-storage organ evolution. The phylogenetic reconstruction supported several gains and losses of spermathecae. Moreover, a complex spermatheca was associated with the occurrence of love darts or other kinds of auxiliary copulatory organs, the presence of a long penial flagellum, and cross-fertilization as the predominant mating system. However, our results also suggest associations of carrefour complexity with body size, reproductive strategy (semelparity versus iteroparity), reproductive mode (oviparity versus ovoviviparity), and habitat type. Carrefour length in 17 snail species possessing a spermatheca was positively correlated with sperm length. Our results indicate that postcopulatory sexual selection as well as life history and habitat specificity may have influenced the evolution of female sperm-storage organs in hermaphroditic gastropods.     

Sexual selection in fungi

The significance of sexual selection, the component of natural selection associated with variation in mating success, is well established for the evolution of animals and plants, but not for the evolution of fungi. Even though fungi do not have separate sexes, most filamentous fungi mate in a hermaphroditic fashion, with distinct sex roles, that is, investment in large gametes (female role) and fertilization by other small gametes (male role). Fungi compete to fertilize, analogous to ‘male‐male’ competition, whereas they can be selective when being fertilized, analogous to female choice. Mating types, which determine genetic compatibility among fungal gametes, are important for sexual selection in two respects. First, genes at the mating‐type loci regulate different aspects of mating and thus can be subject to sexual selection. Second, for sexual selection, not only the two sexes (or sex roles) but also the mating types can form the classes, the members of which compete for access to members of the other class. This is significant if mating‐type gene products are costly, thus signalling genetic quality according to Zahavi's handicap principle. We propose that sexual selection explains various fungal characteristics such as the observed high redundancy of pheromones at the B mating‐type locus of Agaricomycotina, the occurrence of multiple types of spores in Ascomycotina or the strong pheromone signalling in yeasts. Furthermore, we argue that fungi are good model systems to experimentally study fundamental aspects of sexual selection, due to their fast generation times and high diversity of life cycles and mating systems.     

Low rates of bindin codon evolution in lecithotrophic Heliocidaris sea urchins

Life-history variables including egg size affect the evolutionary response to sexual selection in broadcast-spawning sea urchins and other marine animals. Such responses include high or low rates of codon evolution at gamete recognition loci that encode sperm- and egg-surface peptides. Strong positive selection on such loci affects intraspecific mating success and interspecific reproductive divergence (and may play a role in speciation). Here, we analyze adaptive codon evolution in the sperm acrosomal protein bindin from a brooding sea urchin (Heliocidaris bajulus, with large eggs and nonfeeding or lecithotrophic larval development) and compare our results to previously published data for two closely related congeners. Purifying selection and low relative rates of bindin nonsynonymous substitution in H. bajulus were significantly different from selectively neutral bindin evolution in H. erythrogramma despite similar large egg size in those two species, but were similar to the background rate of nonsynonymous bindin substitution for other closely related sea urchins (including H. tuberculata, all with small egg size and feeding planktonic larval development). Bindin evolution is not driven by egg size variation among Heliocidaris species, but may be more consistent with an alternative mechanism based on the effects of high or low spatial density of conspecific mates.     

Sexually antagonistic evolution caused by male–male competition in the pistil

Although sexual selection and sexual conflict are important evolutionary forces in animals, their significance in plants is uncertain. In hermaphroditic organisms, such as many plants, sexual conflict may occur both between mating partners (interlocus conflict) and between male and female sex roles within an individual (intralocus conflict). We performed experimental evolution, involving lines that were crossed with either one or two pollen donors (monogamous or polyandrous lines), in the hermaphroditic plant (Collinsia heterophylla) where early fertilizations are associated with female fitness costs (reduced seed set). Artificial polyandry for four generations resulted in enhanced pollen performance and increased female fitness costs compared to the monogamous and source (starting material) lines. Female fitness was also reduced in the monogamous line, indicating a possible trade‐off between sex roles, resulting from early pollination. We performed a second experiment to investigate a potential harming effect of pollen performance on seed set. We found that high siring success of early arriving pollen competing with later‐arriving pollen was associated with high female fitness costs, consistent with an interlocus sexual conflict. Our study provides evidence for the importance of sexual selection in shaping evolution of plant reproductive strategies, but also pinpoints the complexity of sexual conflict in hermaphroditic species.