SEX ALLOCATION ADJUSTMENT TO MATING GROUP SIZE IN A SIMULTANEOUS HERMAPHRODITE

Sex allocation theory is considered as a touchstone of evolutionary biology, providing some of the best supported examples for Darwinian adaptation. In particular, Hamilton's local mate competition theory has been shown to generate precise predictions for extraordinary sex ratios observed in many separate‐sexed organisms. In analogy to local mate competition, Charnov's mating group size model predicts how sex allocation in simultaneous hermaphrodites is affected by the mating group size (i.e., the number of mating partners plus one). Until now, studies have not directly explored the relationship between mating group size and sex allocation, which we here achieve in the simultaneously hermaphroditic flatworm Macrostomum lignano. Using transgenic focal worms with ubiquitous expression of green‐fluorescent protein (GFP), we assessed the number of wild‐type mating partners carrying GFP+ sperm from these focal worms when raised in different social group sizes. This allowed us to test directly how mating group size was related to the sex allocation of focal worms. We find that the proportion of male investment initially increases with increasing mating group size, but then saturates as predicted by theory. To our knowledge, this is the first direct test of the mating group size model in a simultaneously hermaphroditic animal.     

Sperm transfer is affected by mating history in the simultaneously hermaphroditic snail Lymnaea stagnalis

Abstract. Males are predicted to strategically allocate sperm across mating partners in order to maximize their chances of paternity. This requires that males have the ability to detect aspects of their partner's mating history or the number of potential mates. We investigated whether simultaneous hermaphrodites mating in the male role strategically adjust sperm transfer depending on rearing conditions. The pond snail Lymnaea stagnalis (Basommatophora) is known to donate sperm repeatedly to different partners during a breeding season and store received sperm for >3 months. The rearing conditions of the donor as well as the recipient affect the amount of sperm transferred. Sperm donors raised in isolation transfer more sperm than those raised in groups. Furthermore, isolated sperm donors transfer less sperm to partners that were raised in groups than to those raised in isolation, i.e., virgins. These findings suggest that snails raised in isolation shift their sex allocation toward the male function and indicate that they can somehow assess the mating status of their partner.     

Social situation,sperm competition and sex allocation in a simultaneous hermaphrodite parasite,the cestode Schistocephalus solidus

Evolutionary theory predicts an influence of mating group size on sex allocation in simultaneous hermaphrodites. We experimentally manipulated the social situation during reproduction in a simultaneous hermaphrodite parasite, the tapeworm Schistocephalus solidus, by placing worms as singles, pairs or triplets into an in vitro system that replaces the final host. We then determined the reproductive allocation patterns after 24 h (i.e. before the start of egg release) and after 72 h (i.e. around the peak of egg release rate) using stereology. After 24 h, sex allocation strongly depended on worm volume (which is determined in the second intermediate host), but was not significantly affected by the social situation experienced during reproduction. After 72 h, worms in groups had less vesicular sperm (i.e. sperm to be used in future inseminations) than singles. They also stored significantly more received sperm in their seminal receptacles than singles, suggesting that more sperm had been transferred in groups. Moreover, worms in triplets stored significantly more received sperm than worms in pairs, suggesting that they either mated more often and/or transferred more sperm per mating. This suggests a behavioural response to the increased risk of sperm competition in triplets. We further discuss the relative importance of sex allocation decisions at different life‐history stages.     

No Plastic Responses to Experimental Manipulation of Sperm Competition per se in a Free‐Living Flatworm

In the absence of sperm competition evolutionary theory predicts low mating rates and low ejaculate expenditure per mating, and sex allocation theory for simultaneous hermaphrodites predicts a strongly female‐biased sex allocation. In the presence of sperm competition a shift towards a more male‐biased sex allocation and a higher ejaculate expenditure are predicted. The free‐living flatworm Macrostomum lignano has been shown to respond plastically in mating rate, testis size, and sperm transfer to manipulation of the social group size, a proxy of the strength of sperm competition. However, manipulation of social group size may manipulate not only sperm competition, but also other factors, such as food supply and metabolite concentration. In this study we therefore manipulated sperm competition per se by repeatedly exposing individuals to partners that have either mated with rivals or not, while keeping the social group size constant. Our results suggest that M. lignano does not have the ability to detect sperm competition per se, as worms experimentally exposed to the presence or absence of sperm competition did not differ in sex allocation, sperm transfer or mating behavior. A response to our manipulation would have required individual recognition, the ability to detect self‐referencing tags, or tags or traces left by rivals on or in the mating partners. We first discuss the possibility that highly efficient sperm displacement may have decreased the difference between the treatment groups and then propose three alternative cues that may allow M. lignano to respond plastically to the social group size manipulation used in earlier studies: assessment of the mating rate, chemical cues, or tactile cues.     

High multiple paternity and low last-male sperm precedence in a hermaphroditic planarian flatworm: consequences for reciprocity patterns

It is difficult to predict a priori how mating success translates into fertilization success in simultaneous hermaphrodites with internal fertilization. Whereas insemination decisions will be determined by male interests, fertilization will depend on female interests, possibly leading to discrepancies between insemination and fertilization patterns. The planarian flatworm Schmidtea polychroa, a simultaneous hermaphrodite in which mating partners trade sperm was studied. Sperm can be stored for months yet individuals mate frequently. Using microsatellites, maternity and paternity data were obtained from 748 offspring produced in six groups of 10 individuals during four weeks. Adults produced young from four mates on average. Reciprocal fertilization between two mates was found in only 41 out of 110 registered mate combinations, which is clearly less than what is predicted from insemination patterns. Multiple paternity was high: > 80% of all cocoons had two to five fathers for only three to five offspring per cocoon. Because animals were collected from a natural population, 28% of all hatchlings were sired by unknown sperm donors in the field, despite a 10-day period of acclimatization and within-group mating. This percentage decreased only moderately throughout the experiment, showing that sperm can be stored and used for at least a month, despite frequent mating and sperm digestion. The immediate paternity a sperm donor could expect to obtain was only about 25%. Male reproductive success increased linearly with the number of female partners, providing support for Bateman's principle in hermaphrodites. Our results suggest that hermaphrodites do not trade fertilizations when trading sperm during insemination, lending support to the view that such conditional sperm exchange is driven by exchange of resources.     

Mating frequency and resource allocation to male and female function in the simultaneous hermaphrodite land snail Arianta arbustorum

Sex allocation theory predicts that mating frequency and long‐term sperm storage affect the relative allocation to male and female function in simultaneous hermaphrodites. We examined the effect of mating frequency on male and female reproductive output (number of sperm delivered and eggs deposited) and on the resources allocated to the male and female function (dry mass, nitrogen and carbon contents of spermatophores and eggs) in individuals of the simultaneous hermaphrodite land snail Arianta arbustorum. Similar numbers of sperm were delivered in successive copulations. Consequently, the total number of sperm transferred increased with increasing number of copulations. In contrast, the total number of eggs produced was not influenced by the number of copulations. Energy allocation to gamete production expressed as dry mass, nitrogen or carbon content was highly female‐biased (>95% in all estimates). With increasing number of copulations the relative nitrogen allocation to the male function increased from 1.7% (one copulation) to 4.7% (three copulations), but the overall reproductive allocation remained highly female‐biased. At the individual level, we did not find any trade‐off between male and female reproductive function. In contrast, there was a significant positive correlation between the resources allocated to the male and female function. Snails that delivered many sperm also produced a large number of eggs. This finding contradicts current theory of sex allocation in simultaneous hermaphrodites.     

Different effects of mating group size as male and as female on sex allocation in a simultaneous hermaphrodite

Sex allocation theory predicts that the optimal sexual resource allocation of simultaneous hermaphrodites is affected by mating group size (MGS). Although the original concept assumes that the MGS does not differ between male and female functions, the MGS in the male function (MGSm; i.e., the number of sperm recipients the focal individual can deliver its sperm to plus one) and that in the female function (MGSf; the number of sperm donors plus one) do not always coincide and may differently affect the optimal sex allocation. Moreover, reproductive costs can be split into “variable” (e.g., sperm and eggs) and “fixed” (e.g., genitalia) costs, but these have been seldom distinguished in empirical studies. We examined the effects of MGSm and MGSf on the fixed and variable reproductive investments in the sessilian barnacle Balanus rostratus. The results showed that MGSm had a positive effect on sex allocation, whereas MGSf had a nearly significant negative effect. Moreover, the “fixed” cost varied with body size and both aspects of MGS. We argue that the two aspects of MGS should be distinguished for organisms with unilateral mating.     

The effect of cryptic female choice on sex allocation in simultaneous hermaphrodites

Sex allocation theory for simultaneous hermaphrodites has focused primarily on the effects of sperm competition, but the role of mate choice has so far been neglected. We present a model to study the coevolution of cryptic female choice and sex allocation in simultaneous hermaphrodites. We show that the mechanism of cryptic female choice has a strong effect on the evolutionary outcome: if individuals remove a fixed proportion of less-preferred sperm, the optimal sex allocation is more female biased (i.e. more biased towards egg production) than without cryptic female choice; conversely, if a fixed amount of sperm is removed, sex allocation is less female-biased than without cryptic female choice, and can easily become male biased (i.e. biased towards sperm production). Under male-biased sex allocation, hermaphroditism can become unstable and the population can split into pure males and hermaphrodites with a female-biased allocation. We discuss the idea that the evolution of sex allocation may depend on the outcome of sexual conflict over the fate of received sperm: the sperm donor may attempt to manipulate or by-pass cryptic female choice and the sperm recipient is expected to resist such manipulation. We conclude that cryptic female choice can have a strong influence on sex allocation in simultaneous hermaphrodites and strongly encourage empirical work on this question.     

Multiple paternity and mate competition in non-selfing,monogamous, egg-trading hermaphrodites

In animals in which the two sexes invest relatively similar amounts of resources in their young, the number of mates is expected to affect male and female reproductive success similarly and gender conflicts on the number of mates may not arise. Correspondingly, in non-selfing, simultaneous hermaphrodites with long-term monogamy, the two partners are expected to alternate repeatedly their sexual roles and invest similarly in their offspring. Therefore, the gender conflict on the number of mating partners should not arise. However, when >2 conspecifics are present, hermaphrodites are known to plastically adjust their behavior and sex allocation and compete for mating repeatedly in the male role. We tested whether this leads to multiple paternities of single egg clutches in experimental replicates of small and large groups of non-selfing, egg-trading, behaviorally monogamous polychaete worms (Ophryotrocha diadema) by using neutral genetic markers to estimate paternity. Multiply fertilized egg cocoons were common in these worms; two or more individuals succeeded in fertilizing the same egg cocoon and mate competition increased with group size. Multiply fertilized egg cocoons had a higher proportion of eggs developing into mature worms than singly fertilized egg cocoons. Possibly singly fertilized cocoons had a lower fertilization rate owing to low sperm counts and aflagellate sperm.     

Sex allocation in a simultaneously hermaphroditic marine shrimp

Two fundamental questions dealing with simultaneous hermaphrodites are how resources are optimally allocated to the male and female function and what conditions determine shifts in optimal sex allocation with age or size. In this study, I explored multiple factors that theoretically affect fitness gain curves (that depict the relationship between sex-specific investment and fitness gains) to predict and test the overall and size-dependent sex allocation in a simultaneously hermaphroditic brooding shrimp with an early male phase. In Lysmata wurdemanni, sperm competition is absent as hermaphrodites reproducing in the female role invariably mated only once with a single other shrimp. Shrimps acting as females preferred small over large shrimps as male mating partners, male mating ability was greater for small compared to large hermaphrodites, and adolescent males were predominant in the population during the breeding season. In addition, brooding constraints were not severe and varied linearly with body size whereas the ability to acquire resources increased markedly with body size. Using sex allocation theory as a framework, the findings above permitted to infer the shape of the male and female fitness gain curves for the hermaphrodites. The absence of sperm competition and the almost unconstrained brooding capacity imply that both curves saturate, however the male curve levels off much more quickly than the female curve with increasing level of investment. In turn, the predominance of adolescent males in the population implies that the absolute gain of the female curve is greater than that of the male curve. Last, the size-dependent female preference and male mating ability of hermaphrodites determines that the absolute gain of the male curve is greater for small than for large hermaphrodites. Taking into consideration the inferred shape of the fitness gain curves, two predictions with respect to the optimal sex allocation were formulated. First, overall sex allocation should be female biased; it permits hermaphrodites to profit from the female function that provides a greater fitness return than the male function. Second, sex allocation should be size-dependent with smaller hermaphrodites allocating more than proportionally resources to male reproduction than larger ones. This size-dependent sex allocation permits hermaphrodites to profit from male mating opportunities that are the greatest at small body sizes. Size-dependent sex allocation is also expected because the male fitness gain curve decelerates more quickly than the female gain curve and experiments indicated that resources are greater for large than small hermaphrodites. These two predictions were tested when determining the sex allocation of hermaphrodites by dissecting their gonad and quantifying ovaries versus testes mass. Supporting the predictions above, hermaphrodites allocated, on average, 118 times more to the female than to the male gonad and the proportion of resources devoted to male function was higher in small than in large hermaphrodites. A trade-off between male and female allocation is assumed by theory but no negative correlation between male and female reproductive investment was observed. In L. wurdemanni, the relationship between sex-specific investment and fitness changes during ontogeny in a way that is consistent with an adjustment of sex allocation to improve size-specific reproductive success.     

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.     

Bigger testes increase paternity in a simultaneous hermaphrodite,independently of the sperm competition level

Hermaphroditic animals face the fundamental evolutionary optimization problem of allocating their resources to their male vs. female reproductive function (e.g. testes and sperm vs. ovaries and eggs), and this optimal sex allocation can be affected by both pre‐ and post‐copulatory sexual selection. For example, local sperm competition (LSC) – the competition between related sperm for the fertilization of a partner's ova – occurs in small mating groups and can favour a female‐biased sex allocation, because, under LSC, investment into sperm production is predicted to show diminishing fitness returns. Here, we test whether higher testis investment increases an individual's paternity success under sperm competition, and whether the strength of this effect diminishes when LSC is stronger, as predicted by sex allocation theory. We created two subsets of individuals of the simultaneously hermaphroditic flatworm Macrostomum lignano – by sampling worms from either the highest or lowest quartile of the testis investment distribution – and estimated their paternity success in group sizes of either three (strong LSC) or eight individuals (weak LSC). Specifically, using transgenic focal individuals expressing a dominant green‐fluorescent protein marker, we showed that worms with high testis investment sired 22% more offspring relative to those with low investment, corroborating previous findings in M. lignano and other species. However, the strength of this effect was not significantly modulated by the experienced group size, contrasting theoretical expectations of more strongly diminishing fitness returns under strong LSC. We discuss the possible implications for the evolutionary maintenance of hermaphroditism in M. lignano.     

Sex allocation predicts mating rate in a simultaneous hermaphrodite

Sexual selection theory for separate-sexed animals predicts that the sexes differ in the benefit they can obtain from multiple mating. Conventional sex roles assume that the relationship between the number of mates and the fitness of an individual is steeper in males compared with females. Under these conditions, males are expected to be more eager to mate, whereas females are expected to be choosier. Here we hypothesize that the sex allocation, i.e. the reproductive investment devoted to the male versus female function, can be an important predictor of the mating strategy in simultaneous hermaphrodites. We argue that within-species variation in sex allocation can cause differences in the proportional fitness gain derived through each sex function. Individuals should therefore adjust their mating strategy in a way that is more beneficial to the sex function that is relatively more pronounced. To test this, we experimentally manipulated the sex allocation in a simultaneously hermaphroditic flatworm and investigated whether this affects the mating behaviour. The results demonstrate that individuals with a more male-biased sex allocation (i.e. relatively large testes and small ovaries) are more eager to mate compared with individuals with a more female-biased sex allocation (i.e. relatively small testes and large ovaries). We argue that this pattern is comparable to conventional gender roles in separate-sexed organisms.     

Phenotypic plasticity in sex allocation for a simultaneously hermaphroditic coral reef fish

Phenotypic plasticity can facilitate reproductive strategies that maximize mating success in variable environments and lead to differences in sex allocation among populations. For simultaneous hermaphrodites with sperm competition, including Serranus tortugarum a small coral reef fish, proportional male allocation (testis in total gonad) is often greater where local density or mating group size is higher. We tested whether S. tortugarum reduced male allocation when transplanted from a higher density site to a lower density site. After 4 months, transplants mirrored the sex-allocation patterns of the resident population on their new reef. Transplants had significantly lower male allocation than representatives from their source population, largely as a result of reduced testis mass relative to body size.     

Multiple mating, paternity, and body size in a simultaneous hermaphrodite, Aplysia californica

Sperm displacement and sperm competition prove difficult tomeasure, but are crucial elements in predicting sex allocationstrategies of sperm-storing hermaphrodites. Body size is predictedto affect sex allocation so that within a population, largeanimals invest a greater proportion of resources in female functionthan do small animals. These mating strategies depend on spermdisplacement abilities and lead to similar levels of paternityacross body sizes despite differences in resource level. Thepresent study investigated mating patterns, multiple paternity,and sperm competition in a field population of a simultaneouslyhermaphroditic sea slug, Aplysia californica (California seahare). Animals mating in the female role were larger than themean for the population, indirectly supporting theoretical predictionsfor increased investment in female function with body size.However, contrary to predictions, animals mating in the malerole were not different in size from the population mean orthe animals they inseminated. Individual tagging revealed thatsea slugs are capable of moving across distances that allowfor the sampling of many potential mates, and that they materepeatedly in both sexual roles. Microsatellite paternity analysisdemonstrated that multiple mating in the field leads to multiplepaternity, and last-sperm donors achieve high levels of paternity.There was no effect of body size on paternity. Further paternitystudies are needed to reveal the mechanisms of sperm precedencepatterns in A. californica.     

Differential sperm expenditure reveals a possible role for post‐copulatory sexual selection in a lekking moth

Male reproductive success in the lesser wax moth Achroia grisella is strongly determined by pre‐copulatory mate choice, during which females choose among males aggregated in small leks based on the attractiveness of ultrasonic songs. Nothing is known about the potential of post‐copulatory mechanisms to affect male reproductive success. However, there is evidence that females at least occasionally remate with a second male and that males are unable to produce ejaculates quickly after a previous copulation. Here we investigated the effects of mating history on ejaculate size and demonstrate that the number of transferred sperm significantly decreased from first (i.e., virgin) to second (i.e., nonvirgin) copulation within individual males. For males of identical age, the number of sperm transferred was higher in virgin than in nonvirgin copulations, too, demonstrating that mating history, is responsible for the decrease in sperm numbers transferred and not the concomitant age difference. Furthermore, the number of transferred sperm was significantly repeatable within males. The demonstrated variation in ejaculate size both between subsequent copulations as well as among individuals suggests that there is allocation of a possibly limited amount of sperm. Because female fecundity is not limited by sperm availability in this system, post‐copulatory mechanisms, in particular sperm competition, may play a previously underappreciated role in the lesser wax moth mating system.     

Sex allocation and investment into pre- and post-copulatory traits in simultaneous hermaphrodites: the role of polyandry and local sperm competition

Sex allocation theory predicts the optimal allocation to male and female reproduction in sexual organisms. In animals, most work on sex allocation has focused on species with separate sexes and our understanding of simultaneous hermaphrodites is patchier. Recent theory predicts that sex allocation in simultaneous hermaphrodites should strongly be affected by post-copulatory sexual selection, while the role of pre-copulatory sexual selection is much less clear. Here, we review sex allocation and sexual selection theory for simultaneous hermaphrodites, and identify several strong and potentially unwarranted assumptions. We then present a model that treats allocation to sexually selected traits as components of sex allocation and explore patterns of allocation when some of these assumptions are relaxed. For example, when investment into a male sexually selected trait leads to skews in sperm competition, causing local sperm competition, this is expected to lead to a reduced allocation to sperm production. We conclude that understanding the evolution of sex allocation in simultaneous hermaphrodites requires detailed knowledge of the different sexual selection processes and their relative importance. However, little is currently known quantitatively about sexual selection in simultaneous hermaphrodites, about what the underlying traits are, and about what drives and constrains their evolution. Future work should therefore aim at quantifying sexual selection and identifying the underlying traits along the pre- to post-copulatory axis.     

Phenotypically flexible sex allocation in a simultaneous hermaphrodite

Previous studies on sex allocation in simultaneous hermaphrodites have typically focused either on evolutionary or one-time, ontogenetic optimization of sex allocation, ignoring variation within an individual's lifetime. Here, we study whether hermaphrodites also possess facultative sex allocation, that is, a phenotypic flexibility, allowing them to distribute resources to either sex in an opportunistic way during their adult lifetime. We used the simultaneously hermaphroditic free-living flatworm Macrostomum lignano and raised individuals in pairs and groups of eight worms (further called octets) until sexual maturity was reached and sex allocation for the current conditions was expected to be set. Treatment groups were subsequently transferred to the alternative group size, that is, from pairs to octets or from octets to pairs, and compared to two control groups, which were transferred without changing group size. The results show that worms in treatment groups responded as expected by the local mate competition theory for simultaneous hermaphrodites: increasing group size resulted in a shift toward a more male-biased sex allocation and vice versa. These findings reveal that sex allocation in these animals is not fixed during ontogeny, but remains flexible after maturation. We argue that phenotypically flexible sex allocation in hermaphroditic animals may help us to understand the evolution and ecology of hermaphroditism.     

Size‐dependent sex allocation in a simultaneous hermaphrodite parasite

Most models of sex allocation distinguish between sequential and simultaneous hermaphrodites, although an intermediate sexual pattern, size‐dependent sex allocation, is widespread in plants. Here we investigated sex allocation in a simultaneous hermaphrodite animal, the tapeworm Schistocephalus solidus, in which adult size is highly variable. Sex allocation was determined using stereological techniques, which allow measuring somatic and reproductive tissues in a common currency, namely volume. We investigated the relationships between individual volume and allocation to different reproductive tissues using an allometric model. One measure of female allocation, yolk gland volume, increased more than proportionally with individual volume. This is in contrast to the measure of male allocation, testis volume, which showed a strong tendency to increase less than proportionally with individual volume. Together these patterns led to sex allocation being strongly related to individual volume, with large individuals being more biased towards female allocation. We discuss these findings in the light of current ideas about size‐dependent sex allocation in, primarily, plants and try to extend them to simultaneous hermaphrodite animals.