BEST OF BOTH WORLDS? ASSOCIATION BETWEEN OUTCROSSING AND PARASITE LOADS IN A SELFING FISH

Mixed-mating strategies (i.e., intermediate levels of self-fertilization and outcrossing in hermaphrodites) are relatively common in plants and animals, but why self-fertilization (selfing) rates vary so much in nature has proved difficult to explain. We tested the hypothesis that parasites help maintain mixed-mating using a partially selfing fish (Kryptolebias marmoratus) as a model. We show that outcrossed progeny in the wild are genetically more diverse and less susceptible to multiple parasite infections than their selfed counterparts. Given that outcrossing in K. marmoratus can only be attained by male-hermaphrodite matings, our data provide an explanation for the coexistence of males and hermaphrodites in androdioecious species where hermaphrodites are unable to outcross among themselves. Moreover, our study provides evidence that parasites contribute to maintaining mixed-mating in a natural animal population.     

A mixed-mating strategy in a hermaphroditic vertebrate

Mixed-mating systems, in which hermaphrodites can either self-fertilize or outcross, are common in many species of plants and invertebrates and have been informative models for studying the selective forces that can maintain both inbreeding and outbreeding in populations. Here, we document a remarkable instance of evolutionary convergence to an analogous mixed mating system by a vertebrate, the mangrove killifish (Kryptolebias marmoratus). In this androdioecious species, most individuals are simultaneous hermaphrodites that characteristically self-fertilize, resulting in local populations that consist of (nearly) homozygous lines. Most demes are also genetically diverse, an observation traditionally attributed to de novo mutation coupled with high levels of inter-site migration. However, data presented here, from a survey of 35 microsatellite loci in Floridian populations, show that genotypic diversity also stems proximally from occasional outcross events that release 'explosions' of transient recombinant variation. The result is a local population genetic pattern (of extensive genotypic variety despite low but highly heterogeneous intra-individual heterozygosities) that differs qualitatively from the genetic architectures known in any other vertebrate species. Advantages of a mixed-mating strategy in K. marmoratus probably relate to this fish's solitary lifestyle and its ability to colonize new habitats.     

The role of dyadic interactions in the mixed-mating strategies of the mangrove rivulus Kryptolebias marmoratus

The mangrove rivulus Kryptolebias marmoratus a small cyprinodont fish native to tropical and subtropical waters from Florida to Brazil, is one of two known self-fertilizing, hermaphroditic vertebrates of which K. marmoratus displays androdioecy, a complex system of reproduction in which hermaphrodites and males are present. This study describes the behavioral repertoires observed during dyadic interactions in the laboratory. Kryptolebias marmoratus exhibited 23 distinctive acts or behaviors. Acts were divided into four categories: aggressive, submissive, neutral, and reproductive. Leading and following behaviors played important roles in the behavioral repertoires of these fish. In hermaphrodite-male dyads, males exclusively initiated the reproductive process and actively pursued hermaphrodites. When hermaphrodites were paired, there was no evidence that they behaved like other simultaneous hermaphrodites that alternate sexual roles (e.g. serranids). Hermaphrodites were extremely aggressive towards one another, and the aggressor established dominance rapidly. Male-male dyads were divided into two subdivisions based on the presence or absence of the caudal ocellus on one fish. A caudal ocellus on one male appeared to signal the possibility of a potential mating partner to males lacking it. Pairings of males without an ocellus were similar to hermaphrodite-hermaphrodite dyads in that both members of the pair were aggressive towards one another. These observations may be indicative of interactions taking place in natural communities or assemblages of fish in which both males and hermaphrodites occur and provide evidence on the role of dyadic interactions in the mixed-mating strategies [Current Zoology 56 (1): 6-17, 2010].     

Density-dependent self-fertilization and male versus hermaphrodite siring success in an androdioecious plant

Models of mating-system evolution emphasize the importance of frequency-dependent interactions among mating partners. It is also known that outcross siring success and the selfing rate in self-compatible hermaphrodites can be density dependent. Here, we use array experiments to show that the mating system (i.e., the outcrossing rate) and the siring success of morphs with divergent sex allocation strategies are both density dependent and frequency dependent in androdioecious populations of the wind-pollinated, annual plant Mercurialis annua. In particular, the outcrossing rate is a decreasing function of the mean interplant distance, regulated by a negative exponential pollen fall-off curve. Our results indicate that pollen dispersed from a male inflorescence are over 60% more likely to sire outcrossed progeny than equivalent pollen dispersed from hermaphrodites, likely due to the fact that males, but not hermaphrodites, disperse their pollen from erect inflorescence stalks. Because of this difference, and because males of M. annua produce much more pollen than hermaphrodites, the presence of males in the experimental arrays reduced both the selfing rate and the outcross siring success of hermaphrodites. We use our results to infer a density threshold below which males are unable to persist with hermaphrodites but above which they can invade hermaphroditic populations. We discuss our findings in the context of a metapopulation model, in which males can only persist in well-established populations but are excluded from small, sparse populations, for example, in the early stages of colonization.     

The evolution from females to hermaphrodites results in a sexual conflict over mating in androdioecious nematode worms and clam shrimp

The nematode worm Caenorhabditis elegans and the clam shrimp Eulimnadia texana are two well‐studied androdioecious species consisting mostly of self‐fertilizing hermaphrodites and few males. To understand how androdioecy can evolve, a simple two‐step mathematical model of the evolutionary pathway from a male–female species to a selfing‐hermaphrodite species is constructed. First, the frequency of mutant females capable of facultative self‐fertilization increases if the benefits of reproductive assurance exceed the cost. Second, hermaphrodites become obligate self‐fertilizers if the fitness of selfed offspring exceeds one‐half the fitness of outcrossed offspring. Genetic considerations specific to C. elegans and E. texana show that males may endure as descendants of the ancestral male–female species. These models combined with an extensive literature review suggest a sexual conflict over mating in these androdioecious species: selection favours hermaphrodites that self and males that outcross. The strength of selection on hermaphrodites and males differs, however. Males that fail to outcross suffer a genetic death. Hermaphrodites may never encounter a rare male, and those that do and outcross only bear less fecund offspring. This asymmetric sexual conflict results in an evolutionary stand‐off: rare, but persistent males occasionally fertilize common, but reluctant hermaphrodites. A consequence of this stand‐off may be an increase in the longevity of the androdioecious mating system.     

Diversity in mating behavior of hermaphroditic and male-female Caenorhabditis nematodes

In this study, we addressed why Caenorhabditis elegans males are inefficient at fertilizing their hermaphrodites. During copulation, hermaphrodites generally move away from males before they become impregnated. C. elegans hermaphrodites reproduce by internal self-fertilization, so that copulation with males is not required for species propagation. The hermaphroditic mode of reproduction could potentially relax selection for genes that optimize male mating behavior. We examined males from hermaphroditic and gonochoristic (male-female copulation) Caenorhabditis species to determine if they use different sensory and motor mechanisms to control their mating behavior. Instead, we found through laser ablation analysis and behavioral observations that hermaphroditic C. briggsae and gonochoristic C. remanei and Caenorhabditis species 4, PB2801 males produce a factor that immobilizes females during copulation. This factor also stimulates the vulval slit to widen, so that the male copulatory spicules can easily insert. C. elegans and C. briggsae hermaphrodites are not affected by this factor. We suggest that sensory and motor execution of mating behavior have not significantly changed among males of different Caenorhabditis species; however, during the evolution of internal self-fertilization, hermaphrodites have lost the ability to respond to the male soporific-inducing factor.     

Is sexual selection operating in the androdioecious clam shrimp,Eulimnadia texana (Crustacea: Conchostraca)?

Experiments were performed to document the existence of intersexual or intrasexual selection in the clam shrimp,Eulimnadia texana. Individuals within this species are either males or hermaphrodites. Hermaphrodites can self their own eggs or outcross with a male, but they cannot outcross with other hermaphrodites. Theoretical considerations suggest that both intrasexual and intersexual selection could be occurring on the part of the hermaphrodites and the males. When males were given a choice between two non-gravid hermaphrodites of different sizes, they did not exhibit a mating preference based upon size. When two males of different sizes were isolated with a single nongravid receptive hermaphrodite, the hermaphrodite showed no preference between the two males. There was evidence, however, of male-male competition for receptive hermaphrodites and of mate guarding on the part of the males. During aggressive encounters between twp males, the larger of the two had a significant advantage over the smaller, and larger males were always the victors hermaphrodite takeovers occurred as a result of male-male conflict. Hermaphrodites appear to control the mating process both by struglling with males when they are not receptive to them and by selfing in the presence of males. This suggests that hermaphrodites withhold receptivity cues from males, or produce non-receptivity cues, when they are going to self. Though hermaphrodites do not appear to select males based upon size, they make a selection between selfing and outcrossing by controlling the use of receptivity signals.     

Association behaviour of the self-fertilizing Kryptolebias marmoratus (Poey): the influence of microhabitat use on the potential for a complex mating system

Preliminary observations were conducted to identify conspicuous body postures and movements of males and hermaphrodites in the mangrove killifish Kryptolebias marmoratus . These behaviours were used quantitatively to examine the social interactions for experimental pairings of K. marmoratus of different sexual states ( i.e. simultaneous hermaphrodite and male) in an aquarium with an open-water area and simulated crab burrows. This allowed observation of behaviours that could not be observed in the field. Kryptolebias marmoratus , regardless of sexual state and experimental treatment, spent 40% of the time in the burrow. Hermaphrodites exhibited a preference for associating with males rather than other hermaphrodites. The observed complexity of displayed behaviours and interactions between paired conspecifics indicate that K. marmoratus has a rich repertoire of social behaviour not predicted for a strictly selfing species. Also, land crab burrows play an important role in their social interactions.     

Sampling from natural populations with RNAI reveals high outcrossing and population structure in Caenorhabditis elegans

Despite a nearly worldwide distribution in nature, Caenorhabditis elegans exhibits low levels of genetic polymorphism, possibly as an indirect consequence of low levels of outcrossing. In the laboratory, Caenorhabditis elegans males are produced at low rates and are steadily eliminated from cultures, so that reproduction happens largely through self-fertilization in hermaphrodites. C. elegans is increasingly the focus of evolutionary research; however, natural outcrossing rates are difficult to measure because mating tests with laboratory strains are usually required to identify C. elegans. We sampled natural populations of C. elegans with an RNA interference (RNAi) assay. Heterozygosities and polymorphism patterns revealed surprisingly high levels of population structure and outcrossing (approximately 22% of individuals are estimated to be the result of outcrossing and not self-fertilization). The finding of strong local population structure, together with low levels of diversity on local and global scales, suggests a metapopulation model of frequent extinction and recolonization of local populations. The occurrence of substantial outcrossing suggests that the extinction of local populations is probably not driven by the accumulation of harmful mutations.     

Isolation and characterization of 13 polymorphic microsatellite loci from the clam shrimp Eulimnadia texana (Crustacea: Spinicaudata)

Thirteen polymorphic microsatellite loci were isolated and characterized from the clam shrimp Eulimnadia texana. In analyses of 20–50 individuals from two populations the number of alleles ranged from two to seven with observed heterozygosity ranging between 0.00 and 0.37. The low values for heterozygosity were not unexpected for a group characterized by its unusual androdioecious mating system, in which males compete with self‐compatible hermaphrodites for offspring production. These microsatellites are likely to be useful for further evolutionary investigations of this rare mating system in these crustaceans.     

Do males facilitate the spread of novel phenotypes within populations of the androdioecious nematode Caenorhabditis elegans?

In the androdioecious nematode Caenorhabditis elegans, self-fertilization is the predominant mode of reproduction. Nevertheless, males do occur, and it is still unclear if these represent a selective advantage or merely an evolutionary relict. In this study, we first tested the hypothesis that the production of males might benefit invaders to resident populations. We added single, GFP-marked worms to established laboratory populations and followed GFP frequencies over time. Mated hermaphrodites and also males were more successful in invading resident populations if compared to single, unmated hermaphrodites. The observed higher frequencies should increase the likelihood that any of the associated invading alleles persist. Second, we tested the hypothesis that males and, thus, higher outcrossing rates, are specifically favored under changing environmental conditions. After an outbred population was subjected to changing stress or to control laboratory conditions, we measured the male maintenance of the resulting populations. Interestingly all populations, experimental and control alike, showed high male maintenance, suggesting that persistence of males is also favored under standard laboratory conditions.     

Male–Male Competition in a Mixed‐Mating Fish

Mating systems that comprise a mixture of pure males and self‐fertilising hermaphrodites remain an evolutionary enigma. In particular, our understanding of the sexual selection pressures associated with such mating systems is nascent. Males can only reproduce by fertilising hermaphrodites’ eggs, but hermaphrodites can also fertilise their own eggs and gain a genetic advantage by doing so. Consequently, there should be intense competition among males to access hermaphrodites. Here, we test the importance of male size, colour and heterozygosity in predicting the outcome of male–male competition using the mangrove rivulus, which has a male‐hermaphrodite mixed‐mating system. We pitted males against one another in dyadic laboratory trials to develop a dominance score for each male. We then correlated these scores with male length, several components of male colour, and heterozygosity. Male size was the only significant correlate of dominance: larger males dominated smaller males, implying selection for large male size. However, male mangrove rivulus are similar in size to hermaphrodites, indicating that directional selection for large body size in males is no greater than it is in hermaphrodites. Across all trials, colour was unrelated to dominance, but contests between similarly sized males were usually won by more colourful individuals. As mangrove rivulus are dichromatic, we suspect that male colour may prove to be more important in mate choice than we found it to be in intrasexual competition. Heterozygosity did not explain dominance directly, but correlated strongly with male size, implying an indirect role in intrasexual competition.     

Intersexual conflict in androdioecious clam shrimp: Do androdioecious hermaphrodites evolve to avoid mating with males?

A recent sexual conflict model posits that a form of intersexual conflict may explain the persistence of males in androdioecious (males + hermaphrodites) populations of animals that are being selected to transition from dioecious (gonochoristic) mating to self‐compatible hermaphroditism. During the evolutionary spread of a self‐compatible hermaphrodite to replace females, the selective pressures on males to outcross are in conflict with the selective pressures on hermaphrodites to self. According to this model, the unresolved conflict interferes with the evolutionary trajectory from dioecy to hermaphroditism, slowing or halting that transition and strengthening the otherwise “transitory” breeding system of androdioecy into a potentially stable breeding strategy. Herein, we assess this model using two dioecious and two androdioecious clam shrimp (freshwater crustaceans) to ask two questions: (1) Have hermaphrodites evolved so that males cannot effectively recognize them?; and (2) Do androdioecious hermaphrodites avoid males? Androdioecious males made more mistakes than dioecious males when guarding potential mates suggesting that androdioecious males were less effective at finding hermaphrodites than dioecious males were at finding females. Similarly, in a three‐chambered experiment, focal hermaphrodites chose to aggregate with their same sex, whereas focal dioecious males chose to aggregate with the alternate sex. Together, these two experiments support the sexual conflict model of the maintenance of androdioecy and suggest that hermaphrodites are indeed evolving to avoid and evade males.     

Sexuality and hermaphroditism in fishes. I. Synchronous functional hermaphroditism in the serranid fish Serranus scriba L

Anatomical and histological examination of Serranus scriba L. showed the existence of primary females (67%), hermaphrodites (31%) and primary males (2%). Synchronous functional hermaphroditism is described on the basis of an anatomical and histological study of the gonads. Although they function simultaneously, the testicular and ovarian parts of hermaphrodite gonads have completely separate ducts. Females and hermaphrodites have the same annual reproduction cycle. In hermaphrodites, the testicular part matures one month sooner than the ovarian part. Cross fertilization between primary females and hermaphrodite individuals and between two different hermaphrodites probably occurs, while self-fertilization is less likely. The testicular tissues of primary males are of the acinar type and those of hermaphrodites are of the radial type. It is possible that primary males do not take part in reproduction. Serranus scriba in Egyptian Mediterranean waters is a longperiod spawner, which spawns from June to the end of October, i.e. it is a summer-autumn spawner.     

ECOLOGICAL GENETICS OF GYNODIOECY IN SILENE VULGARIS: RELATIVE FITNESS OF FEMALES AND HERMAPHRODITES DURING THE COLONIZATION PROCESS

Recent theoretical models have addressed the influence of metapopulation dynamics on the fitness of females and hermaphrodites in gynodioecious plants. In particular, selection is thought to favor hermaphrodites during population establishment because that sex should be less prone to pollen limitation, especially if self-fertilization is possible. However, inbreeding depression could limit this advantage. In this experimental study of Silene vulgaris, a weedy gynodioecious plant, the fitness of females and hermaphrodites was estimated from seed production in both mixed-sex populations and for individuals isolated from these populations by 20, 40, 80, or 160 m. In mixed populations females display statistically significant greater per capita seed production owing to higher capsule production and higher rates of seed germination. The fitness of both sexes declines with increasing isolation, but at different rates, such that in the 160-m treatment hermaphrodites are by far the more fit sex. Allozyme studies suggest that this differential decline is because the selfing rate in hermaphrodites increases as a function of isolation, at least partially compensating for a decline in the availability of outcross pollen. Overall, the negative effects of pollen limitation on females far outweighs the negative effects of inbreeding depression following selfing in hermaphrodites. Thus, extinction/recolonization dynamics would appear to favor hermaphrodites as long as seed dispersal events exceed some critical distance.     

Genetic variation for outcrossing among Caenorhabditis elegans isolates

The evolution of breeding systems results from the existence of genetic variation and selective forces favoring different outcrossing rates. In this study we determine the extent of genetic variation for characters directly related to outcrossing, such as male frequency, male mating ability, and male reproductive success, in several wild isolates of the nematode Caenorhabditis elegans. This species is characterized by an androdioecious breeding system in which males occur with hermaphrodites that can either self-fertilize or outcross with males. We find genetic variation for all characters measured, but also find that environmental variation is a large fraction of the total phenotypic variance. We further determine the existence of substantial genetic variation for population competitive performance in several laboratory environments. However, these measures are uncorrelated with outcrossing characters. The data presented here contribute to an understanding of male maintenance in natural populations through their role in outcrossing.     

Selfing versus outcrossing in the androdioecious clam shrimp,Eulimnadia texana (Crustacea,Conchostraca)

The clam shrimpEulimnadia texana is an androdioecious crustacean in which hermaphrodites may self fertilize or outcross with males but cannot outcross with other hermaphrodites. Outcrossing is maintained within most populations of this species despite the high genetic cost of sex, suggesting that compensating factors provide an advantage to outcrossing. We hypothesized that one such benefit would be the production of larger clutch sizes resulting from outcrossed matings. To test this prediction, we recorded the body sizes and clutch sizes of hermaphrodites which mated via selfing or bia outcrossing. Clutch sizes showed significant, almost exponential, increases as body size increased in both selfing and outcrossing hernmaphrodites. The rate of this increase was the same for both groups, and there was no significant difference in clutch size when body size was controlled for between the two fertilization types.     

Mixed mating in androdioecious Mercurialis annua inferred using progeny arrays and diploid-acting microsatellite loci in a hexaploid background

Background and Aims

The frequency at which males can be maintained with hermaphrodites in androdioecious populations is predicted to depend on the selfing rate, because self-fertilization by hermaphrodites reduces prospective siring opportunities for males. In particular, high selfing rates by hermaphrodites are expected to exclude males from a population. Here, the first estimates are provided of the mating system from two wild hexaploid populations of the androdioecious European wind-pollinated plant M. annua with contrasting male frequencies.

Methods

Four diploid microsatellite loci were used to genotype 19–20 progeny arrays from two populations of M. annua, one with males and one without. Mating-system parameters were estimated using the program MLTR.

Key Results

Both populations had similar, intermediate outcrossing rates (t_m = 0·64 and 0·52 for the population with and without males, respectively). The population without males showed a lower level of correlated paternity and biparental inbreeding and higher allelic richness and gene diversity than the population with males.

Conclusions

The results demonstrate the utility of new diploid microsatellite loci for mating system analysis in a hexaploid plant. It would appear that androdioecious M. annua has a mixed-mating system in the wild, an uncommon finding for wind-pollinated species. This study sets a foundation for future research to assess the relative importance of the sexual system, plant-density variation and stochastic processes for the regulation of male frequencies in M. annua over space and time.     

Density-dependent regulation of the sex ratio in an annual plant

Sex ratios are subject to strong frequency-dependent selection regulated by the mating system and the relative male versus female investment. In androdioecious plant populations, where males co-occur with hermaphrodites, the sex ratio depends on the rate of self-fertilization by hermaphrodites and on the relative pollen production of males versus hermaphrodites. Here, we report evolutionary changes in the sex ratio from experimental mating arrays of the androdioecious plant Mercurialis annua. We found that the progeny sex ratio depended strongly on density, with fewer males in the progeny of plants grown under low density. This occurred in part because of a plastic adjustment in pollen production by hermaphrodites, which produced more pollen when grown at low density than at high density. Our results provide support for the prediction that environmental conditions govern sex ratios through their effects on the relative fertility of unisexual versus hermaphrodite individuals.