Evolution of outcrossing in experimental populations of Caenorhabditis elegans

Caenorhabditis elegans can reproduce exclusively by self-fertilization. Yet, males can be maintained in laboratory populations, a phenomenon that continues to puzzle biologists. In this study we evaluated the role of males in facilitating adaptation to novel environments. For this, we contrasted the evolution of a fitness component exclusive to outcrossing in experimental populations of different mating systems. We introgressed a modifier of outcrossing into a hybrid population derived from several wild isolates to transform the wild-type androdioecious mating system into a dioecious mating system. By genotyping 375 single-nucleotide polymorphisms we show that the two populations had similar standing genetic diversity available for adaptation, despite the occurrence of selection during their derivation. We then performed replicated experimental evolution under the two mating systems from starting conditions of either high or low levels of diversity, under defined environmental conditions of discrete non-overlapping generations, constant density at high population sizes (N = 10⁴), no obvious spatial structure and abundant food resources. During 100 generations measurements of sex ratios and male competitive performance showed: 1) adaptation to the novel environment; 2) directional selection on male frequency under androdioecy; 3) optimal outcrossing rates of 0.5 under androdioecy; 4) the existence of initial inbreeding depression; and finally 5) that the strength of directional selection on male competitive performance does not depend on male frequencies. Taken together, these results suggest that androdioecious males are maintained at intermediate frequencies because outcrossing is adaptive.     

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.     

The proximate determinants of sex ratio in C. elegans populations

The soil nematode Caenorhabditis elegans is an example of a species in which self-fertilizing hermaphrodites predominate, but functional males continue to persist--allowing outcrossing to persevere at low levels. Hermaphrodites can produce male progeny as a consequence of sex chromosome non-disjunction or via outcrossing with males. Consequently, the genetics of sex determination coupled with the efficiency by which males find, inseminate and obtain fertilizations with hermaphrodites will influence the frequency at which males and outcrossing occurs in such populations. Behavioural and physiological traits with a heritable basis, as well as ecological characters, may influence male reproductive success and therefore sex ratio. Because sex ratio is tied to male reproductive success, sex ratio greatly affects outcrossing rates, patterns of genetic variation, and the ability of natural selection to act within populations. In this paper we explore the determinants of male frequency in C. elegans with a mathematical model and experimental data. We address the role of the genetic machinery of sex determination via sex chromosome non-disjunction on sex ratio and the influence of physiological components of C. elegans' life history that contribute to variation in sex ratio by way of male reproductive success. Finally, we discuss the short-term and long-term factors that are likely to affect sex ratio and breeding system evolution in species like C. elegans.     

Does inbreeding avoidance maintain gender dimorphism in Wurmbea dioica (Colchicaceae)?

The maintenance of females in gender dimorphic populations requires that they have a fitness advantage to compensate for their loss of male reproductive function. We assess whether inbreeding avoidance provides this advantage in two subdioecious Wurmbea dioica populations by estimating seed production, outcrossing rates and inbreeding depression. Fruiting males produced less than half as many seeds as females, owing to low outcrossing rates and early acting inbreeding depression. Inbreeding coefficients of fruiting males demonstrated that progeny were more inbred than their parents, implying that few selfed progeny reach maturity, as confirmed by inbreeding depression estimates that exceeded 0.85. In a glasshouse experiment, open-pollinated females exhibited a fitness advantage of 3.7 relative to fruiting males, but when we increased fruiting male outcrossing rate, female advantage was only 1.4. This reduced advantage is insufficient to maintain females if nuclear genes control sex. Thus, inbreeding avoidance could maintain females at high frequencies, although this is contingent upon high frequencies of fruiting males, which can be altered by environmentally determined gender plasticity.     

High local genetic diversity and low outcrossing rate in Caenorhabditis elegans natural populations

BACKGROUND: Caenorhabditis elegans is a major model system in biology, yet very little is known about its biology outside the laboratory. In particular, its unusual mode of reproduction with self-fertile hermaphrodites and facultative males raises the question of its frequency of outcrossing in natural populations. RESULTS: We describe the first analysis of C. elegans individuals sampled directly from natural populations. C. elegans is found predominantly in the dauer stage and with a very low frequency of males versus hermaphrodites. Whereas C. elegans was previously shown to display a low worldwide genetic diversity, we find by comparison a surprisingly high local genetic diversity of C. elegans populations; this local diversity is contributed in great part by immigration of new alleles rather than by mutation. Our results on heterozygote frequency, male frequency, and linkage disequilibrium furthermore show that selfing is the predominant mode of reproduction in C. elegans natural populations but that infrequent outcrossing events occur, at a rate of approximately 1%. CONCLUSIONS: Our results give a first insight in the biology of C. elegans in the natural populations. They demonstrate that local populations of C. elegans are genetically diverse and that a low frequency of outcrossing allows for the recombination of these locally diverse genotypes.     

Barriers to outcrossing success in the primarily self-fertilizing clam shrimp, Eulimnadia texana (Crustacea, Branchiopoda)

Abstract. The expected proportion of males in androdioecious populations (those comprised of males and hermaphrodites) largely depends on the fertilization opportunities of males. If male mating opportunities are low due to restricted access to hermaphroditic eggs, then populations will be hermaphrodite-biased. Hermaphrodites have two mechanisms available to limit male mating success: (1) pre-mating barriers to outcrossing, in which hermaphrodites choose not to pair with males and (2) post-mating barriers to outcrossing, in which hermaphrodite sperm has greater access to eggs than male sperm. In this study, we measured male mating success in the androdioecious clam shrimp Eulimnadia texana when pre-mating barriers to outcrossing were removed. These branchiopod crustaceans are small (5–8 mm), filter feeders that live in ephemeral pools in the deserts of the southwestern United States. Using genetic markers, we measured male mating success in laboratory experiments in two populations of these shrimp. We correlated mating success with clasping time, clasping during egg transfer, and male thrusting during egg transfer. Males fertilized an average of 24–40% of the hermaphrodites' eggs. Outcrossing success was positively correlated with clasping duration, and was nearly an order of magnitude higher for males thrusting during egg transfer relative to thrusting at other times during pairing. Because these estimates of mating success were similar to previously reported estimates (in which both pre- and post-mating barriers to outcrossing were potentially important), we deduced that pre-mating barriers to outcrossing do not greatly decrease male outcrossing success in E. texana ; the low fertilization (25–50% of available eggs) by males is thus due to post-mating barrier(s) to outcrossing.     

Two's company, three's a crowd: experimental evaluation of the evolutionary maintenance of trioecy in Mercurialis annua (Euphorbiaceae)

Trioecy is an uncommon sexual system in which males, females, and hermaphrodites co-occur as three clearly different gender classes. The evolutionary stability of trioecy is unclear, but would depend on factors such as hermaphroditic sex allocation and rates of outcrossing vs. selfing. Here, trioecious populations of Mercurialis annua are described for the first time. We examined the frequencies of females, males and hermaphrodites across ten natural populations and evaluated the association between the frequency of females and plant densities. Previous studies have shown that selfing rates in this species are density-dependent and are reduced in the presence of males, which produce substantially more pollen than hermaphrodites. Accordingly, we examined the evolutionary stability of trioecy using an experiment in which we (a) indirectly manipulated selfing rates by altering plant densities and the frequency of males in a fully factorial manner across 20 experimental plots and (b) examined the effect of these manipulations on the frequency of the three sex phenotypes in the next generation of plants. In the parental generation, we measured the seed and pollen allocations of hermaphrodites and compared them with allocations by unisexual plants. In natural populations, females occurred at higher frequencies in denser patches, a finding consistent with our expectations. Under our experimental conditions, however, no combination of plant densities and male frequencies was associated with increased frequencies of females. Our results suggest that the factors that regulate female frequencies in trioecious populations of M. annua are independent of those regulating male frequencies (density), and that the stable co-existence of all three sex phenotypes within populations is unlikely.     

Adult nutrition,but not inbreeding,affects male primary sexual traits in the leaf‐footed cactus bug Narnia femorata (Hemiptera: Coreidae)

Adverse conditions may be the norm rather than the exception in natural populations. Many populations experience poor nutrition on a seasonal basis. Further, brief interludes of inbreeding can be common as population density fluctuates and because of habitat fragmentation. Here, we investigated the effects of poor nutrition and inbreeding on traits that can be very important to reproductive success and fitness in males: testes mass, sperm concentration, and sperm viability. Our study species was Narnia femorata, a species introduced to north‐central Florida in the 1950s. This species encounters regular, seasonal changes in diet that can have profound phenotypic effects on morphology and behavior. We generated inbred and outbred individuals through a single generation of full‐sibling mating or outcrossing, respectively. All juveniles were provided a natural, high‐quality diet of Opuntia humifusa cactus cladode with fruit until they reached adulthood. New adult males were put on a high‐ or low‐quality diet for at least 21 days before measurements were taken. As expected, the low‐quality diet led to significantly decreased testes mass in both inbred and outbred males, although there were surprisingly no detectable effects on sperm traits. We did not find evidence that inbreeding affected testes mass, sperm concentration, and sperm viability. Our results highlight the immediate and overwhelming effects of nutrition on testes mass, while suggesting that a single generation of inbreeding might not be detrimental for primary sexual traits in this particular population.     

Effect of outbreeding on weight and growth rate of captive infant rhesus macaques

The introduction of unrelated conspecifics into captive groups of primate species is desirable to inhibit inbreeding. However, for many species, such groups increasingly represent highly inbred isolate populations that are analogous to regionally isolated populations of the same species among whom the effects of outcrossing on fitness are unknown. A study of potentially adverse effects of cross-breeding between regionally isolated populations of rhesus macaques was conducted to assess the maximum advantage or disadvantage to be expected for cross-breeding such populations of other cercopithecoid primates. For this purpose, the infant weights and rates of growth of hybrid Chinese/Indian rhesus macaques were compared to those of their nonhybrid Indian peers and to those of consanguineously inbred Indian rhesus macaques from several other captive breeding groups. Neither adverse nor advantageous effects of such hybridization were detected, suggesting that outcrossing between isolates of other cercopithecoid primates with a similar social structure and mating pattern should not affect the fitness of resulting offspring. Since levels of inbreeding are roughly inversely proportional to levels of genetic diversity within populations, such outcrossing should be intensified by zoos and other captive breeding centers to ensure the continued survival of captive species, especially those that are endangered or otherwise irreplaceable.     

THE EVOLUTION OF SELF-FERTILIZATION AND INBREEDING DEPRESSION IN PLANTS. II. EMPIRICAL OBSERVATIONS

A bimodal distribution of outcrossing rates was observed for natural plant populations, with more primarily selfing and primarily outcrossing species, and fewer species with intermediate outcrossing rate than expected by chance. We suggest that this distribution results from selection for the maintenance of outcrossing in historically large, outcrossing populations with substantial inbreeding depression, and from selection for selfing when increased inbreeding, due to pollinator failure or population bottlenecks, reduces the level of inbreeding depression. Few species or populations are fixed at complete selfing or complete outcrossing. A low level of selfing in primarily outcrossing species is unlikely to be selectively advantageous, but will not reduce inbreeding depression to the level where selfing is selectively favored, particularly if accompanied by reproductive compensation. Similarly, occasional outcrossing in primarily selfing species is unlikely to regularly provide sufficient heterosis to maintain selection for outcrossing through individual selection. Genetic, morphological and ecological constraints may limit the potential for outcrossing rates in selfers to be reduced below some minimum level.     

Sex differences in host defence interfere with parasite‐mediated selection for outcrossing during host–parasite coevolution

The Red Queen hypothesis proposes that coevolving parasites select for outcrossing in the host. Outcrossing relies on males, which often show lower immune investment due to, for example, sexual selection. Here, we demonstrate that such sex differences in immunity interfere with parasite‐mediated selection for outcrossing. Two independent coevolution experiments with Caenorhabditis elegans and its microparasite Bacillus thuringiensis produced decreased yet stable frequencies of outcrossing male hosts. A subsequent systematic analysis verified that male C. elegans suffered from a direct selective disadvantage under parasite pressure (i.e. lower resistance, decreased sexual activity, increased escape behaviour), which can reduce outcrossing and thus male frequencies. At the same time, males offered an indirect selective benefit, because male‐mediated outcrossing increased offspring resistance, thus favouring male persistence in the evolving populations. As sex differences in immunity are widespread, such interference of opposing selective constraints is likely of central importance during host adaptation to a coevolving parasite.     

Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost?

Heterostyly is a genetic polymorphism in which plant populations are composed of two or more morphs that differ in stigma and anther heights. The polymorphism promotes intermorph pollen transfer, thus outcrossing. Heterostyly has been reported in 28 angiosperm families and is frequently lost in heterostylous lineages. To assess ecological factors related to shifts from heterostyly to monomorphy, we examined the reproductive ecology of heterostylous tropical shrubs of Psychotria . Among 21 species at Barro Colorado Island and the nearby Parque Nacional Soberania, Panama, 14 species were heterostylous while seven were monomorphic. A molecular phylogeny and the existence elsewhere of heterostylous populations indicated that the breakdown of heterostyly had occurred independently. Heterostylous and monomorphic species were visited by the same bee species, although visit frequencies were lower in monomorphic species. Monomorphic species had significantly lower population density and greater fruit set than did heterostylous species. Autonomous autogamy made a large contribution to fruit set in monomorphic species and was only rarely observed in heterostylous species. The results indicate monomorphic and heterostylous species produce more seeds through selfing and outcrossing, respectively. The limitation of outcrossing as a result of low population density may be related to the breakdown of heterostyly and the evolution of selfing.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 125–134.     

Offspring sex ratio under inbreeding and outbreeding in a gynodioecious plant

Silene vulgaris is a gynodioecious plant native to Eurasia and now found throughout much of North America. Using hermaphrodite plants from three geographic regions (Stamford, NY; Broadway,VA; and Giles Co., VA) and four local populations within each region, we employed a hierarchical crossing design to explore the geographic structure of sex determining genes. Sex determination in this species is cytonuclear involving multiple cytoplasmic male sterility and nuclear restorer loci. Due to dominance effects within nuclear restorer loci, self-fertilization of hermaphrodites heterozygous at restorer loci should produce some homozygous recessive female offspring. Female offspring may also result from outcrossing among related individuals. At greater geographic and genetic distances, mismatches between cytoplasmic and nuclear sex determining genes should also produce high frequencies of female offspring if coevolution between cytoplasmic and nuclear sex determining alleles occurs independently among widely separated populations. We found evidence of dominance effects among nuclear restorer loci but no evidence of nuclear-cytoplasmic mismatches at the regional level. Of 63 maternal lines, 55 produced at least one female offspring when self-fertilized. Outcrossing within populations produced significantly fewer female offspring than self-fertilization. Outcrossing among regions produced the lowest proportion of female offspring, significantly fewer than outcrossing among populations within regions. Regions responded differently to among-region outcrossing with pollen donors from the two Virginia regions producing far fewer female offspring with New York dams than crosses among New York populations. These results indicate that nuclear restoration is complex, involving multiple loci with epistatic interactions and that most hermaphrodites in nature are heterozygous at one or more restorer locus. Further, regional differences in restorer frequencies indicate significant genetic structure for sex determining genes at large geographic scales, perhaps reflecting invasion history.     

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.     

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.     

An alpha-tubulin mutant demonstrates distinguishable functions among the spindle assembly checkpoint genes in Saccharomyces cerevisiae

The free-living nematode worm Caenorhabditis elegans reproduces primarily as a self-fertilizing hermaphrodite, yet males are maintained in wild-type populations at low frequency. To determine the role of males in C. elegans, we develop a mathematical model for the genetic system of hermaphrodites that can either self-fertilize or be fertilized by males and we perform laboratory observations and experiments on both C. elegans and a related dioecious species C. remanei. We show that the mating efficiency of C. elegans is poor compared to a dioecious species and that C. elegans males are more attracted to C. remanei females than they are to their conspecific hermaphrodites. We postulate that a genetic mutation occurred during the evolution of C. elegans hermaphrodites, resulting in the loss of an attracting sex pheromone present in the ancestor of both C. elegans and C. remanei. Our findings suggest that males are maintained in C. elegans because of the particular genetic system inherited from its dioecious ancestor and because of nonadaptive spontaneous nondisjunction of sex chromosomes, which occurs during meiosis in the hermaphrodite. A theoretical argument shows that the low frequency of male mating observed in C. elegans can support male-specific genes against mutational degeneration. This results in the continuing presence of functional males in a 99.9% hermaphroditic species in which outcrossing is disadvantageous to hermaphrodites.     

Variation in the breeding behavior of the dry forest tree Enterolobium cyclocarpum (guanacaste) in Costa Rica

We studied the breeding systems of four populations of Enterolobium cyclocarpum (guanacaste, earpod tree) in Costa Rica. Multilocus estimates of the outcrossing rate indicate that E. cyclocarpum is a predominant outcrossing species (t(m) ranged between 0.881 and 0.901) and biparental inbreeding is low (range between 0.058 and 0.079). Overall, our analyses showed significant differences in the gene frequencies between pollen and ovules and significant differences in pollen gene frequencies between the four populations. We also found significant differences in the pollen gene frequencies calculated for single trees in the same population. Outcrossing rates and pollen gene frequencies varied in two consecutive years in two populations of E. cyclocarpum. The correlated mating model revealed that there are differences in the correlation of paternity between populations and years. These findings indicate that there is variation in the average number of trees that father the seed crop of each tree and/or that some fathers are overrepresented in the seed crop of each tree. The implication of these findings for the development of strategies for conservation and management of this species are discussed.     

Fitness Effects of Ems-Induced Mutations on the X Chromosome of DROSOPHILA MELANOGASTER. II. Hemizygous Fitness Effects

X chromosomes mutagenized with EMS were tested for their effects on the fitness of hemizygous carriers. The tests were carried out in populations in which treated and untreated X chromosomes segregated from matings between males and attached-X females; the populations were maintained for several generations, during which time changes in the frequencies of the treated and untreated chromosomes were observed. From the rates at which the frequencies changed, the fitness effects of the treated chromosomes were determined. It was found that flies hemizygous for a mutagenized chromosome were 1.7% less fit per mM EMS treatment than those hemizygous for an untreated chromosome. Since the same flies were only 0.5% per mM less viable than their untreated counterparts, the total fitness effect of an X chromosome carrying EMS-induced mutants is three to four times greater than its viability effect. By comparing the heterozygous effect of a mutagenized X chromosome on fitness with the corresponding hemizygous effect, the dominance value for the chromosome is estimated to be about 0.25.     

Mating system variation along a successional gradient in the allogamous and colonizing plant Crepis sancta (Asteraceae)

We analysed mating system in an annual and colonizing plant, Crepis sancta, that occupies different successional stages in the French Mediterranean region. Based on a previous experiment, we hypothesized that low inbreeding depression measured in young successional stages should select for selfing whereas higher inbreeding depression in old stages should select for outcrossing. Nine populations of C. sancta (Asteraceae) from contrasting successional stages were used to analyse (1) Seed set after autonomous and enforced selfing in controlled conditions and (2) outcrossing rates in natural conditions using allozymes (progeny array analysis). We found that C. sancta possesses a pseudo‐self‐incompatibility system and that mating system varies among populations. Allozymes revealed that the population multilocus outcrossing rates vary from 0.77 to 0.99. The lowest outcrossing rates occur in the youngest successional stages and complete outcrossing is found in old stages. The data partially agree with the predictions we made and the results are more generally discussed in the light of factors changing during succession. We did not find any evidence of reproductive assurance in the nine populations, contrary to what is often assumed as a major factor governing mating system evolution in colonizing species. We propose that mating system variation can be interpreted as the result of the balance between the cost of outcrossing and inbreeding depression in a metapopulation context.     

Genetic rescue of small inbred populations: meta‐analysis reveals large and consistent benefits of gene flow

Many species have fragmented distribution with small isolated populations suffering inbreeding depression and/or reduced ability to evolve. Without gene flow from another population within the species (genetic rescue), these populations are likely to be extirpated. However, there have been only ~ 20 published cases of such outcrossing for conservation purposes, probably a very low proportion of populations that would potentially benefit. As one impediment to genetic rescues is the lack of an overview of the magnitude and consistency of genetic rescue effects in wild species, I carried out a meta‐analysis. Outcrossing of inbred populations resulted in beneficial effects in 92.9% of 156 cases screened as having a low risk of outbreeding depression. The median increase in composite fitness (combined fecundity and survival) following outcrossing was 148% in stressful environments and 45% in benign ones. Fitness benefits also increased significantly with maternal ΔF (reduction in inbreeding coefficient due to gene flow) and for naturally outbreeding versus inbreeding species. However, benefits did not differ significantly among invertebrates, vertebrates and plants. Evolutionary potential for fitness characters in inbred populations also benefited from gene flow. There are no scientific impediments to the widespread use of outcrossing to genetically rescue inbred populations of naturally outbreeding species, provided potential crosses have a low risk of outbreeding depression. I provide revised guidelines for the management of genetic rescue attempts.