Genetic Causes and Genomic Consequences of Breakdown of Distyly in Linum trigynum.

Distyly is an iconic floral polymorphism governed by a supergene, which promotes efficient pollen transfer and outcrossing through reciprocal differences in the position of sexual organs in flowers, often coupled with heteromorphic self-incompatibility. Distyly has evolved convergently in multiple flowering plant lineages, but has also broken down repeatedly, often resulting in homostylous, self-compatible populations with elevated rates of self-fertilization. Here, we aimed to study the genetic causes and genomic consequences of the shift to homostyly in Linum trigynum, which is closely related to distylous Linum tenue. Building on a high-quality genome assembly, we show that L. trigynum harbors a genomic region homologous to the dominant haplotype of the distyly supergene conferring long stamens and short styles in L. tenue, suggesting that loss of distyly first occurred in a short-styled individual. In contrast to homostylous Primula and Fagopyrum, L. trigynum harbors no fixed loss-of-function mutations in coding sequences of S-linked distyly candidate genes. Instead, floral gene expression analyses and controlled crosses suggest that mutations downregulating the S-linked LtWDR-44 candidate gene for male self-incompatibility and/or anther height could underlie homostyly and self-compatibility in L. trigynum. Population genomic analyses of 224 whole-genome sequences further demonstrate that L. trigynum is highly self-fertilizing, exhibits significantly lower genetic diversity genome-wide, and is experiencing relaxed purifying selection and less frequent positive selection on nonsynonymous mutations relative to L. tenue. Our analyses shed light on the loss of distyly in L. trigynum, and advance our understanding of a common evolutionary transition in flowering plants.

Detecting directional epistasis and dominance from cross-line analyses in alpine populations of Arabidopsis thaliana.

The contribution of non-additive genetic effects to the genetic architecture of fitness and to the evolutionary potential of populations has been a topic of theoretical and empirical interest for a long time. Yet, the empirical study of these effects in natural populations remains scarce, perhaps because measuring dominance and epistasis relies heavily on experimental line crosses. In this study, we explored the contribution of dominance and epistasis in natural alpine populations of Arabidopsis thaliana for 2 fitness traits, the dry biomass and the estimated number of siliques, measured in a greenhouse. We found that, on average, crosses between inbred lines of A. thaliana led to mid-parent heterosis for dry biomass but outbreeding depression for an estimated number of siliques. While heterosis for dry biomass was due to dominance, we found that outbreeding depression for an estimated number of siliques could be attributed to the breakdown of beneficial epistatic interactions. We simulated and discussed the implication of these results for the adaptive potential of the studied populations, as well as the use of line-cross analyses to detect non-additive genetic effects.

Fisher's automatic advantage of self-fertilization does not apply in cleistogamous species.

PREMISE: In hermaphroditic plants, the evolution of self-fertilization is driven by two major forces; the cost of outcrossing or Fisher's automatic advantage of selfing and inbreeding depression. Seminal theoretical works have established that an inbreeding depression threshold of 0.5 governs the evolution. Below that threshold, selfing evolves, above that, outcrossing evolves. Does this threshold apply to cleistogamous plants? METHODS: I developed a model using a Lloydian approach to analyze the evolution of cleistogamy. RESULTS: I showed that the inbreeding depression threshold does not apply in cleistogamous species, and that because cleistogamous (closed) flowers do not export pollen, Fisher's advantage of selfing is totally cancelled. CONCLUSIONS: In line with model predictions, I discuss the fact that cleistogamous species often exhibit low inbreeding depression in empirical studies.

Support for Baker's law: Facultative self-fertilization ability decreases pollen limitation in experimental colonization.

PREMISE: The ability to self-fertilize is predicted to provide an advantage in colonization because a single individual can reproduce and establish a next generation in a new location regardless of the density of mates. While there is theoretical and correlative support for this idea, the strength of mate limitation as a selective agent has not yet been delineated from other factors that can also select for self-fertilization in colonization of new habitats. We used known mating-system variation in the American bellflower (Campanula americana) to explore how plants' ability to self-fertilize can mitigate density-dependent reproduction and impact colonization success. METHODS: We created experimental populations of single individuals or a small number of plants to emulate isolated colonization events. These populations were composed of plants that differed in their ability to self-fertilize. We compared pollen limitation of the single individuals to that of small populations. RESULTS: Experimental populations of plants that readily self-fertilize produced consistent seed numbers regardless of population size, whereas plants with lower ability to self-fertilize had density-dependent reproduction with greater seed production in small populations than in populations composed of a single individual. CONCLUSIONS: We experimentally isolated the effect of mate limitation in colonization and found that it can select for increased self-fertilization. We show the benefit of self-fertilization in colonization, which helps to explain geographic patterns of self-fertilization and shows support for Baker's law, a long-held hypothesis in the field of mating-system evolution.

Population Rescue through an Increase in the Selfing Rate under Pollen Limitation: Plasticity versus Evolution.

AbstractIncreased rates of self-fertilization offer reproductive assurance when plant populations experience pollen limitation, but self-fertilization may reduce fitness by exposing deleterious mutations. If an environmental change responsible for pollen limitation also induces plastic mating system shifts toward self-pollination, the reproductive assurance benefit and inbreeding depression cost of increased self-fertilization occur immediately, while the benefit and cost happen more gradually when increased self-fertilization occur through evolution. I built eco-evolutionary models to explore the demographic and genetic conditions in which higher self-fertilization by plasticity and/or evolution rescues populations, following deficits due to a sudden onset of pollen limitation. Rescue is most likely under an intermediate level of selfing rate increase, either through plasticity or evolution, and this critical level of selfing rate increase is higher under stronger pollen limitation. Generally, rescue is more likely through plasticity than through evolution. Under weak pollen limitation, rescue by enhanced self-fertilization may mainly occur through purging of deleterious mutations rather than reproductive assurance. The selfing rate increase conferring the highest rescue probability is lower when the initial population size is smaller. This article shows the importance of plasticity during plant population rescue and offers insights for future studies of the evolution of mating system plasticity.

Honeybees (Apis mellifera) decrease the fitness of plants they pollinate.

Most flowering plants require animal pollination and are visited by multiple pollinator species. Historically, the effects of pollinators on plant fitness have been compared using the number of pollen grains they deposit, and the number of seeds or fruits produced following a visit to a virgin flower. While useful, these methods fail to consider differences in pollen quality and the fitness of zygotes resulting from pollination by different floral visitors. Here we show that, for three common native self-compatible plants in Southern California, super-abundant, non-native honeybees (Apis mellifera L.) visit more flowers on an individual before moving to the next plant compared with the suite of native insect visitors. This probably increases the transfer of self-pollen. Offspring produced after honeybee pollination have similar fitness to those resulting from hand self-pollination and both are far less fit than those produced after pollination by native insects or by cross-pollination. Because honeybees often forage methodically, visiting many flowers on each plant, low offspring fitness may commonly result from honeybee pollination of self-compatible plants. To our knowledge, this is the first study to directly compare the fitness of offspring resulting from honeybee pollination to that of other floral visitors.

Multiple introductions, polyploidy and mixed reproductive strategies are linked to genetic diversity and structure in the most widespread invasive plant across Southern Ocean archipelagos.

Biological invasions in remote areas that experience low human activity provide unique opportunities to elucidate processes responsible for invasion success. Here we study the most widespread invasive plant species across the isolated islands of the Southern Ocean, the annual bluegrass, Poa annua. To analyse geographical variation in genome size, genetic diversity and reproductive strategies, we sampled all major sub-Antarctic archipelagos in this region and generated microsatellite data for 470 individual plants representing 31 populations. We also estimated genome sizes for a subset of individuals using flow cytometry. Occasional events of island colonization are expected to result in high genetic structure among islands, overall low genetic diversity and increased self-fertilization, but we show that this is not the case for P. annua. Microsatellite data indicated low population genetic structure and lack of isolation by distance among the sub-Antarctic archipelagos we sampled, but high population structure within each archipelago. We identified high levels of genetic diversity, low clonality and low selfing rates in sub-Antarctic P. annua populations (contrary to rates typical of continental populations). In turn, estimates of selfing declined in populations as genetic diversity increased. Additionally, we found that most P. annua individuals are probably tetraploid and that only slight variation exists in genome size across the Southern Ocean. Our findings suggest multiple independent introductions of P. annua into the sub-Antarctic, which promoted the establishment of genetically diverse populations. Despite multiple introductions, the adoption of convergent reproductive strategies (outcrossing) happened independently in each major archipelago. The combination of polyploidy and a mixed reproductive strategy probably benefited P. annua in the Southern Ocean by increasing genetic diversity and its ability to cope with the novel environmental conditions.

Genomic insights into inter- and intraspecific mating system shifts in Primulina.

The mating system shift from outcrossing to selfing is one of the most frequent evolutionary trends in flowering plants. However, the genomic consequences of this shift remain poorly understood. Specifically, the relative importance of the demographic and genetic processes causing changes in genetic variation and selection efficacy associated with the evolution of selfing is unclear. Here we sequenced the genomes of two Primulina species with contrasting mating systems: P. eburnea (outcrossing) versus P. tabacum (outcrossing, mixed-mating and selfing populations). Whole-genome resequencing data were used to investigate the genomic consequences of mating system shifts within and between species. We found that highly selfing populations of P. tabacum display loss of genetic diversity, increased deleterious mutations, higher genomic burden and fewer adaptive substitutions. However, compared with outcrossing populations, mixed-mating populations did not display loss of genetic diversity and accumulation of genetic load. We find no evidence of population bottlenecks associated with the shift to selfing, which suggests that the genetic effects of selfing on Ne and possibly linked selection, rather than demographic history, are the primary drivers of diversity reduction in highly selfing populations. Our results highlight the importance of distinguishing the relative contribution of mating system and demography on the genomic consequences associated with mating system evolution in plants.

Phylogenomics reveals extensive introgression and a case of mito-nuclear discordance in the killifish genus Kryptolebias.

Introgression is a widespread evolutionary process leading to phylogenetic inconsistencies among distinct parts of the genomes, particularly between mitochondrial and nuclear-based phylogenetic reconstructions (e.g., mito-nuclear discordances). Here, we used mtDNA and genome-wide nuclear sites to provide the first phylogenomic-based hypothesis on the evolutionary relationships within the killifish genus Kryptolebias. In addition, we tested for evidence of past introgression in the genus given the multiple reports of undergoing hybridization between its members. Our mtDNA phylogeny generally agreed with the relationships previously proposed for the genus. However, our reconstruction based on nuclear DNA revealed an unknown lineage - Kryptolebias sp. 'ESP' - as the sister group of the self-fertilizing mangrove killifishes, K. marmoratus and K. hermaphroditus. All individuals sequenced of Kryptolebias sp. 'ESP' had the same mtDNA haplotype commonly observed in K. hermaphroditus, demonstrating a clear case of mito-nuclear discordance. Our analysis further confirmed extensive history of introgression between Kryptolebias sp. 'ESP' and K. hermaphroditus. Population genomics analyses indicate no current gene flow between the two lineages, despite their current sympatry and history of introgression. We also confirmed introgression between other species pairs in the genus that have been recently reported to form hybrid zones. Overall, our study provides a phylogenomic reconstruction covering most of the Kryptolebias species, reveals a new lineage hidden in a case of mito-nuclear discordance, and provides evidence of multiple events of ancestral introgression in the genus. These findings underscore the importance of investigating different genomic information in a phylogenetic framework, particularly in taxa where introgression is common as in the sexually diverse mangrove killifishes.

Polyploidization as an opportunistic mutation: The role of unreduced gametes formation and genetic drift in polyploid establishment.

It is broadly assumed that polyploidy success reflects an increase in fitness associated with whole-genome duplication (WGD), due to higher tolerance to stressful conditions. Nevertheless, WGD also arises with several costs in neo-polyploid lineages, like genomic instability, or cellular mis-management. In addition to these costs, neo-polyploid individuals also face frequency dependent selection because of frequent low-fitness triploids formed by cross-ploidy pollinations when tetraploids are primarily rare in the population. Interestingly, the idea that polyploidy can be fixed by genetic drift as a neutral or deleterious mutation is currently underexplored in the literature. To test how and when polyploidy can fix in a population by chance, we built a theoretical model in which autopolyploidization occurs through the production of unreduced gametes, a trait modelled as a quantitative trait that is allowed to vary through time. We found that when tetraploid individuals are less or as fit as their diploid progenitors, fixation of polyploidy is only possible when genetic drift is stronger than natural selection. The necessity of drift for tetraploid fixation holds even when polyploidy confers a selective advantage, except for scenarios where tetraploids are much fitter than diploids. Finally, we found that self-fertilization is less beneficial for tetraploid establishment than previously thought, notably when polyploids harbour an initial decrease in fitness. Our results bring a novel, non-exclusive explanation for the unequal temporal and spatial distribution of polyploid species.

Evolution of sex allocation plasticity in a hermaphroditic flatworm genus.

Sex allocation theory in simultaneous hermaphrodites predicts that optimal sex allocation is influenced by local sperm competition, which occurs when related sperm compete to fertilize a given set of eggs. Different factors, including the mating strategy and the ability to self-fertilize, are predicted to affect local sperm competition and hence the optimal SA. Moreover, since the local sperm competition experienced by an individual can vary temporally and spatially, this can favour the evolution of sex allocation plasticity. Here, using seven species of the free-living flatworm genus Macrostomum, we document interspecific variation in sex allocation, but neither their mating strategy nor their ability to self-fertilize significantly predicted sex allocation among these species. Since we also found interspecific variation in sex allocation plasticity, we further estimated standardized effect sizes for plasticity in response to (i) the presence of mating partners (i.e. in isolation vs. with partners) and (ii) the strength of local sperm competition (i.e. in small vs. large groups). We found that self-fertilization predicted sex allocation plasticity with respect to the presence of mating partners, with plasticity being lower for self-fertilizing species. Finally, we showed that interspecific variation in sex allocation is higher than intraspecific variation due to sex allocation plasticity. Our study suggests that both sex allocation and sex allocation plasticity are evolutionarily labile, with self-fertilization predicting the latter in Macrostomum.

Simulated herbivory enhances leaky sex expression in the dioecious herb Mercurialis annua.

BACKGROUND AND AIMS: Plant reproductive traits are widely understood to be responsive to the selective pressures exerted by pollinators, but there is also increasing evidence for an important role for antagonists such as herbivores in shaping these traits. Many dioecious species show leaky sex expression, with males and females occasionally producing flowers of the opposite sex. Here, we asked to what extent leakiness in sex expression in Mercurialis annua (Euphorbiaceae) might also be plastically responsive to simulated herbivory. This is important because enhanced leakiness in dioecious populations could lead to a shift in both the mating system and in the conditions for transitions between combined and separate sexes. METHODS: We examined the effect of simulated herbivory on the sexual expression of males and females of M. annua in two experiments in which different levels of simulated herbivory led to enhanced leakiness in both sexes. KEY RESULTS: We showed that leaky sex expression in both males and females of the wind-pollinated dioecious herb M. annua is enhanced in response to simulated herbivory, increasing the probability for and the degree of leakiness in both sexes. We also found that leakiness was greater in larger females but not in larger males. CONCLUSIONS: We discuss hypotheses for a possible functional link between herbivory and leaky sex expression, and consider what simulated herbivory-induced leakiness might imply for the evolutionary ecology of plant reproductive systems, especially the breakdown of dioecy and the evolution of hermaphroditism.

Systematics and geographical distribution of Galba species, a group of cryptic and worldwide freshwater snails.

Cryptic species can present a significant challenge to the application of systematic and biogeographic principles, especially if they are invasive or transmit parasites or pathogens. Detecting cryptic species requires a pluralistic approach in which molecular markers facilitate the detection of coherent taxonomic units that can then be analyzed using various traits (e.g., internal morphology) and crosses. In asexual or self-fertilizing species, the latter criteria are of limited use. We studied a group of cryptic freshwater snails (genus Galba) from the family Lymnaeidae that have invaded almost all continents, reproducing mainly by self-fertilization and transmitting liver flukes to humans and livestock. We aim to clarify the systematics, distribution, and phylogeny of these species with an integrative approach that includes morphology, molecular markers, wide-scale sampling across America, and data retrieved from GenBank (to include Old World samples). Our phylogenetic analysis suggests that the genus Galba originated ca. 22 Myr ago and today comprises six species or species complexes. Four of them show an elongated-shell cryptic phenotype and exhibit wide variation in their genetic diversity, geographic distribution, and invasiveness. The remaining two species have more geographically restricted distributions and exhibit a globose-shell cryptic phenotype, most likely phylogenetically derived from the elongated one. We emphasize that no Galba species should be identified without molecular markers. We also discuss several hypotheses that can explain the origin of cryptic species in Galba, such as convergence and morphological stasis.

Extensive hybridization and past introgression between divergent lineages in a quasi-clonal hermaphroditic fish: Ramifications for species concepts and taxonomy.

Extreme inbreeding is expected to reduce the incidence of hybridization, serving as a prezygotic barrier. Mangrove rivulus is a small killifish that reproduces predominantly by self-fertilization, producing highly homozygous lines throughout its geographic range. The Bahamas and Caribbean are inhabited by two highly diverged phylogeographic lineages of mangrove rivulus, Kryptolebias marmoratus and a 'Central clade' closely related to K. hermaphroditus from Brazil. The two lineages are largely allopatric, but recently were found in syntopy on San Salvador, Bahamas, where a single hybrid was reported. To better characterize the degree of hybridization and the possibility of secondary introgression, here we conducted a detailed genetic analysis of the contact zone on San Salvador. Two mixed populations were identified, one of which contained sexually mature hybrids. The distribution of heterozygosity at diagnostic microsatellite loci in hybrids showed that one of these hybrids was an immediate offspring from the K. marmoratus x Central clade cross, whereas the remaining five hybrids were products of reproduction by self-fertilization for 1-3 generations following the initial cross. Two hybrids had mitochondrial haplotypes of K. marmoratus and the remaining four hybrids had a haplotype of the Central clade, indicating that crosses go in both directions. In hybrids, alleles of parental lineages were represented in equal proportions suggesting lack of recent backcrossing to either of the parental lineages. However, sympatric populations of two lineages were less diverged than allopatric populations, consistent with introgression. Results are discussed in terms of applicability of the biological species concept for isogenic, effectively clonal, organisms.

Selfing rate variation within species is unrelated to life-history traits or geographic range position.

PREMISE: In plants, populations and species vary widely along the continuum from outcrossing to selfing. Life-history traits and ecological circumstances influence among-species variation in selfing rates, but their general role in explaining intraspecific variation is unknown. Using a database of plant species, we test whether life-history traits, geographic range position, or abundance predict selfing rate variation among populations. METHODS: We identified species where selfing rates were estimated in at least three populations at known locations. Two key life-history traits (generation time and growth form) were used to predict within-species selfing rate variation. Populations sampled within a species' native range were assessed for proximity to the nearest edge and abundance. Finally, we conducted linear and segmented regressions to determine functional relationships between selfing rate and geographic range position within species. RESULTS: Selfing rates for woody species varied less than for herbs, which is explained by the lower average selfing rate of woody species. Relationships between selfing and peripherality or abundance significantly varied among species in their direction and magnitude. However, there was no general pattern of increased selfing toward range edges. A power analysis shows that tests of this hypothesis require studying many (i.e., 40+) populations. CONCLUSIONS: Intraspecific variation in plant mating systems is often substantial yet remains difficult to explain. Beyond sampling more populations, future tests of biogeographic hypotheses will benefit from phylogeographic information concerning specific range edges, the study of traits influencing mating system (e.g., herkogamy), and measures of abundance at local scales (e.g., population density).

Demography and mating system shape the genome-wide impact of purifying selection in Arabis alpina.

Plant mating systems have profound effects on levels and structuring of genetic variation and can affect the impact of natural selection. Although theory predicts that intermediate outcrossing rates may allow plants to prevent accumulation of deleterious alleles, few studies have empirically tested this prediction using genomic data. Here, we study the effect of mating system on purifying selection by conducting population-genomic analyses on whole-genome resequencing data from 38 European individuals of the arctic-alpine crucifer Arabis alpina We find that outcrossing and mixed-mating populations maintain genetic diversity at similar levels, whereas highly self-fertilizing Scandinavian A. alpina show a strong reduction in genetic diversity, most likely as a result of a postglacial colonization bottleneck. We further find evidence for accumulation of genetic load in highly self-fertilizing populations, whereas the genome-wide impact of purifying selection does not differ greatly between mixed-mating and outcrossing populations. Our results demonstrate that intermediate levels of outcrossing may allow efficient selection against harmful alleles, whereas demographic effects can be important for relaxed purifying selection in highly selfing populations. Thus, mating system and demography shape the impact of purifying selection on genomic variation in A. alpina These results are important for an improved understanding of the evolutionary consequences of mating system variation and the maintenance of mixed-mating strategies.

The Evolutionary Interplay between Adaptation and Self-Fertilization.

Genome-wide surveys of nucleotide polymorphisms, obtained from next-generation sequencing, have uncovered numerous examples of adaptation in self-fertilizing organisms, especially regarding changes to climate, geography, and reproductive systems. Yet existing models for inferring attributes of adaptive mutations often assume idealized outcrossing populations, which risks mischaracterizing properties of these variants. Recent theoretical work is emphasizing how various aspects of self-fertilization affects adaptation, yet empirical data on these properties are lacking. We review theoretical and empirical studies demonstrating how self-fertilization alters the process of adaptation, illustrated using examples from current sequencing projects. We propose ideas for how future research can more accurately quantify aspects of adaptation in self-fertilizers, including incorporating the effects of standing variation, demographic history, and polygenic adaptation.

Hidden genetic variance contributes to increase the short-term adaptive potential of selfing populations.

Standing genetic variation is considered a major contributor to the adaptive potential of species. The low heritable genetic variation observed in self-fertilizing populations has led to the hypothesis that species with this mating system would be less likely to adapt. However, a non-negligible amount of cryptic genetic variation for polygenic traits, accumulated through negative linkage disequilibrium, could prove to be an important source of standing variation in self-fertilizing species. To test this hypothesis, we simulated populations under stabilizing selection subjected to an environmental change. We demonstrate that, when the mutation rate is high (but realistic), selfing populations are better able to store genetic variance than outcrossing populations through genetic associations, notably due to the reduced effective recombination rate associated with predominant selfing. Following an environmental shift, this diversity can be partially remobilized, which increases the additive variance and adaptive potential of predominantly (but not completely) selfing populations. In such conditions, despite initially lower observed genetic variance, selfing populations adapt as readily as outcrossing ones within a few generations. For low mutation rates, purifying selection impedes the storage of diversity through genetic associations, in which case, as previously predicted, the lower genetic variance of selfing populations results in lower adaptability compared to their outcrossing counterparts. The population size and the mutation rate are the main parameters to consider, as they are the best predictors of the amount of stored diversity in selfing populations. Our results and their impact on our knowledge of adaptation under high selfing rates are discussed.

The Effect of Pollen Limitation on the Evolution of Mating System and Seed Size in Hermaphroditic Plants.

Pollen limitation, when inadequate pollen receipt results in a plant setting fewer seeds and fruits, can reduce plant reproductive success and promote the evolution of self-fertilization as a mechanism of reproductive assurance. However, the effect of pollen limitation on the joint evolution of mating system and seed size is not known. Using an evolutionarily stable strategy resource allocation model, we show that where moderate pollen limitation and strong inbreeding depression select for complete outcrossing, pollen limitation should also increase the optimal seed size. In contrast, pollen limitation should not affect the optimal seed size under complete selfing, in which case ovule fertilization is certain. Under intermediate conditions, a mixed mating system evolves if the probability of ovule fertilization declines as more ovules are produced, so that a selfed seed with inbreeding depression provides equal marginal fitness returns to a larger outcrossed seed that may result from pollen limitation. Under mixed mating, outcrossed seeds should be larger than selfed seeds, and pollen limitation should not affect the optimal size of either outcrossed or selfed seeds. Our results identify a novel pathway through which pollen limitation selects for mixed mating and provide an adaptive explanation as to why selfed seeds are often smaller than outcrossed seeds.

The evolutionary response of mating system to heterosis.

Isolation allows populations to diverge and to fix different alleles. Deleterious alleles that reach locally high frequencies contribute to genetic load, especially in inbred or selfing populations, in which selection is relaxed. In the event of secondary contact, the recessive portion of the genetic load is masked in the hybrid offspring, producing heterosis. This advantage, only attainable through outcrossing, should favour evolution of greater outcrossing even if inbreeding depression has been purged from the contributing populations. Why, then, are selfing-to-outcrossing transitions not more common? To evaluate the evolutionary response of mating system to heterosis, we model two monomorphic populations of entirely selfing individuals, introduce a modifier allele that increases the rate of outcrossing and investigate whether the heterosis among populations is sufficient for the modifier to invade and fix. We find that the outcrossing mutation invades for many parameter choices, but it rarely fixes unless populations harbour extremely large unique fixed genetic loads. Reversions to outcrossing become more likely as the load becomes more polygenic, or when the modifier appears on a rare background, such as by dispersal of an outcrossing genotype into a selfing population. More often, the outcrossing mutation instead rises to moderate frequency, which allows recombination in hybrids to produce superior haplotypes that can spread without the mutation's further assistance. The transience of heterosis can therefore explain why secondary contact does not commonly yield selfing-to-outcrossing transitions.