Using runs of homozygosity and machine learning to disentangle sources of inbreeding and infer self-fertilization rates.

Runs of homozygosity (ROHs) are indicative of elevated homozygosity and inbreeding due to mating of closely related individuals. Self-fertilization can be a major source of inbreeding which elevates genome-wide homozygosity and thus should also create long ROHs. While ROHs are frequently used to understand inbreeding in the context of conservation and selective breeding, as well as for consanguinity of populations and their demographic history, it remains unclear how ROH characteristics are altered by selfing and if this confounds expected signatures of inbreeding due to demographic change. Using simulations, we study the impact of the mode of reproduction and demographic history on ROHs. We apply random forests to identify unique characteristics of ROHs, indicative of different sources of inbreeding. We pinpoint distinct features of ROHs that can be used to better characterize the type of inbreeding the population was subjected to and to predict outcrossing rates and complex demographic histories. Using additional simulations and four empirical datasets, two from highly selfing species and two from mixed-maters, we predict the selfing rate and validate our estimations. We find that self-fertilization rates are successfully identified even with complex demography. Population genetic summary statistics improve algorithm accuracy particularly in the presence of additional inbreeding, e.g., from population bottlenecks. Our findings highlight the importance of ROHs in disentangling confounding factors related to various sources of inbreeding and demonstrate situations where such sources cannot be differentiated. Additionally, our random forest models provide a novel tool to the community for inferring selfing rates using genomic data.

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.

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.

Outcrossing in Caenorhabditis elegans increases in response to food limitation.

Theory predicts that organisms should diversify their offspring when faced with a stressful environment. This prediction has received empirical support across diverse groups of organisms and stressors. For example, when encountered by Caenorhabditis elegans during early development, food limitation (a common environmental stressor) induces the nematodes to arrest in a developmental stage called dauer and to increase their propensity to outcross when they are subsequently provided with food and enabled to develop to maturity. Here we tested whether food limitation first encountered during late development/early adulthood can also induce increased outcrossing propensity in C. elegans. Previously well-fed C. elegans increased their propensity to outcross when challenged with food limitation during the final larval stage of development and into early adulthood, relative to continuously well-fed (control) nematodes. Our results thus support previous research demonstrating that the stress of food limitation can induce increased outcrossing propensity in C. elegans. Furthermore, our results expand on previous work by showing that food limitation can still increase outcrossing propensity even when it is not encountered until late development, and this can occur independently of the developmental and gene expression changes associated with dauer.

Latitudinal trends in mating system traits in the highly self-fertilizing Lobelia inflata revealed by community science.

Mating systems in angiosperms range from obligate outcrossing to highly self-fertilizing. The belief that obligate selfing does not exist is contradicted by genetic evidence in several populations of L. inflata, in which selfing is enforced by the anthers enclosing the style. However, whether the mating systems of these populations are typical, or an extreme across the species range is unknown. Such trends are hypothesized to result from selection for reproductive assurance under mate limitation at range margins. Here, we use ~7500 iNaturalist community science images, in which stylar exsertion can be observed, to test this hypothesis in L. inflata and, for comparison, in four typical congeneric Lobelias that express a staminate, then a pistillate phase (protandry). Specifically, we analyzed the effects of latitude and range marginality on the frequency of stylar exsertion and number of exserted flowers. Outcrossing capacity in L. inflata increased at low latitudes and near the overall range center, supporting our hypothesis, with exsertion frequencies significantly lower than in congenerics. Interestingly, in outcrossing capable individuals, the number of style-exserted flowers was consistent across the species range and among species, indicating outcrossing capable L. inflata individuals resemble congenerics. These findings suggest that variation in stylar exsertion is expressed among individuals rather than by all individuals within populations. However, whether this is a result of differences in exsertion allele frequencies or of differentiation in the induction of a threshold trait requires further study. Moreover, the trends in outcrossing capability revealed here imply the potential for geographic variation in L. inflata mating system.

Influence of the mutation load on the genomic composition of hybrids between outcrossing and self-fertilizing species.

Hybridization is a natural process whereby two diverging evolutionary lineages reproduce and create offspring of mixed ancestry. Differences in mating systems (e.g., self-fertilization and outcrossing) are expected to affect the direction and extent of hybridization and introgression in hybrid zones. Among other factors, selfers and outcrossers are expected to differ in their mutation loads. This has been studied both theoretically and empirically; however, conflicting predictions have been made on the effects mutation loads of parental species with different mating systems can have on the genomic composition of hybrids. Here, we develop a multi-locus, selective model to study how the different mutation load built up in selfers and outcrossers as a result of selective interference and homozygosity impact the long-term genetic composition of hybrid populations. Notably, our results emphasize that genes from the parental population with lesser mutation load get rapidly overrepresented in hybrid genomes, regardless of the hybrids own mating system. When recombination tends to be more important than mutation, outcrossers' genomes tend to be of higher quality and prevail. When recombination rates are low, however, selfers' genomes may reach higher quality than outcrossers' genomes and prevail in the hybrids. Taken together, these results provide concrete insights into one of the multiple factors influencing hybrid genome ancestry and introgression patterns in hybrid zones containing species with different mating systems.

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.

Population bottleneck associated with but likely preceded the recent evolution of self-fertilization in a coastal dune plant.

Evolution of self-fertilization may be initiated by a historical population bottleneck, which should diagnostically reduce lineage-wide genetic variation. However, selfing can also strongly reduce genetic variation after it evolves. Distinguishing process from pattern is less problematic if mating system divergence is recent and geographically simple. Dramatically reduced diversity is associated with the transition from outcrossing to selfing in the Pacific coastal endemic Abronia umbellata that includes large-flowered, self-incompatible populations (var. umbellata) south of San Francisco Bay and small-flowered, autogamous populations (var. breviflora) to the north. Compared to umbellata, synonymous nucleotide diversity across 10 single-copy nuclear genes was reduced by 94% within individual populations and 90% across the whole selfing breviflora lineage, which contained no unique polymorphisms. The geographic pattern of genetic variation is consistent with a single origin of selfing that occurred recently (7-28 kya). These results are best explained by a historical bottleneck, but the two most northerly umbellata populations also contained little variation and clustered with selfing populations, suggesting that substantial diversity loss preceded the origin of selfing. A bottleneck may have set the stage for the eventual evolution of selfing by purging genetic load that prevents the spread of selfing.

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.

Generation of Self-Fertilizing Hermaphroditic Fish from Gonochoristic Fish, Medaka (Oryzias latipes).

INTRODUCTION: Hermaphroditism is a mode of reproduction involving an individual animal that possesses both a testis and an ovary either sequentially or simultaneously. The mechanism creating hermaphrodites remains unknown. Previously, we identified foxl3 as the germline sex determination gene in a gonochoristic fish, medaka (Oryzias latipes). foxl3 loss-of-function (foxl3-/-) females produce functional sperm as well as eggs in the ovary. However, these two gametes are not self-fertilizing because of the histological separation of each gamete production. In this study, we attempted to generate self-fertilizing medaka from female medaka by modifying germline sex using foxl3-/- mutants and by using exogenous androgen to induce partial sex reversal of somatic cells. METHODS: foxl3-/- XX females were treated with 11-ketotestosterone (11-KT), a potent teleost fish androgen, at the sexually mature stage for 30 days (90-120 dph). Then, the fish were kept under normal conditions until they were either being dissected or crossed with infertile males. RESULTS AND DISCUSSION: We showed that the foxl3-/- XX female medaka can be transformed into a self-fertilizing hermaphrodite by inducing the formation of a male-like structure with exogenous 11-KT. Self-fertilization occurs in either the ovarian cavity, the oviduct, or both where sperm is released from a tubule-like structure which is likely derived from germinal epithelium, suggesting that timely modification of 2 independent mechanisms, regulation of germline sex and partial sex reversal of somatic cells, are critical to change the reproduction mode. Our results will provide insights in developmental and evolutional occurrence of hermaphrodite vertebrates, facilitate an innovative technique to improve the efficient selection of fish with desirable traits, and contribute to the rescue of endangered species.

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.

Additive and non-additive epigenetic signatures of natural hybridization between fish species with different mating systems.

Hybridization is a major source of evolutionary innovation. In plants, epigenetic mechanisms can help to stabilize hybrid genomes and contribute to reproductive isolation, but the relationship between genetic and epigenetic changes in animal hybrids is unclear. We analysed the relationship between genetic background and methylation patterns in natural hybrids of two genetically divergent fish species with different mating systems, Kryptolebias hermaphroditus (self-fertilizing) and K. ocellatus (outcrossing). Co-existing parental species displayed highly distinct genetic (SNPs) and methylation patterns (37,000 differentially methylated cytosines). Hybrids had predominantly intermediate methylation patterns (88.5% of the sites) suggesting additive effects, as expected from hybridization between genetically distant species. The large number of differentially methylated cytosines between hybrids and parental species (n = 5,800) suggests that hybridization may play a role in increasing genetic and epigenetic variation. Although most of the observed epigenetic variation was additive and had a strong genetic component, we also found a small percentage of non-additive, potentially stochastic, methylation differences that might act as an evolutionary bet-hedging strategy and increase fitness under environmental instability.

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.

Pollinator loss causes rapid adaptive evolution of selfing and dramatically reduces genome-wide genetic variability.

Although selfing populations harbor little genetic variation limiting evolutionary potential, the causes are unclear. We experimentally evolved large, replicate populations of Mimulus guttatus for nine generations in greenhouses with or without pollinating bees and studied DNA polymorphism in descendants. Populations without bees adapted to produce more selfed seed yet exhibited striking reductions in DNA polymorphism despite large population sizes. Importantly, the genome-wide pattern of variation cannot be explained by a simple reduction in effective population size, but instead reflects the complicated interaction between selection, linkage, and inbreeding. Simulations demonstrate that the spread of favored alleles at few loci depresses neutral variation genome wide in large populations containing fully selfing lineages. It also generates greater heterogeneity among chromosomes than expected with neutral evolution in small populations. Genome-wide deviations from neutrality were documented in populations with bees, suggesting widespread influences of background selection. After applying outlier tests to detect loci under selection, two genome regions were found in populations with bees, yet no adaptive loci were otherwise mapped. Large amounts of stochastic change in selfing populations compromise evolutionary potential and undermine outlier tests for selection. This occurs because genetic draft in highly selfing populations makes even the largest changes in allele frequency unremarkable.

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.