HD-Zip proteins modify floral structures for self-pollination in tomato.

Cleistogamy is a type of self-pollination that relies on the formation of a stigma-enclosing floral structure. We identify three homeodomain-leucine zipper IV (HD-Zip IV) genes that coordinately promote the formation of interlocking trichomes at the anther margin to unite neighboring anthers, generating a closed anther cone and cleistogamy (flower morphology necessitating strict self-pollination). These HD-Zip IV genes also control style length by regulating the transition from cell division to endoreduplication. The expression of these HD-Zip IV genes and their downstream gene, Style 2.1, was sequentially modified to shape the cleistogamy morphology during tomato evolution and domestication. Our results provide insights into the molecular basis of cleistogamy in modern tomato and suggest targets for improving fruit set and preventing pollen contamination in genetically modified crops.

Early-acting inbreeding depression can evolve as an inbreeding avoidance mechanism.

Despite the potential for mechanical, developmental and/or chemical mechanisms to prevent self-fertilization, incidental self-fertilization is inevitable in many predominantly outcrossing species. In such cases, inbreeding can compromise individual fitness. Unquestionably, much of this inbreeding depression is maladaptive. However, we show that when reproductive compensation allows for the replacement of inviable embryos lost early in development, selection can favour deleterious recessive variants that induce 'self-sacrificial' death of inbred embryos. Our theoretical results provide numerous testable predictions which could challenge the assumption that inbreeding depression is always maladaptive. Our work is applicable any species that cannot fully avoid inbreeding, exhibits substantial inbreeding depression, and has the potential to compensate embryos lost early in development. In addition to its general applicability, our theory suggests that self-sacrificial variants might be responsible for the remarkably low realized selfing rates of gymnosperms with high primary selfing rates, as gymnosperms exhibit strong inbreeding depression, have effective reproductive compensation mechanisms, and cannot evolve chemical self-incompatibility.

Digest: Mating systems, intragenomic conflict, and speciation.

Conflict over the degree of maternal investment in an offspring can exist between an offspring's maternally inherited and paternally inherited alleles. Such conflict is not expected under self-fertilization. A new study led by Rifkin and Ostevik suggests that divergence in the degree of conflict between closely related outcrossing and selfing species can lead to aberrant early development of hybrids in morning glories. This dynamic represents a potentially powerful driver of reproductive incompatibility and thus speciation.

Parallel evolution of morphological and genomic selfing syndromes accompany the breakdown of heterostyly.

Evolutionary transitions from outcrossing to selfing in flowering plants have convergent morphological and genomic signatures and can involve parallel evolution within related lineages. Adaptive evolution of morphological traits is often assumed to evolve faster than nonadaptive features of the genomic selfing syndrome. We investigated phenotypic and genomic changes associated with transitions from distyly to homostyly in the Primula oreodoxa complex. We determined whether the transition to selfing occurred more than once and investigated stages in the evolution of morphological and genomic selfing syndromes using 22 floral traits and both nuclear and plastid genomic data from 25 populations. Two independent transitions were detected representing an earlier and a more recently derived selfing lineage. The older lineage exhibited classic features of the morphological and genomic selfing syndrome. Although features of both selfing syndromes were less developed in the younger selfing lineage, they exhibited parallel development with the older selfing lineage. This finding contrasts with the prediction that some genomic changes should lag behind adaptive changes to morphological traits. Our findings highlight the value of comparative studies on the timing and extent of transitions from outcrossing to selfing between related lineages for investigating the tempo of morphological and molecular evolution.

Purging due to self-fertilization does not prevent accumulation of expansion load.

As species expand their geographic ranges, colonizing populations face novel ecological conditions, such as new environments and limited mates, and suffer from evolutionary consequences of demographic change through bottlenecks and mutation load accumulation. Self-fertilization is often observed at species range edges and, in addition to countering the lack of mates, is hypothesized as an evolutionary advantage against load accumulation through increased homozygosity and purging. We study how selfing impacts the accumulation of genetic load during range expansion via purging and/or speed of colonization. Using simulations, we disentangle inbreeding effects due to demography versus due to selfing and find that selfers expand faster, but still accumulate load, regardless of mating system. The severity of variants contributing to this load, however, differs across mating system: higher selfing rates purge large-effect recessive variants leaving a burden of smaller-effect alleles. We compare these predictions to the mixed-mating plant Arabis alpina, using whole-genome sequences from refugial outcrossing populations versus expanded selfing populations. Empirical results indicate accumulation of expansion load along with evidence of purging in selfing populations, concordant with our simulations, suggesting that while purging is a benefit of selfing evolving during range expansions, it is not sufficient to prevent load accumulation due to range expansion.

Joint inference of evolutionary transitions to self-fertilization and demographic history using whole-genome sequences.

The evolution from outcrossing to selfing occurred recently across the eukaryote tree of life in plants, animals, fungi, and algae. Despite short-term advantages, selfing is hypothetically an evolutionary dead-end reproductive strategy. The tippy distribution on phylogenies suggests that most selfing species are of recent origin. However, dating such transitions is challenging yet central for testing this hypothesis. We build on previous theories to disentangle the differential effect of past changes in selfing rate or from that of population size on recombination probability along the genome. This allowed us to develop two methods using full-genome polymorphisms to (1) test if a transition from outcrossing to selfing occurred and (2) infer its age. The teSMC and tsABC methods use a transition matrix summarizing the distribution of times to the most recent common ancestor along the genome to estimate changes in the ratio of population recombination and mutation rates overtime. First, we demonstrate that our methods distinguish between past changes in selfing rate and demographic history. Second, we assess the accuracy of our methods to infer transitions to selfing approximately up to 2.5Ne generations ago. Third, we demonstrate that our estimates are robust to the presence of purifying selection. Finally, as a proof of principle, we apply both methods to three Arabidopsis thaliana populations, revealing a transition to selfing approximately 600,000 years ago. Our methods pave the way for studying recent transitions to self-fertilization and better accounting for variation in mating systems in demographic inferences.

Stigma receptors control intraspecies and interspecies barriers in Brassicaceae.

Flowering plants have evolved numerous intraspecific and interspecific prezygotic reproductive barriers to prevent production of unfavourable offspring1. Within a species, self-incompatibility (SI) is a widely utilized mechanism that rejects self-pollen2,3 to avoid inbreeding depression. Interspecific barriers restrain breeding between species and often follow the SI × self-compatible (SC) rule, that is, interspecific pollen is unilaterally incompatible (UI) on SI pistils but unilaterally compatible (UC) on SC pistils1,4-6. The molecular mechanisms underlying SI, UI, SC and UC and their interconnections in the Brassicaceae remain unclear. Here we demonstrate that the SI pollen determinant S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11)2,3 or a signal from UI pollen binds to the SI female determinant S-locus receptor kinase (SRK)2,3, recruits FERONIA (FER)7-9 and activates FER-mediated reactive oxygen species production in SI stigmas10,11 to reject incompatible pollen. For compatible responses, diverged pollen coat protein B-class12-14 from SC and UC pollen differentially trigger nitric oxide, nitrosate FER to suppress reactive oxygen species in SC stigmas to facilitate pollen growth in an intraspecies-preferential manner, maintaining species integrity. Our results show that SRK and FER integrate mechanisms underlying intraspecific and interspecific barriers and offer paths to achieve distant breeding in Brassicaceae crops.

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.

No detectable changes in reproductive behaviour of Caenorhabditis elegans males after 97 generations under obligatory outcrossing.

In Caenorhabditis elegans, a species reproducing mostly via self-fertilization, numerous signatures of selfing syndrome are observed, including differences in reproductive behaviour compared to related obligatory outcrossing species. In this study we investigated the effect of nearly 100 generations of obligatory outcrossing on several characteristics of male reproductive behaviour. A genetically uniform ancestral population carrying a mutation changing the reproductive system to obligatory outcrossing was split into four independent populations. We predicted that the transition from the natural reproductive system, where males were extremely rare, to obligatory outcrossing, where males comprise 50% of the population and are necessary for reproduction, will increase the selection pressure on higher effectiveness of mating behaviour. Several characteristics of male mating behaviour during a 15 min interaction as well as copulation success were compared between the ancestral and evolved populations. No significant differences in male mating behaviour or fertilization success were detected between generations 1 and 97 of obligatory outcrossing populations. We found, however, that longer contact with females increased chances of successful copulation, although this effect did not differ between populations. We conclude that either selection acting on male mating behaviour has not been strong enough, or mutational input of new adaptive variants has not been sufficient to cause noticeable behavioural differences after 97 generations of evolution starting from genetically uniform population.

The effects of selfing on multi-step adaptation.

Macroevolutionary studies have estimated higher extinction rates of self-compatible lineages than self-incompatible ones. A leading explanation is that selfing may prevent adaptation, since models show that selfing can inhibit the fixation of adaptive alleles at a single locus (1-step adaptation). However, adaptation often involves changes at multiple loci (multi-step adaption), but the effects of selfing remain unclear because selfing increases homozygosity, which affects selection intensity, the effective population size, and the effective recombination rate. By modeling using population genetic models, I investigate the effects of selfing on adaption requiring fixation of 2 adaptive alleles, I show that intermediate selfing rates generally promote adaption, by increasing the fixation probability of the double-mutant haplotype once it is generated. In constant-sized populations, selfing increases the rate of adaptation through the fixation of new mutations even when both alleles are dominant. In demographically declining populations, the rescue probability rises sharply as the selfing rate increases from zero, but quickly drops to be low when it approaches 1.0. These findings are at odds with the hypothesis that higher extinction rates of self-compatible lineages result from reduced adaptive potential but may help explain why some studies have failed to detect relaxation of selection in selfers and also the prevalence of mixed-mating systems.

Interpopulation variation in inbreeding is primarily driven by tolerance of mating with relatives in a spermcasting invertebrate.

The degree to which individuals inbreed is a fundamental aspect of population biology shaped by both passive and active processes. Yet, the relative influences of random and non-random mating on the overall magnitude of inbreeding are not well characterized for many taxa. We quantified variation in inbreeding among qualitatively accessible and isolated populations of a sessile marine invertebrate (the colonial ascidian Lissoclinum verrilli) in which hermaphroditic colonies cast sperm into the water column for subsequent uptake and internal fertilization. We compared estimates of inbreeding to simulations predicting random mating within sites to evaluate if levels of inbreeding were (1) less than expected because of active attempts to limit inbreeding, (2) as predicted by genetic subdivision and passive inbreeding tolerance, or (3) greater than simulations due to active attempts to promote inbreeding via self-fertilization or a preference for related mates. We found evidence of restricted gene flow and significant differences in the genetic diversity of L. verrilli colonies among sites, indicating that on average colonies were weakly related in accessible locations, but their levels of relatedness matched that of first cousins or half-siblings on isolated substrates. Irrespective of population size, progeny arrays revealed variation in the magnitude of inbreeding across sites that tracked with the mean relatedness of conspecifics. Biparental reproduction was confirmed in most offspring (86%) and estimates of total inbreeding largely overlapped with simulations of random mating, suggesting that interpopulation variation in mother-offspring resemblance was primarily due to genetic subdivision and passive tolerance of related mates. Our results highlight the influence of demographic isolation on the genetic composition of populations, and support theory predicting that tolerance of biparental inbreeding, even when mates are closely related, may be favoured under a broad set of ecological and evolutionary conditions.

Genetic and demographic signatures accompanying the evolution of the selfing syndrome in Daphne kiusiana, an evergreen shrub.

BACKGROUND AND AIMS: The evolution of mating systems from outcrossing to self-fertilization is a common transition in flowering plants. This shift is often associated with the 'selfing syndrome', which is characterized by less visible flowers with functional changes to control outcrossing. In most cases, the evolutionary history and demographic dynamics underlying the evolution of the selfing syndrome remain poorly understood. METHODS: Here, we characterize differences in the demographic genetic consequences and associated floral-specific traits between two distinct geographical groups of a wild shrub, Daphne kiusiana, endemic to East Asia; plants in the eastern region (southeastern Korea and Kyushu, Japan) exhibit smaller and fewer flowers compared to those of plants in the western region (southwestern Korea). Genetic analyses were conducted using nuclear microsatellites and chloroplast DNA (multiplexed phylogenetic marker sequencing) datasets. KEY RESULTS: A high selfing rate with significantly increased homozygosity characterized the eastern lineage, associated with lower levels of visibility and herkogamy in the floral traits. The two lineages harboured independent phylogeographical histories. In contrast to the western lineage, the eastern lineage showed a gradual reduction in the effective population size with no signs of a severe bottleneck despite its extreme range contraction during the last glacial period. CONCLUSIONS: Our results suggest that the selfing-associated morphological changes in D. kiusiana are of relatively old origin (at least 100 000 years ago) and were driven by directional selection for efficient self-pollination. We provide evidence that the evolution of the selfing syndrome in D. kiusiana is not strongly associated with a severe population bottleneck.

Recent speciation associated with range expansion and a shift to self-fertilization in North American Arabidopsis.

The main processes classically evoked for promoting reproductive isolation and speciation are geographic separation reducing gene flow among populations, divergent selection, and chance genomic change. In a case study, we present evidence that the additional factors of climate change, range expansion and a shift in mating towards inbreeding can initiate the processes leading to parapatric speciation. At the end of the last Pleistocene glaciation cycle, the North American plant Arabidopsis lyrata expanded its range and concomitantly lost its reproductive mode of outcrossing multiple times. We show that in one of the newly colonized areas, the self-fertilizing recolonization lineage of A. lyrata gave rise to selfing A. arenicola, which expanded its range to subarctic and arctic Canada and Greenland, while the parental species remained restricted to temperate North America. Despite the vast range expansion by the new species, mutational load did not increase, probably because of selfing and quasi-clonal selection. We conclude that such peripheral parapatric speciation combined with range expansion and inbreeding may be an important but so far overlooked mode of speciation.

How broad is the selfing syndrome? Insights from convergent evolution of gene expression across species and tissues in the Capsella genus.

The shift from outcrossing to selfing is one of the main evolutionary transitions in plants. It is accompanied by profound effects on reproductive traits, the so-called selfing syndrome. Because the transition to selfing also implies deep genomic and ecological changes, one also expects to observe a genomic selfing syndrome. We took advantage of the three independent transitions from outcrossing to selfing in the Capsella genus to characterize the overall impact of mating system change on RNA expression, in flowers but also in leaves and roots. We quantified the extent of both selfing and genomic syndromes, and tested whether changes in expression corresponded to adaptation to selfing or to relaxed selection on traits that were constrained in outcrossers. Mating system change affected gene expression in all three tissues but more so in flowers than in roots and leaves. Gene expression in selfing species tended to converge in flowers but diverged in the two other tissues. Hence, convergent adaptation to selfing dominates in flowers, whereas genetic drift plays a more important role in leaves and roots. The effect of mating system transition is not limited to reproductive tissues and corresponds to both adaptation to selfing and relaxed selection on previously constrained traits.

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.

The western redcedar genome reveals low genetic diversity in a self-compatible conifer.

We assembled the 9.8-Gbp genome of western redcedar (WRC; Thuja plicata), an ecologically and economically important conifer species of the Cupressaceae. The genome assembly, derived from a uniquely inbred tree produced through five generations of self-fertilization (selfing), was determined to be 86% complete by BUSCO analysis, one of the most complete genome assemblies for a conifer. Population genomic analysis revealed WRC to be one of the most genetically depauperate wild plant species, with an effective population size of approximately 300 and no significant genetic differentiation across its geographic range. Nucleotide diversity, π, is low for a continuous tree species, with many loci showing zero diversity, and the ratio of π at zero- to fourfold degenerate sites is relatively high (approximately 0.33), suggestive of weak purifying selection. Using an array of genetic lines derived from up to five generations of selfing, we explored the relationship between genetic diversity and mating system. Although overall heterozygosity was found to decline faster than expected during selfing, heterozygosity persisted at many loci, and nearly 100 loci were found to deviate from expectations of genetic drift, suggestive of associative overdominance. Nonreference alleles at such loci often harbor deleterious mutations and are rare in natural populations, implying that balanced polymorphisms are maintained by linkage to dominant beneficial alleles. This may account for how WRC remains responsive to natural and artificial selection, despite low genetic diversity.

Climate change is associated with increased allocation to potential outcrossing in a common mixed mating species.

PREMISE: Although the balance between cross- and self-fertilization is driven by the environment, no long-term study has documented whether anthropogenic climate change is affecting reproductive strategy allocation in species with mixed mating systems. Here, we test whether the common blue violet (Viola sororia; Violaceae) has altered relative allocation to the production of potentially outcrossing flowers as the climate has changed throughout the 20th century. METHODS: Using herbarium records spanning from 1875 to 2015 from the central United States, we quantified production of obligately selfing cleistogamous (CL) flowers and potentially outcrossing chasmogamous (CH) flowers by V. sororia, coupled these records with historic temperature and precipitation data, and tested whether changes to the proportion of CL flowers correlate with temporal climate trends. RESULTS: We find that V. sororia progressively produced lower proportions of CL flowers across the past century and in environments with lower mean annual temperature and higher total annual precipitation. We also find that both CL and CH flower phenology has advanced across this time period. CONCLUSIONS: Our results suggest that V. sororia has responded to lower temperatures and greater water availability by shifting reproductive strategy allocation away from selfing and toward potential outcrossing. This provides the first long-term study of how climate change may affect relative allocation to potential outcrossing in species with mixed mating systems. By revealing that CL flowering is associated with low water availability and high temperature, our results suggest the production of obligately selfing flowers is favored in water limited environments.

Removal of the block to self-fertilization by low-calcium artificial seawater in the ascidian Ciona intestinalis.

Ascidians (Urochordate) are hermaphroditic marine invertebrates that release sperm and eggs to the surrounding seawater. However, several ascidians, including Ciona intestinalis and Halocynthia roretzi, show strict self-sterility due to a self/nonself-recognition mechanism in the interaction between sperm and the vitelline coat (VC) of the eggs. We have previously reported that sperm intracellular Ca2+ level drastically increased immediately after sperm binding to the VC of self eggs but not nonself eggs in C. intestinalis type A, which was potently inhibited by lowering the external Ca2+ concentration, suggesting that sperm Ca2+ influx occurs after sperm self-recognition on the VC. Here, we investigated whether self-sterility was abolished by lowering the external Ca2+ concentration in C. intestinalis. The results showed that the block to self-fertilization was removed by low-Ca2+ (∼1 mM) seawater without decreasing the fertilization rate. Such an effect was not observed with Mg2+ or K+. These results led us to conclude that a low-Ca2+ environment is sufficient to block the self-recognition signal upon fertilization. As low-Ca2+ seawater showed no effect on H. roretzi self-sterility, we propose that the mechanism of self-sterility in Ciona must be distinctive from that in Halocynthia.

The selfing syndrome and beyond: diverse evolutionary consequences of mating system transitions in plants.

The shift from outcrossing to self-fertilization (selfing) is considered one of the most prevalent evolutionary transitions in flowering plants. Selfing species tend to share similar reproductive traits in morphology and function, and such a set of traits is called the 'selfing syndrome'. Although the genetic basis of the selfing syndrome has been of great interest to evolutionary biologists, knowledge of the causative genes or mutations was limited until recently. Thanks to advances in population genomic methodologies combined with high-throughput sequencing technologies, several studies have successfully unravelled the molecular and genetic basis for evolution of the selfing syndrome in Capsella, Arabidopsis, Solanum and other genera. Here we first introduce recent research examples that have explored the loci, genes and mutations responsible for the selfing syndrome traits, such as reductions in petal size or in pollen production, that are mainly relevant to pre-pollination processes. Second, we review the relationship between the evolution of selfing and interspecific pollen transfer, highlighting the findings of post-pollination reproductive barriers at the molecular level. We then discuss the emerging view of patterns in evolution of the selfing syndrome, such as the pervasive involvement of loss-of-function mutations and the relative importance of selection versus neutral degradation. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

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.