Unveiling the genome-wide consequences of range expansion and mating-system transitions in Primula vulgaris.

Genetic diversity is heterogeneously distributed among populations of the same species, due to the joint effects of multiple demographic processes, including range contractions and expansions, and mating-systems shifts. Here, we ask how both processes shape genomic diversity in space and time in the classical Primula vulgaris model. This perennial herb originated in the Caucasus region and was hypothesized to have expanded westward following glacial retreat in the Quaternary. Moreover, this species is a long-standing model for mating-system transitions, exemplified by shifts from heterostyly to homostyly. Leveraging a high-quality reference genome of the closely related Primula veris and whole genome resequencing data from both heterostylous and homostylous individuals from populations encompassing a wide distribution of P. vulgaris, we reconstructed the demographic history of P. vulgaris. Results are compatible with the previously proposed hypothesis of range expansion from the Caucasus region approximately 79,000 years ago and suggests later shifts to homostyly following rather than preceding post-glacial colonization of England. Furthermore, in accordance with population genetic theoretical predictions, both processes are associated with reduced genetic diversity, increased linkage disequilibrium, and reduced efficacy of purifying selection. A novel result concerns the contrasting effects of range expansion vs. shift to homostyly on Transposable Elements (TE), for the former process is associated with changes in TE genomic content, while the latter is not. Jointly, our results elucidate how the interactions among range expansion, transitions to selfing, and Quaternary climatic oscillations shape plant evolution.

Ecological correlates of population genetics in Linum suffruticosum, an heterostylous polyploid and taxonomic complex endemic to the Western Mediterranean Basin.

Linum suffruticosum s.l. is a taxonomic complex widespread in the Western Mediterranean basin. The complex is characterized by a high phenotypic and cytogenetic diversity, and by a unique three-dimensional heterostyly system that makes it an obligate outcrosser. We studied the patterns of genetic diversity and structure of populations throughout the entire distribution of L. suffruticosum s.l. with microsatellite markers. We analysed their relationships with various biological and ecological variables, including the morph ratio and sex organ reciprocity of populations measured with a novel multi-dimensional method. Populations consistently showed an approximate 1:1 morph ratio with high sex organ reciprocity and high genetic diversity. We found high genetic differentiation of populations, showing a pattern of isolation by distance. The Rif mountains in NW Africa were the most important genetic barrier. The taxonomic treatment within the group was not related to the genetic differentiation of populations, but to their environmental differentiation. Genetic diversity was unrelated to latitude, elevation, population size, niche suitability or breeding system. However, there was a clear influence of ploidy level on the genetic diversity of populations, and a seeming centre-periphery pattern in its distribution. Our results suggest that polyploidization events, high outcrossing rates, isolation by distance and important geographical barriers to gene flow have played major roles in the microevolutionary history of this species complex.

The Primula edelbergii S-locus is an example of a jumping supergene.

Research on supergenes, non-recombining genomic regions housing tightly linked genes that control complex phenotypes, has recently gained prominence in genomics. Heterostyly, a floral heteromorphism promoting outcrossing in several angiosperm families, is controlled by the S-locus supergene. The S-locus has been studied primarily in closely related Primula species and, more recently, in other groups that independently evolved heterostyly. However, it remains unknown whether genetic architecture and composition of the S-locus are maintained among species that share a common origin of heterostyly and subsequently diverged across larger time scales. To address this research gap, we present a chromosome-scale genome assembly of Primula edelbergii, a species that shares the same origin of heterostyly with Primula veris (whose S-locus has been characterized) but diverged from it 18 million years ago. Comparative genomic analyses between these two species allowed us to show, for the first time, that the S-locus can 'jump' (i.e. translocate) between chromosomes maintaining its function in controlling heterostyly. Additionally, we found that four S-locus genes were conserved but reshuffled within the supergene, seemingly without affecting their expression, thus we could not detect changes explaining the lack of self-incompatibility in P. edelbergii. Furthermore, we confirmed that the S-locus is not undergoing genetic degeneration. Finally, we investigated P. edelbergii evolutionary history within Ericales in terms of whole genome duplications and transposable element accumulation. In summary, our work provides a valuable resource for comparative analyses aimed at investigating the genetics of heterostyly and the pivotal role of supergenes in shaping the evolution of complex phenotypes.

DNA methylation analysis of floral parts revealed dynamic changes during the development of homostylous Fagopyrum tataricum and heterostylous F. esculentum flowers.

BACKGROUND: Proper flower development is essential for plant reproduction, a crucial aspect of the plant life cycle. This process involves precisely coordinating transcription factors, enzymes, and epigenetic modifications. DNA methylation, a ubiquitous and heritable epigenetic mechanism, is pivotal in regulating gene expression and shaping chromatin structure. Fagopyrum esculentum demonstrates anti-hypertensive, anti-diabetic, anti-inflammatory, cardio-protective, hepato-protective, and neuroprotective properties. However, the heteromorphic heterostyly observed in F. esculentum poses a significant challenge in breeding efforts. F. tataricum has better resistance to high altitudes and harsh weather conditions such as drought, frost, UV-B radiation damage, and pests. Moreover, F. tataricum contains significantly higher levels of rutin and other phenolics, more flavonoids, and a balanced amino acid profile compared to common buckwheat, being recognised as functional food, rendering it an excellent candidate for functional food applications. RESULTS: This study aimed to compare the DNA methylation profiles between the Pin and Thrum flower components of F. esculentum, with those of self-fertile species of F. tataricum, to understand the potential role of this epigenetic mechanism in Fagopyrum floral development. Notably, F. tataricum flowers are smaller than those of F. esculentum (Pin and Thrum morphs). The decline in DNA methylation levels in the developed open flower components, such as petals, stigmas and ovules, was consistent across both species, except for the ovule in the Thrum morph. Conversely, Pin and Tartary ovules exhibited a minor decrease in DNA methylation levels. The highest DNA methylation level was observed in Pin stigma from closed flowers, and the most significant decrease was in Pin stigma from open flowers. In opposition, the nectaries of open flowers exhibited higher levels of DNA methylation than those of closed flowers. The decrease in DNA methylation might correspond with the downregulation of genes encoding methyltransferases. CONCLUSIONS: Reduced overall DNA methylation and the expression of genes associated with these epigenetic markers in fully opened flowers of both species may indicate that demethylation is necessary to activate the expression of genes involved in floral development.

Asymmetrical disassortative pollination mediated by long-/short-tongued pollinators in a distylous Limonium myrianthum (Plumbaginaceae) with a short corolla tubular small flower.

In heterostylous plants, short-tongued pollinators are often ineffective/inefficient owing to the limitations imposed by a long corolla tube. However, it is unclear how disassortative pollen transfer is achieved in small flowers. We investigated the pollination pattern and floral morph variation by analyzing heterostylous syndrome, pollinator groups, and pollen deposition after a single visitation in two Limonium myrianthum populations with short-corolla-tubular small flowers. The predominant pollinators in the Hutubi population were pollen-seeking short-tongued syrphids, which can only transfer pollen between high-level sexual organs. In the Xishan population, nectar-seeking short-tongued insects were efficient pollinators with symmetrical disassortative pollen transfer between high- and low-level sexual organs, whereas long-tongued pollinators had a low efficiency between high-level sexual organs due to the low contact probability with the stigma of long-styled flowers (L-morph), which no longer offered the same advantage observed in tubular flowers. Asymmetrical disassortative pollination may cause the female fitness of short-styled (S-morph) individuals in the Hutubi and L-morph individuals in the Xishan population to suffer greater selection pressure and exhibit a higher degree of floral morph variation. Limonium myrianthum exhibits an unusual pollination pattern in which the small flowers with short corolla tubes make it possible for short-tongued insects to become effective pollinators. However, factors such as the position of stigma-anther within the flower, pollinator species and their preference further caused asymmetrical disassortative pollen transfer. Therefore, more factors should be considered when evaluating the effectiveness of short- and long-tongued insects in pollination service.

Floral morph variation mediated by clonal growth and pollinator functional groups of Limonium otolepis in a heterostylous fragmented population.

Abstract. Heterostyly, a genetic style polymorphism, is linked to symmetric pollen transfer, vital for its maintenance. Clonal growth typically impacts sexual reproduction by influencing pollen transfer. However, the floral morph variation remains poorly understood under the combined effects of pollinators and clonal growth in heterostyly characterized by negative frequency-dependent selection and disassortative mating. We estimated morph ratios, ramets per genet and heterostylous syndrome and quantified legitimate pollen transfer via clonal growth, pollinators and reciprocal herkogamy between floral morphs in Limonium otolepis, a fragmented population composed of five subpopulations in the desert environment of northwestern China, with small flower and large floral morph variation. All subpopulations but one exhibited pollen-stigma morphology dimorphism. The compatibility between mating types with different pollen-stigma morphologies remained consistent regardless of reciprocal herkogamy. Biased ratios and ramets per genet of the two mating types with distinct pollen-stigma morphologies caused asymmetric pollen flow and varying fruit sets in all subpopulations. Short-tongued insects were the primary pollinators due to small flower sizes. However, pollen-feeding Syrphidae sp. triggered asymmetry in pollen flow between high and low sex organs, with short-styled morphs having lower stigma pollen depositions and greater variation. Clonal growth amplified this variation by reducing intermorph pollen transfer. All in all, pollinators and clonal growth jointly drive floral morph variation. H-morphs with the same stigma-anther position and self-incompatibility, which mitigate the disadvantages of sunken low sex organs with differing from the classical homostyly, might arise from long- and short-styled morphs through a 'relaxed selection'. This study is the first to uncover the occurrence of the H-morph and its associated influencing factors in a distylous plant featuring clonal growth, small flowers and a fragmented population.

A hemizygous supergene controls homomorphic and heteromorphic self-incompatibility systems in Oleaceae.

Self-incompatibility (SI) has evolved independently multiple times and prevents self-fertilization in hermaphrodite angiosperms. Several groups of Oleaceae such as jasmines exhibit distylous flowers, with two compatibility groups each associated with a specific floral morph.1 Other Oleaceae species in the olive tribe have two compatibility groups without associated morphological variation.2,3,4,5 The genetic basis of both homomorphic and dimorphic SI systems in Oleaceae is unknown. By comparing genomic sequences of three olive subspecies (Olea europaea) belonging to the two compatibility groups, we first locate the genetic determinants of SI within a 700-kb hemizygous region present only in one compatibility group. We then demonstrate that the homologous hemizygous region also controls distyly in jasmine. Phylogenetic analyses support a common origin of both systems, following a segmental genomic duplication in a common ancestor. Examination of the gene content of the hemizygous region in different jasmine and olive species suggests that the mechanisms determining compatibility groups and floral phenotypes (whether homomorphic or dimorphic) in Oleaceae rely on the presence/absence of two genes involved in gibberellin and brassinosteroid regulation.

Genomic analyses elucidate S-locus evolution in response to intra-specific losses of distyly in Primula vulgaris.

Distyly, a floral dimorphism that promotes outcrossing, is controlled by a hemizygous genomic region known as the S-locus. Disruptions of genes within the S-locus are responsible for the loss of distyly and the emergence of homostyly, a floral monomorphism that favors selfing. Using whole-genome resequencing data of distylous and homostylous individuals from populations of Primula vulgaris and leveraging high-quality reference genomes of Primula we tested, for the first time, predictions about the evolutionary consequences of transitions to selfing on S-genes. Our results reveal a previously undetected structural rearrangement in CYPᵀ associated with the shift to homostyly and confirm previously reported, homostyle-specific, loss-of-function mutations in the exons of the S-gene CYPᵀ. We also discovered that the promoter and intronic regions of CYPᵀ in distylous and homostylous individuals are conserved, suggesting that down-regulation of CYPᵀ via mutations in its promoter and intronic regions is not a cause of the shift to homostyly. Furthermore, we found that hemizygosity is associated with reduced genetic diversity in S-genes compared with their paralogs outside the S-locus. Additionally, the shift to homostyly lowers genetic diversity in both the S-genes and their paralogs, as expected in primarily selfing plants. Finally, we tested, for the first time, long-standing theoretical models of changes in S-locus genotypes during early stages of the transition to homostyly, supporting the assumption that two copies of the S-locus might reduce homostyle fitness.

PfPIN5 promotes style elongation by regulating cell length in Primula forbesii Franch.

BACKGROUND AND AIMS: Style dimorphism is one of the polymorphic characteristics of flowers in heterostylous plants, which have two types of flowers: the pin morph, with long styles and shorter anthers, and the thrum morph, with short styles and longer anthers. The formation of dimorphic styles has received attention in the plant world. Previous studies showed that CYP734A50 in Primula determined style length and limited style elongation and that the brassinosteroid metabolic pathway was involved in regulation of style length. However, it is unknown whether there are other factors affecting the style length of Primula. METHODS: Differentially expressed genes highly expressed in pin morph styles were screened based on Primula forbesii transcriptome data. Virus-induced gene silencing was used to silence these genes, and the style length and anatomical changes were observed 20 days after injection. KEY RESULTS: PfPIN5 was highly expressed in pin morph styles. When PfPIN5 was silenced, the style length was shortened in pin and long-homostyle plants by shortening the length of style cells. Moreover, silencing CYP734A50 in thrum morph plants increased the expression level of PfPIN5 significantly, and the style length increased. The results indicated that PfPIN5, an auxin efflux transporter gene, contributed to regulation of style elongation in P. forbesii. CONCLUSIONS: The results implied that the auxin pathway might also be involved in the formation of styles of P. forbesii, providing a new pathway for elucidating the molecular mechanism of style elongation in P. forbesii.

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.

Long-and short-billed hummingbirds as pollinators of Palicourea demissa, a distylous treelet of Neotropical cloud forests.

Distyly has been interpreted as a mechanism that promotes cross-pollination between floral morphs. According to this hypothesis, pollen from anthers positioned at different heights could adhere to different body parts of the pollinator that would correspond to those points where stigmas of compatible morph contact the animal. In this regard, hummingbird species with different bill sizes may play different roles as pollinators of each morph. If pollinators mobilize more legitimate pollen towards one of the two morphs, gender specialization may occur. This work aimed to assess experimentally the role of long- and short-billed hummingbirds as pollinators of short-style (SS) and long-style (LS) flowers of Palicourea demissa, a distylous, hummingbird-pollinated treelet in Venezuelan cloud forests. Flowers were emasculated and exposed to a single visit of the hummingbird Coeligena torquata (long-billed), Heliangelus spencei (short-billed) or Adelomyia melanogenys (short-billed). Later, stigmas were removed, and pollen load counted under a microscope to calculate the probability of legitimate- and illegitimate-pollen transfer by hummingbirds. The probability analyses of pollen transference showed that short-billed hummingbirds have higher pollination probabilities from SS-anthers to LS- and SS-stigmas, and from LS-anther to LS-stigmas than from LS-anther to SS-stigmas. In contrast, long-billed hummingbirds have higher probabilities of pollen transference from LS-anthers to SS-stigmas than in other directions. A deeper view of the sexual expression of each morph in P. demissa will depend on future studies that determine possible morpho-differences in the biological function of male and female floral structures, and the role played by less frequent floral visitors as mediators of legitimate pollination between floral morphs.

Drivers of diversification in Linum (Linaceae) by means of chromosome evolution: correlations with biogeography, breeding system and habit.

BACKGROUND AND AIMS: Chromosome evolution leads to hybrid dysfunction and recombination patterns and has thus been proposed as a major driver of diversification in all branches of the tree of life, including flowering plants. In this study we used the genus Linum (flax species) to evaluate the effects of chromosomal evolution on diversification rates and on traits that are important for sexual reproduction. Linum is a useful study group because it has considerable reproductive polymorphism (heterostyly) and chromosomal variation (n = 6-36) and a complex pattern of biogeographical distribution. METHODS: We tested several traditional hypotheses of chromosomal evolution. We analysed changes in chromosome number across the phylogenetic tree (ChromEvol model) in combination with diversification rates (ChromoSSE model), biogeographical distribution, heterostyly and habit (ChromePlus model). KEY RESULTS: Chromosome number evolved across the Linum phylogeny from an estimated ancestral chromosome number of n = 9. While there were few apparent incidences of cladogenesis through chromosome evolution, we inferred up to five chromosomal speciation events. Chromosome evolution was not related to heterostyly but did show significant relationships with habit and geographical range. Polyploidy was negatively correlated with perennial habit, as expected from the relative commonness of perennial woodiness and absence of perennial clonality in the genus. The colonization of new areas was linked to genome rearrangements (polyploidy and dysploidy), which could be associated with speciation events during the colonization process. CONCLUSIONS: Chromosome evolution is a key trait in some clades of the Linum phylogeny. Chromosome evolution directly impacts speciation and indirectly influences biogeographical processes and important plant traits.

Development of microsatellite markers for sister species Linum suffruticosum and Linum tenuifolium in their overlapping ranges.

BACKGROUND: Microsatellite markers were developed for distylous Linum suffruticosum and tested in the monomorphic sister species Linum tenuifolium. These species are perennial herbs endemic to the western and northwestern Mediterranean, respectively, with a partially overlapping distribution area. METHODS AND RESULTS: We developed 12 microsatellite markers for L. suffruticosum using next generation sequencing, and assessed their polymorphism and genetic diversity in 152 individuals from seven natural populations. The markers displayed high polymorphism, with two to 16 alleles per locus and population, and average observed and expected heterozygosities of 0.833 and 0.692, respectively. All loci amplified successfully in the sister species L. tenuifolium, and 150 individuals from seven populations were also screened. The polymorphism exhibited was high, with two to ten alleles per locus and population, and average observed and expected heterozygosities of 0.77 and 0.62, respectively. CONCLUSIONS: The microsatellite markers identified in L. suffruticosum and tested in L. tenuifolium are a powerful tool to facilitate future investigations of the population genetics, mating patterns and hybridization between both Linum species in their contact zone.

Complementary roles of hummingbirds and bees: Pollen pickup, pollen deposition, and fruit production in the distylous Palicourea rigida.

PREMISE: Distyly is a condition in which individual plants in a population express two floral morphs, L- and S-morph, characterized by reciprocal placements of anthers and stigmas between morphs. The function of distyly requires that pollinators collect pollen from L- and S-morphs on different parts along their bodies to then deposit it on the stigmas of the opposite morph, known as legitimate pollination. However, different pollinator groups might differ in the ability to transfer pollen legitimately. METHODS: We investigated patterns of pollen pickup along the body of different functional groups (hummingbirds and bees) using preserved specimens to analyze their role in the reproductive success of Palicourea rigida. We measured pollen deposition on the body of pollinators, on stigmas, and fruit production after a single visit. RESULTS: Pollen from L- and S-flowers appeared segregated on different body parts of the hummingbird and bee used in the study. S-pollen was deposited primarily on the proximal regions (near the head), and L-pollen was placed in the distal regions (tip of the proboscis and bill). Hummingbirds were more efficient at legitimate pollination than bees, particularly to S-stigmas. However, fruit formation after single visits by both pollinators was similar. CONCLUSIONS: The morphology of distylous flowers allows the segregated placement of L-and S-pollen on different body parts of the animal specimens used, facilitating the promotion of legitimate pollen transfer, an observation consistent between the two functional pollinator groups. Also, the results show that full fruit set requires more than one visit.

Incomplete partitioning of pollinators by Linum suffruticosum and its coflowering congeners.

PREMISE: Linum suffruticosum shows variations in pollinator fit, pollen pickup, and local pollinators that predict pollen deposition rates. The species often coflowers with other Linum species using the same pollinators. We investigated whether L. suffruticosum trait variation could be explained by local patterns of pollinator sharing and associated evolution to reduce interspecific pollen transfer. METHODS: Pollinator observations were made in different localities (single species, coflowering with other congeners). Floral traits were measured to detect differences across populations and from coflowering species. Reproductive costs were quantified using interspecific hand pollinations and measures of pollen-tube formation, combined with observations of pollen arrival on stigmas and pollen-tube formation after natural pollination in allopatric and sympatric localities. RESULTS: The size and identity of the most important pollinator of L. suffruticosum and whether there was pollinator sharing with coflowering species appeared to explain floral trait variation related to pollinator fit. The morphological overlap of the flowers of L. suffruticosum with those of coflowering species varied, depending on coflowering species identity. A post-pollination incompatibility system maintains reproductive isolation, but conspecific pollen-tube formation was lower after heterospecific pollination. Under natural pollination at sites of coflowering with congeners, conspecific pollen-tube formation was lower than at single-species localities. CONCLUSIONS: Trait variation in L. suffruticosum appears to respond to the most important local pollinator. Locally, incomplete pollinator partitioning might cause interspecific pollination, imposing reproductive costs. These reproductive costs may generate selection on floral traits for reduced morphological overlap with coflowering congeners, leading to the evolution of pollination ecotypes.

Skewed morph ratios lead to lower genetic diversity of the heterostylous Primula veris in fragmented grasslands.

Populations of heterostylous plant species are ideally composed of equal frequencies of two (distylous) or three (tristylous) morphologically different floral morphs. Intra-morph incompatibility helps to avoid inbreeding and to maintain genetic diversity, supporting plant fitness and long-term viability. Habitat fragmentation can lead to skewed morph ratios and thereby reduce the abundance of compatible mates. This, in turn, can result in a loss of genetic diversity. We tested whether the genetic diversity of heterostylous plants is affected by morph ratio bias using populations of the distylous grassland plant Primula veris in recently fragmented grasslands. We recorded morph frequencies and population sizes in 30 study populations of P. veris on two Estonian islands characterised by different degrees of habitat fragmentation. Examining variation of thousands of single nucleotide polymorphisms (SNPs) and heterostyly-specific genetic markers, we quantified overall and morph-specific genetic diversity and differentiation in these populations. Morph frequencies deviated more in smaller populations. Skewed morph ratios had a negative effect on the genetic diversity of P. veris in more fragmented grasslands. In the populations of better-connected grassland systems, genetic differentiation among S-morphs was higher than among L-morphs. Our study shows that deviations from morph balance are stronger in small populations and have a negative impact on the genetic diversity of the distylous plant P. veris. Together with the direct negative effects of habitat loss and decreased population size on the genetic diversity of plants, morph ratio bias may intensify the process of genetic erosion, thus exacerbating the local extinction of heterostylous species.

Comprehensive transcriptomic profiling reveals complex molecular mechanisms in the regulation of style-length dimorphism in Guettarda speciosa (Rubiaceae), a species with "anomalous" distyly.

Background: The evolution of heterostyly, a genetically controlled floral polymorphism, has been a hotspot of research since the 19th century. In recent years, studies on the molecular mechanism of distyly (the most common form of heterostyly) revealed an evolutionary convergence in genes for brassinosteroids (BR) degradation in different angiosperm groups. This floral polymorphism often exhibits considerable variability that some taxa have significant stylar dimorphism, but anther height differs less. This phenomenon has been termed "anomalous" distyly, which is usually regarded as a transitional stage in evolution. Compared to "typical" distyly, the genetic regulation of "anomalous" distyly is almost unknown, leaving a big gap in our understanding of this special floral adaptation strategy. Methods: Here we performed the first molecular-level study focusing on this floral polymorphism in Guettarda speciosa (Rubiaceae), a tropical tree with "anomalous" distyly. Comprehensive transcriptomic profiling was conducted to examine which genes and metabolic pathways were involved in the genetic control of style dimorphism and if they exhibit similar convergence with "typical" distylous species. Results: "Brassinosteroid homeostasis" and "plant hormone signal transduction" was the most significantly enriched GO term and KEGG pathway in the comparisons between L- and S-morph styles, respectively. Interestingly, homologs of all the reported S-locus genes either showed very similar expressions between L- and S-morph styles or no hits were found in G. speciosa. BKI1, a negative regulator of brassinosteroid signaling directly repressing BRI1 signal transduction, was identified as a potential gene regulating style length, which significantly up-regulated in the styles of S-morph. Discussion: These findings supported the hypothesis that style length in G. speciosa was regulated through a BR-related signaling network in which BKI1 may be one key gene. Our data suggested, in species with "anomalous" distyly, style length was regulated by gene differential expressions, instead of the "hemizygous" S-locus genes in "typical" distylous flowers such as Primula and Gelsemium, representing an "intermediate" stage in the evolution of distyly. Genome-level analysis and functional studies in more species with "typical" and "anomalous" distyly would further decipher this "most complex marriage arrangement" in angiosperms and improve our knowledge of floral evolution.

Annotation of the Turnera subulata (Passifloraceae) Draft Genome Reveals the S-Locus Evolved after the Divergence of Turneroideae from Passifloroideae in a Stepwise Manner.

A majority of Turnera species (Passifloraceae) exhibit distyly, a reproductive system involving both self-incompatibility and reciprocal herkogamy. This system differs from self-incompatibility in Passiflora species. The genetic basis of distyly in Turnera is a supergene, restricted to the S-morph, and containing three S-genes. How supergenes and distyly evolved in Turnera, and the other Angiosperm families exhibiting distyly remain largely unknown. Unraveling the evolutionary origins in Turnera requires the generation of genomic resources and extensive phylogenetic analyses. Here, we present the annotated draft genome of the S-morph of distylous Turnera subulata. Our annotation allowed for phylogenetic analyses of the three S-genes' families across 56 plant species ranging from non-seed plants to eudicots. In addition to the phylogenetic analysis, we identified the three S-genes' closest paralogs in two species of Passiflora. Our analyses suggest that the S-locus evolved after the divergence of Passiflora and Turnera. Finally, to provide insights into the neofunctionalization of the S-genes, we compared expression patterns of the S-genes with close paralogs in Arabidopsis and Populus trichocarpa. The annotation of the T. subulata genome will provide a useful resource for future comparative work. Additionally, this work has provided insights into the convergent nature of distyly and the origin of supergenes.

Diverse mating consequences of the evolutionary breakdown of the sexual polymorphism heterostyly.

Reproductive systems of flowering plants are evolutionarily fluid, with mating patterns changing in response to shifts in abiotic conditions, pollination systems, and population characteristics. Changes in mating should be particularly evident in species with sexual polymorphisms that become ecologically destabilized, promoting transitions to alternative reproductive systems. Here, we decompose female mating portfolios (incidence of selfing, outcross mate number, and intermorph mating) in eight populations of Primula oreodoxa, a self-compatible insect-pollinated herb. This species is ancestrally distylous, with populations subdivided into two floral morphs that usually mate with each other (disassortative mating). Stages in the breakdown of polymorphism also occur, including "mixed" populations of distylous and homostylous (self-pollinating) morphs and purely homostylous populations. Population morph ratios vary with elevation in association with differences in pollinator availability, providing an unusual opportunity to investigate changes in mating patterns accompanying transitions in reproductive systems. Unexpectedly, individuals mostly outcrossed randomly, with substantial disassortative mating in at most two distylous populations. As predicted, mixed populations had higher selfing rates than distylous populations, within mixed populations, homostyles selfed almost twice as much as the distylous morphs, and homostylous populations exhibited the highest selfing rates. Populations with homostyles outcrossed with fewer mates and mate number varied negatively with population selfing rates. These differences indicate maintenance of distyly at low elevation, transition to monomorphic selfing at high elevation, and uncertain, possibly variable fates at intermediate elevation. By quantifying the earliest changes in mating that initiate reproductive transitions, our study highlights the key role of mating in promoting evolutionary divergence.

Different molecular changes underlie the same phenotypic transition: Origins and consequences of independent shifts to homostyly within species.

The repeated transition from outcrossing to selfing is a key topic in evolutionary biology. However, the molecular basis of such shifts has been rarely examined due to lack of knowledge of the genes controlling these transitions. A classic example of mating system transition is the repeated shift from heterostyly to homostyly. Occurring in 28 angiosperm families, heterostyly is characterized by the reciprocal position of male and female sexual organs in two (or three) distinct, usually self-incompatible floral morphs. Conversely, homostyly is characterized by a single, self-compatible floral morph with reduced separation of male and female organs, facilitating selfing. Here, we investigate the origins of homostyly in Primula vulgaris and its microevolutionary consequences by integrating surveys of the frequency of homostyles in natural populations, DNA sequence analyses of the gene controlling the position of female sexual organs (CYPᵀ), and microsatellite genotyping of both progeny arrays and natural populations characterized by varying frequencies of homostyles. As expected, we found that homostyles displace short-styled individuals, but long-style morphs are maintained at low frequencies within populations. We also demonstrated that homostyles repeatedly evolved from short-styled individuals in association with different types of loss-of-function mutations in CYPᵀ. Additionally, homostyly triggers a shift to selfing, promoting increased inbreeding within and genetic differentiation among populations. Our results elucidate the causes and consequences of repeated transitions to homostyly within species, and the putative mechanisms precluding its fixation in P. vulgaris. This study represents a benchmark for future analyses of losses of heterostyly in other angiosperms.