Heterostyly is a floral polymorphism controlled by an S-locus supergene in several angiosperm families. Most heterostylous species are self-incompatible. Here, we investigate the genomic architecture of distyly in self-compatible Cordia subcordata in which incompatibility has apparently been lost. We assembled chromosome-level genomes of floral morphs and conducted population genomic analyses to locate the S-locus region. We used transcriptomic analyses of floral organs and functional validation by gene overexpression to identify genes controlling floral dimorphism. The tempo and mode of origin of S-locus genes was also investigated to determine whether gene duplication facilitated supergene assembly. The candidate S-locus in C. subcordata contained 12 genes, eight of which were restricted to the S-morph. CsGA2ox6 deactivates gibberellins and was exclusively expressed in S-morph pistils. Overexpression of CsGA2ox6 in transgenic tobacco produced flowers with shortened styles and an apparently functioning self-incompatibility system. The genomic locations of paralogs and estimations of duplication age suggested that the S-locus genes may have arisen through stepwise duplications, although an origin via segmental duplication could not be excluded. Our study revealed molecular convergence with several other distylous families in hemizygous structure and possibly in the mode of supergene origins. We also identified a molecular pathway for style-length control, likely through gibberellin deactivation by CsGA2ox6, which may have also controlled the expression of self-incompatibility in transgenic plants.

This review summarizes the research contributions of Spencer C.H. Barrett during the past 50 years on the reproductive biology of plants. Topics covered include sexual polymorphisms, especially heterostyly and dioecy, the ecology and genetics of mating-system transitions, and the mechanisms influencing sex-ratio variation and sex chromosome evolution with examples from diverse angiosperm species. The article commences with a summary of the author’s early life and the influences that later shaped his research at the University of Toronto. Case studies are presented in chronological order and highlight the diversification of research methods, including field experiments, comparative approaches and genomic analyses, that have enabled efforts to address more general questions in evolutionary biology. Two interwoven themes are evident in the review: the importance of natural history observations as a basis for many of the research questions pursued; and the key role of collaborations with trainees and colleagues in Canada and overseas. The author also points out that unexpected discoveries and new research pathways often arise by chance and an open mind can be a key ingredient to a successful research career. The review concludes by identifying several under-studied areas in plant reproductive biology that would be worthwhile to pursue in the future.

The potential for conflict between sexes and life stages while sharing predominantly the same genome has important evolutionary consequences. In dioecious angiosperms, genes beneficial for the haploid pollen stage may reduce the fitness of diploid offspring of both males and females. However, we still lack an understanding of the extent of shared genetic architecture for gene expression between the sexes or life stages in plants, a key component for predicting the potential for conflict. We performed expression quantitative trait loci (eQTL) mapping to test if standing variation affects sexes and life stages differently using a population sample of the dioecious outcrossing plant Rumex hastatulus. We compared effect sizes and allele frequencies of cis-eQTLs in male and female leaf tissues and pollen and tested for genotype-by-sex interactions for gene expression. We found stronger shared genetic architecture between sexes than between life stages, suggesting greater potential for ongoing sexual conflict in leaves, which have been shown to be sexually dimorphic in earlier studies. In contrast, conflict over optimal gene expression between pollen and leaves may be easily resolved due to their distinct genetic architectures. Additionally, a burden of rare variant test suggested a signature of stabilizing selection against extreme gene expression in leaves. Our study highlights the use of eQTLs to investigate selection on gene expression and the evolution of conflict between sexes and life stages in dioecious species.

Heterostyly is a classic Darwinian adaptation promoting disassortative pollination. The polymorphism usually depends on floral tubes to enforce spatial segregation of pollen deposited on pollinators. How this is achieved in species with bowl-shaped flowers and generalised pollinators remains less well-understood. We investigated the mechanistic basis of Darwin's cross-promotion hypothesis in distylous Persicaria jucunda, which lacks a floral tube and is fly-pollinated. We integrated morphological measurements of flowers and flies with high-definition video analysis of foraging behaviour. Exploiting pollen-size dimorphism, we quantified morph-specific pollen placement on pollinator body parts and the composition of naturally pollinated stigmatic pollen loads. A 'morphological fit' was evident with fly proboscis length matching lower-level sex organs and upper-level organs matching the position of the head. This morphological congruence caused L-morph pollen to be deposited primarily on proboscides (76.6%) and S-morph pollen on heads (85.7%), resulting in a significantly higher proficiency of intermorph than intramorph pollen transfer. Despite the absence of a floral tube in P. jucunda, our findings of a morphological fit between flower and pollinator structures and consistent foraging behaviour by flies provide mechanistic support for Darwin's cross-promotion hypothesis and resolve the puzzle of how disassortative pollination occurs in open-flowered heterostylous systems.

The S-locus supergene controlling heterostyly has multiple angiosperm origins and is characterized by convergent evolution in form and function. The genetic architecture of floral polymorphism has been studied in several unrelated families, but not Rubiaceae, which has the largest number of heterostylous species. We assembled genomes of the long- and short-styled morphs of distylous Mussaenda lancipetala and investigated the structure and evolution of the S-locus in three Mussaenda species to evaluate evidence for convergence in genetic architecture and gene function in unrelated families. Genome assemblies and population genomics indicated that the S-locus in M. lancipetala is comprised of three hemizygous genes - MuIAA, MuGA3ox, and MuAPs - present only in the S-morph. In each species, MuIAA, involved in the auxin response pathway, was highly expressed in pistils and floral tubes of the S-morph, and is a likely candidate gene controlling style length. Molecular evolution analysis indicated that the S-haplotype has accumulated repetitive sequences, S-linked genes showed no evidence of relaxed purifying selection, and the evolutionary assembly of the S-locus involved stepwise duplication. Our findings provide support for the prevalence of hemizygosity in S-locus genes and have identified molecular pathways underpinning convergent morphological evolution of clear adaptive significance.

Tristyly is an angiosperm sexual polymorphism characterized by three flower morphs maintained in populations by negative frequency-dependent selection resulting from disassortative mating among morphs. The floral morphs possess reciprocal stigma and anther heights controlled by two epistatically interacting diallelic loci (S and M). Although considerable progress has been made on determining the genetic architecture and genes governing the related heterostylous polymorphism distyly, our understanding of these aspects of the genetic basis of tristyly has not been examined. Here, we address this knowledge gap by investigating the genomic basis of tristyly in Eichhornia paniculata (Pontederiaceae), an annual bee-pollinated herb native to the Neotropics, primarily N.E. Brazil. With chromosome-level genome assemblies of E. paniculata, we dentified the S- and M-loci on either side of a large region of low recombination on the same chromosome. The S-locus consisted of two divergent haplotypes: the S-haplotype (2.51 Mb) with three S-haplotype-specific genes and the s-haplotype (596 kb) with five s-haplotype-specific genes. Two of the S-haplotype-specific genes, LAZY1-S and HRGP-S, were specifically expressed in styles and stamens, respectively, making them candidate tristyly genes and providing evidence for this locus functioning as a hemizygous supergene. The M-locus contained one gene (LAZY1-M), homologous to LAZY1-S, present in the M-haplotype but absent from the m-haplotype. Estimates of gene ages and phylogenetic reconstruction were consistent with the theoretical prediction that the S-locus evolved before the M-locus. Evidence for reuse of the same gene highlights the potential role of gene duplication in the evolution of epistatic multilocus polymorphisms.

PREMISE:Flowering phenology strongly influences reproductive success in plants. Days to first flower is easy to quantify and widely used to characterize phenology, but reproductive fitness depends on the full schedule of flower production over time. We investigated flowering schedules in relation to the onset and duration of flowering and tested for latitudinal clines in schedule shape associated with rapid evolution and range expansion of an invasive plant. METHODS:We examined floral display traits among 13 populations of Lythrum salicaria, sampled along a 10-degree latitudinal gradient in eastern North America. We grew these collections in a common garden field experiment at a mid-latitude site and quantified variation in flowering schedule shape using principal coordinate analysis (PCoA) and quantitative metrics analogous to central moments of probability distributions (i.e., mean, variance, skew, and kurtosis). RESULTS:Consistent with earlier evidence for adaptation to shorter growing seasons, we found that populations from higher latitudes had earlier start and mean flowering day, on average, when compared to populations from southern latitudes. Flowering skew increased with latitude, whereas kurtosis decreased, consistent with a bet-hedging strategy in biotic environments with more herbivores and greater competition for pollinators. CONCLUSIONS:Heritable clines in flowering schedules are consistent with adaptive evolution in response to a predicted shift toward weaker biotic interactions and less variable but more stressful abiotic environments at higher latitudes, potentially contributing to rapid evolution and range expansion of this invasive species.

There is growing interest in the potential role of gene regulatory evolution in the expansion and degeneration of Y chromosomes. Evolutionarily young sex chromosomes, such as those found in the flowering plant Rumex hastatulus, can provide insights into the early stages of this process. Using a new high-quality genome assembly with a well-assembled Y chromosome and a highly replicated transcriptome dataset, we found widespread underexpression of Y genes compared to their homologous counterparts on the X chromosome. We also found evidence for increased expression differences for more distantly diverged gametologs, suggestive of progressive loss of expression of Y linked genes over time. Genes with greater expression loss on the Y chromosome also showed elevated rates of protein evolution, as expected if silencing alleles mask the effects of deleterious mutations on the Y and/or if selective interference drives early degeneration in regulatory sequences. However, in contrast with the predictions of recent models of sex chromosome expansion and degeneration by regulatory evolution, there was no evidence of early dosage compensation. Overall, we conclude that in Rumex Hill-Robertson interference alone may be the main driver of Y chromosome degeneration, although further understanding of the temporal order of regulatory changes could help further untangle cause and effect. ### Competing Interest Statement The authors have declared no competing interest.

Heterostylous plants are defined by the reciprocal positioning of stigmas and anthers in floral morphs—a trait proposed by Darwin to enhance the efficiency of disassortative (intermorph) pollen transfer. This floral polymorphism may also reduce gamete wastage by minimizing sexual interference between male and female reproductive organs. In distylous species, two floral morphs occur: a long‐styled morph with stigmas positioned above the anthers and a short‐styled morph with stigmas below the anthers. A related floral polymorphism, known as stigma‐height dimorphism, involves variation in stigma height but not anther placement. To test how floral architecture influences pollen transfer and reproductive interference, we used 3D‐printed artificial flowers based on Petunia grandiflora , incorporating real styles and anthers from glasshouse‐grown plants. These artificial flowers simulated distyly and two forms of stigma‐height dimorphism. In flight cage experiments, captive bumblebees ( Bombus impatiens ) from commercial colonies facilitated pollen transfer within and between flowers. We measured pollen grain deposition on stigmas and styles, as well as residual pollen in donor anthers. Our results provided partial support for Darwin's hypothesis: in distylous arrays, reciprocal sex‐organ placement enhanced intermorph pollen deposition, especially in the short‐styled morph. Bumblebee foraging time influenced pollen load, with longer visits to long‐styled flowers resulting in increased pollen deposition. Patterns of self‐pollen deposition—a form of reproductive interference—varied with the degree of spatial separation between sexual organs. As expected, stigma‐height dimorphic arrays exhibited higher self‐pollen transfer than distylous arrays. While not conclusive, our findings emphasize the role of floral morphology in shaping pollination dispersal, self‐interference and pollinator behaviour. The use of three‐dimensional printed flowers demonstrates a promising experimental approach for future studies on plant–pollinator interactions and the functional significance of floral design. Read the free Plain Language Summary for this article on the Journal blog.

The maintenance of genetic variation by balancing selection is of considerable interest to evolutionary biologists. An important but understudied potential driver of balancing selection is antagonistic pleiotropy between diploid and haploid stages of the plant life cycle. Despite sharing a common genome, sporophytes (2n) and gametophytes (n) may undergo differential or even opposing selection. Theoretical work suggests antagonistic pleiotropy between life stages can generate balancing selection and maintain genetic variation. Despite the potential for far-reaching consequences of gametophytic selection, empirical tests of its pleiotropic effects (neutral, synergistic, or antagonistic) on sporophytes are generally lacking. Here, we examined the population genomic signals of selection across life stages in the angiosperm Rumex hastatulus and the moss Ceratodon purpureus. We compared gene expression between life stages and sexes, combined with neutral diversity statistics and the analysis of the distribution of fitness effects. In contrast to what would be predicted under balancing selection due to antagonistic pleiotropy, we found that unbiased genes between life stages were under stronger purifying selection, likely explained by a predominance of synergistic pleiotropy between life stages and strong purifying selection on broadly expressed genes. In addition, we found that 30% of candidate genes under balancing selection in R. hastatulus were located within inversion polymorphisms. Our findings provide novel insights into the genome-wide characteristics and consequences of plant gametophytic selection.

Reciprocal herkogamy has evolved multiple times in flowering plants and is thought to enhance cross-pollination and reduce reproductive interference. Mirror-image flowers represent a form of reciprocal herkogamy in which plants have either right-or left-deflected styles (dimorphic enantiostyly) or produce both stylar orientations (monomorphic enantiostyly). The ecological conditions under which these two forms of enantiostyly originate and persist remain poorly understood. Here, we investigate how floral asymmetry affects pollen transfer dynamics and mating outcomes, and under which conditions enantiostyly provides a fitness advantage over straight-styled floral morphologies. We integrate field observations of dimorphic enantiostylous Wachendorfia paniculata with a mathematical model simulating pollen transfer in plants with straight-styled, monomorphic, and dimorphic enantiostylous flowers. The model incorporates pollinator pathways, pollen carryover, and floral display size, and was parameterized using our ecological data. Enantiostylous flowers received more outcrossed pollen than straight-styled flowers, with dimorphism outperforming monomorphism. However, enantiostyly increased stochasticity in pollen transfer due to variation in pollinator pathways. Intrafloral self-pollination was extremely low in enantiostylous flowers and did not differ between monomorphic and dimorphic forms. Dimorphic enantiostylous plants exhibited longer pollen carryover curves and exported more pollen to more mates potentially increasing siring success and mate diversity. Enantiostyly, particularly in dimorphic systems, can provide a selective advantage by improving pollen receipt and export components of fitness through enhanced outcrossing. This mating advantage may be reduced under conditions of pollen limitation or when autogamy in straight-styled flowers is low. Our findings clarify the functional significance of enantiostyly and offer a framework for understanding the evolution of floral asymmetry in angiosperms. ### Competing Interest Statement The authors have declared no competing interest. International Human Frontier Science Program Organization, RGP0036/2021

Chromosomal rearrangements are a major driver of evolutionary innovation, shaping processes including local adaptation, speciation, and sex chromosome evolution. Multispecies synteny datasets are rich in information on the drivers of genomic rearrangement, but statistical approaches that enable insights to be obtained from this information are still in their infancy. Here, we present a novel framework for the application of phylogenetic comparative methods to multispecies synteny datasets. We apply this approach to Rumex, a clade of flowering plants that exhibits rapid karyotypic evolution, including multiple origins of XY sex determination from hermaphroditic ancestors. Leveraging new genome assemblies, we find evidence for accelerated syntenic evolution associated with evolutionary transitions to dioecy, highlighting how explicit phylogenetic hypothesis testing can generate new insights into adaptive hypotheses for rearrangements. ### Competing Interest Statement The authors have declared no competing interest. Natural Sciences and Engineering Research Council, https://ror.org/01h531d29 University of Rochester, https://ror.org/022kthw22

In many flowering plants, male and female reproductive organs mature at different times to avoid self-pollination, a phenomenon termed dichogamy. Most dichogamous species are either protandrous or protogynous, making this strategy difficult to study genetically. However, in the ginger Alpinia mutica, protandrous and protogynous floral morphs co-occur within populations, and the synchronized rhythmic movement of styles and dehiscence of stamens promotes cross-pollination between morphs. Here we demonstrate that a single Mendelian locus with a dominant allele governing protogyny controls sexual polymorphism. We used haplotype-resolved genomes and population genomics to identify the dichogamy-determining region, revealing a large deletion in the protandrous morphotype. We found that the key gene SMPED1, located adjacent to the deletion, governs the timing of anther dehiscence and style movement. SMPED1 is widespread among angiosperms and probably has conserved function. Our findings represent a new genetic characterization of a key mating system gene controlling the synchrony of sex organs in flowering plants. This study reports that the gene SMPED1, located in a previously identified dichogamy-determining region in Alpinia species, controls the timing of sex-organ synchrony, improving our understanding of the evolutionary mechanisms of plant sexual diversity.

Heterostyly is a polymorphic floral adaptation controlled by supergenes. The molecular basis of distyly has been investigated in diploid species from several unrelated families, but information is lacking for polyploid systems. Here, we address this knowledge gap in Schizomussaenda henryi, a tetraploid distylous species of Rubiaceae, the family with the greatest number of heterostylous species. Using chromosome-level genome assemblies and transcriptome profiling, we characterized its tetraploid genome, identified the S-locus region, and performed evolutionary analysis. The S-locus contains four hemizygous genes in the S-morph. SchzAUX22 emerged as a candidate gene potentially regulating both style length and filament growth via auxin signaling. Phylogenetic and k-mer-based analysis suggested a hybrid allopolyploid origin for S. henryi, while no subgenome dominance was detected. Results from the comparison of Ks values indicated that S-locus formation likely occurred through stepwise duplications. This study provides the first comprehensive genomic analysis of distyly in a polyploid species and demonstrates that the S-locus remains intact despite allopolyploidization resulting from hybridization. Our results indicated that polyploidization does not necessitate the breakdown of distyly, which occurs in several other heterostylous lineages.

Water serves diverse functions in plant growth and reproduction. Here, we report a novel strategy in the terrestrial karst plant Hemiboea magnibracteata analogous to amniotic fluid in mammals. Developing flower buds enclosed in 'womb-like' bracts are completely immersed in fluids and protected against excessive temperature and desiccation. The reservoir of water also likely limits drought and calcium stress in the highly specialized abiotic conditions of karst habitats.

The temporal sequence and mechanistic interplay between life-history and mating-system transitions in plants remain poorly understood. Here, we combine crossing experiments with population genomic analyses to investigate these processes in the Incarvillea sinensis complex (Bignoniaceae), which contains annual selfing and perennial outcrossing populations. Crossing experiments revealed complete post-zygotic isolation between annuals and perennials. Genomic data derived from de novo assemblies of annual and perennial individuals, along with SNP and chloroplast sequencing of 126 individuals from 30 populations, demonstrated strong genetic divergence between life histories, with no evidence of contemporary gene flow between them. Compared to perennials, annuals exhibited significantly reduced genetic diversity, elevated differentiation and a greater number of chromosomal rearrangements—particularly translocations. Divergence time estimates indicated that annual and perennial lineages split during the early Pleistocene (ca. 2.22 Mya), whereas the shift to selfing from outcrossing within the annual lineage was a more recent event, occurring during the late Pleistocene (~0.03 Mya). These results indicate that the mating-system shift was not the initial driver of divergence, and we infer that the life-history transition likely occurred earlier, although its precise timing could not be directly determined. Our findings support a two-stage model of divergence in which adaptation to seasonally arid environments first drove the evolution of annuality, initiating speciation through ecological isolation and genetic divergence. The subsequent evolution of selfing then likely further promoted reproductive isolation via both pre- and post-zygotic mechanisms, potentially accelerating rapid genomic differentiation and effectively completing the speciation process.

Many animals and plants show left-right (LR) asymmetry. The LR asymmetry of mirror-image flowers has clear functional significance, with the reciprocal placement of male and female organs in left- versus right-handed flowers promoting cross-pollination. Here, we study how handedness of mirror-image flowers is determined and elaborated during development in the South African geophyte Cyanella alba. Inflorescences of C. alba produce flowers with a largely consistent handedness. However, this handedness has no simple genetic basis and individual plants can switch their predominant handedness between years. Rather, it is the direction of the phyllotactic spiral that predicts floral handedness. Style deflection is driven by increased cell expansion in the adaxial carpel facing the next oldest flower compared to the other adaxial carpel. The more expanding carpel shows transcriptional signatures of increased auxin signaling and auxin application can reverse the orientation of style deflection. We propose that a recently described inherent LR auxin asymmetry in the initiating organs of spiral phyllotaxis determines handedness in C. alba, creating a stable yet non-genetic floral polymorphism. This mechanism links chirality across different levels of plant development and exploits a developmental constraint in a core patterning process to produce morphological variation of ecological relevance.

Heteranthery, the occurrence of functionally and structurally distinct stamens within a flower, represents a striking example of convergent evolution among diverse animal-pollinated lineages. Although the ecological basis of this somatic polymorphism is understood, the developmental and molecular mechanisms are largely unknown. To address this knowledge gap, we selected Monochoria elata (Pontederiaceae) as our study system due to its typical heterantherous floral structure. We constructed a chromosome-level genome assembly of M. elata, conducted transcriptomic analyses and target phytohormone metabolome analysis to explore gene networks and hormones associated with heteranthery. We focused on three key stamen characteristics—colour, spatial patterning, and filament elongation—selected for their significant roles in stamen differentiation and their relevance to the functional diversity observed in heterantherous species. Our analyses suggest that gene networks involving MelLEAFY3, MADS-box, and TCP genes regulate stamen identity, with anthocyanin influencing colour, and lignin contributing to filament elongation. Additionally, variation in jasmonic acid and abscisic acid concentration between feeding and pollinating anthers appears to contribute to their morphological divergence. Our findings highlight gene networks and hormones associated with intra-floral stamen differentiation and indicate that whole genome duplications have likely facilitated the evolution of heteranthery during divergence from other Pontederiaceae without heteranthery.