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Although self-incompatibility in apples (Malus × domestica Borkh.) is regulated by a single S-locus with multiple S-haplotypes that comprise pistil S (S-RNase) and pollen S genes, it is not desirable in commercial orchards because it requires cross-pollination to achieve stable fruit production. Therefore, it is important to identify and characterize self-compatible apple cultivars. However, little is known about self-compatibility (SC) and its underlying molecular mechanisms in apples. In this study, we discovered that 'Vered', an early maturing and low chilling-requiring apple cultivar, exhibits stable SC, which was evaluated via self-pollination tests. The S-genotype of 'Vered' was designated as S 24 S 39 sm . Results of genetic analysis of selfed progeny of 'Vered' revealed that SC is associated with the S 39 sm -haplotype, and molecular analyses indicated that it is caused by alternative splicing and a 205-bp deletion in S 39 sm -RNase. These events induce frameshifts and ultimately produce the defective S 39 sm -RNase isoforms that lack their C-terminal half. These results enabled us to develop a 117-bp DNA marker that can be used to assist in the selection of self-compatible apples with the dysfunctional S 39 sm -RNase. Thus, analysis of 'Vered' provided insights into the molecular mechanism of the very rare trait of natural stylar-part SC. Moreover, 'Vered' is a valuable genetic resource for breeding cultivars with SC and/or low chilling requirement in apple. Our findings contribute to a better understanding of self-compatible molecular mechanisms in apple and provide for the accelerated breeding of self-compatible apple cultivars., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Shifts in pollinator occurrence and their pollen transport effectiveness drive the evolution of mating systems in flowering plants. Understanding the genomic basis of these changes is essential for predicting the persistence of a species under environmental changes. We investigated the genomic changes in Brassica rapa over nine generations of pollination by hoverflies associated with rapid morphological evolution toward the selfing syndrome. We combined a genotyping-by-sequencing (GBS) approach with a genome-wide association study (GWAS) to identify candidate genes, and assessed their functional role in the observed morphological changes by studying mutations of orthologous genes in the model plant Arabidopsis thaliana. We found 31 candidate genes involved in a wide range of functions from DNA/RNA binding to transport. Our functional assessment of orthologous genes in A. thaliana revealed that two of the identified genes in B. rapa are involved in regulating the size of floral organs. We found a protein kinase superfamily protein involved in petal width, an important trait in plant attractiveness to pollinators. Moreover, we found a histone lysine methyltransferase (HKMT) associated with stamen length. Altogether, our study shows that hoverfly pollination leads to rapid evolution toward the selfing syndrome mediated by polygenic changes., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Pollinator-driven evolution of floral traits is thought to be a major driver of angiosperm speciation and diversification. Ophrys orchids mimic female insects to lure male pollinators into pseudocopulation. This strategy, called sexual deception, is species-specific, thereby providing strong premating reproductive isolation. Identifying the genomic architecture underlying pollinator adaptation and speciation may shed light on the mechanisms of angiosperm diversification. Here, we report the 5.2 Gb chromosome-scale genome sequence of Ophrys sphegodes. We find evidence for transposable element expansion that preceded the radiation of the O. sphegodes group, and for gene duplication having contributed to the evolution of chemical mimicry. We report a highly differentiated genomic candidate region for pollinator-mediated evolution on chromosome 2. The Ophrys genome will prove useful for investigations into the repeated evolution of sexual deception, pollinator adaptation and the genomic architectures that facilitate evolutionary radiations., (© 2024. The Author(s).)

Effective dispersal among plant populations is dependent on vector behaviour, landscape features and availability of adequate habitats. To capture landscape feature effects on dispersal, studies must be conducted at scales reflecting single-generation dispersal events (mesoscale). Many studies are conducted at large scales where genetic differentiation is due to dispersal occurring over multiple generations, making it difficult to interpret the effects of specific landscape features on vector behaviour. Genetic structure at the mesoscale may be determined by ecological and evolutionary processes, such as the consequences of vector behaviour on patterns of gene flow. We used chloroplast haplotypes and nuclear genome SNP surveys to identify landscape features influencing seed and pollen dispersal at a mesoscale within the Rogue River Valley in southern Oregon. We evaluated biotic and abiotic vector behaviour by contrasting two annual species with differing dispersal mechanisms; Achyrachaena mollis (Asteraceae) is a self-pollinating and anemochoric species, and Plectritis congesta (Caprifoliaceae) is biotically pollinated with barochoric seeds. Using landscape genetics methods, we identified features of the study region that conduct or restrict dispersal. We found chloroplast haplotypes were indicative of historic patterns of gene flow prior to human modification of landscapes. Seed dispersal of A. mollis was best supported by models of isolation by distance, while seed-driven gene flow of P. congesta was determined by the distribution of preserved natural spaces and quality habitat. Nuclear genetic structure was driven by both pollen and seed dispersal, and both species responded to contemporary landscape changes, such as urban and agricultural conversion, and habitat availability., (© 2024 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Understanding how spatial patterns of mating and gene flow respond to habitat loss and geographical isolation is a crucial aspect of forest fragmentation genetics. Naturally fragmented riparian tree populations exhibit unique characteristics that significantly influence these patterns. In this study, we investigate mating patterns, pollen-mediated gene flow, and genetic diversity in relict populations of Frangula alnus in southern Spain by testing specific hypotheses related to the riparian habitat. We employ a novel approach that combines paternity analysis, particularly suited for small and isolated populations, with complex network theory and Bayesian models to predict mating likelihood among tree pairs. Our findings reveal a prevalence of short-distance pollination, resulting in spatially driven local mating clusters with a distinct subset of trees being highly connected in the mating network. Additionally, we observe numerous pollination events over distances of hundreds of metres and considerable pollen immigration. Local neighbourhood density is the primary factor influencing within-population mating patterns and pollen dispersal; moreover, mating network properties reflect the population's size and spatial configuration. Conversely, among-population pollen dispersal is mainly determined by tree size, which influences floral display. Our results do not support a major role of directional pollen dispersal in longitudinal trends of genetic diversity. We provide evidence that long-term fragmented tree populations persist in unique environments that shape mating patterns and impose constraints to pollen-mediated gene flow. Nevertheless, even seemingly strongly isolated populations can maintain functional connectivity over extended periods, especially when animal-mediated mating networks promote genetic diversity, as in this riparian tree species., (© 2024 John Wiley & Sons Ltd.)

Genome duplication (generating polyploids) is an engine of novelty in eukaryotic evolution and a promising crop improvement tool. Yet newly formed polyploids often have low fertility. Here we report that a severe fertility-compromising defect in pollen tube tip growth arises in new polyploids of Arabidopsis arenosa . Pollen tubes of newly polyploid A. arenosa grow slowly, have aberrant anatomy and disrupted physiology, often burst prematurely, and have altered gene expression. These phenotypes recover in evolved polyploids. We also show that gametophytic (pollen tube) genotypes of two tip-growth genes under selection in natural tetraploid A. arenosa are strongly associated with pollen tube performance in the tetraploid. Our work establishes pollen tube tip growth as an important fertility challenge for neo-polyploid plants and provides insights into a naturally evolved multigenic solution.

Since the insights by Charles Darwin, heterostyly, a floral polymorphism with morphs bearing stigmas and anthers at reciprocal heights, has become a model system for the study of natural selection. Based on his archetypal heterostylous flower, including regular symmetry, few stamens and a tube, Darwin hypothesised that heterostyly evolved to promote outcrossing through efficient pollen transfer between morphs involving different areas of a pollinator's body, thus proposing his seminal pollination-precision hypothesis. Here we update the number of heterostylous and other style-length polymorphic taxa to 247 genera belonging to 34 families, notably expanding known cases by 20%. Using phylogenetic and comparative analyses across the angiosperms, we show numerous independent origins of style-length polymorphism associated with actinomorphic, tubular flowers with a low number of sex organs, stamens fused to the corolla, and pollination by long-tongued insects. These associations provide support for the Darwinian pollination-precision hypothesis as a basis for convergent evolution of heterostyly across angiosperms., (© 2024. The Author(s).)

Polyphenol oxidases (PPOs) are type-3 copper enzymes and are involved in many biological processes. However, the potential functions of PPOs in pollination are not fully understood. In this work, we have screened 13 PPO members in Nicotiana. tabacum (named NtPPO1-13, NtPPOs) to explore their characteristics and functions in pollination. The results show that NtPPOs are closely related to PPOs in Solanaceae and share conserved domains except NtPPO4. Generally, NtPPOs are diversely expressed in different tissues and are distributed in pistil and male gametes. Specifically, NtPPO9 and NtPPO10 are highly expressed in the pistil and mature anther. In addition, the expression levels and enzyme activities of NtPPOs are increased after N. tabacum self-pollination. Knockdown of NtPPOs would affect pollen growth after pollination, and the purines and flavonoid compounds are accumulated in self-pollinated pistil. Altogether, our findings demonstrate that NtPPOs potentially play a role in the pollen tube growth after pollination through purines and flavonoid compounds, and will provide new insights into the role of PPOs in plant reproduction., (© 2024. The Author(s).)

The evolutionary switch to hummingbird pollination exemplifies complex adaptation, requiring evolutionary change in multiple component traits. Despite this complexity, diverse lineages have converged on hummingbird-adapted flowers on a relatively short evolutionary timescale. Here, I review how features of the genetic basis of adaptation contribute to this remarkable evolutionary lability. Large-effect substitutions, large mutational targets for adaptation, adaptive introgression, and concentrated architecture all contribute to the origin and maintenance of hummingbird-adapted flowers. The genetic features of adaptation are likely shaped by the ecological and geographic context of the switch to hummingbird pollination, with implications for future evolutionary trajectories., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Although flavonoids play multiple roles in plant growth and development, the involvement in plant self-incompatibility (SI) have not been reported. In this research, the fertility of transgenic tobacco plants overexpressing the Ginkgo biloba dihydroflavonol 4-reductase gene, GbDFR6 , were investigated. To explore the possible physiological defects leading to the failure of embryo development in transgenic tobacco plants, functions of pistils and pollen grains were examined. Transgenic pistils pollinated with pollen grains from another tobacco plants (either transgenic or wild-type), developed full of well-developed seeds. In contrast, in self-pollinated transgenic tobacco plants, pollen-tube growth was arrested in the upper part of the style, and small abnormal seeds developed without fertilization. Although the mechanism remains unclear, our research may provide a valuable method to create SI tobacco plants for breeding.

Anthropogenic activities are triggering global changes in the environment, causing entire communities of plants, pollinators and their interactions to restructure, and ultimately leading to species declines. To understand the mechanisms behind community shifts and declines, as well as monitoring and managing impacts, a global effort must be made to characterize plant-pollinator communities in detail, across different habitat types, latitudes, elevations, and levels and types of disturbances. Generating data of this scale will only be feasible with rapid, high-throughput methods. Pollen DNA metabarcoding provides advantages in throughput, efficiency and taxonomic resolution over traditional methods, such as microscopic pollen identification and visual observation of plant-pollinator interactions. This makes it ideal for understanding complex ecological networks and their responses to change. Pollen DNA metabarcoding is currently being applied to assess plant-pollinator interactions, survey ecosystem change and model the spatiotemporal distribution of allergenic pollen. Where samples are available from past collections, pollen DNA metabarcoding has been used to compare contemporary and past ecosystems. New avenues of research are possible with the expansion of pollen DNA metabarcoding to intraspecific identification, analysis of DNA in ancient pollen samples, and increased use of museum and herbarium specimens. Ongoing developments in sequencing technologies can accelerate progress towards these goals. Global ecological change is happening rapidly, and we anticipate that high-throughput methods such as pollen DNA metabarcoding are critical for understanding the evolutionary and ecological processes that support biodiversity, and predicting and responding to the impacts of change., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Alpine plant-pollinator communities play an important role in the functioning of alpine ecosystems, which are highly threatened by climate change. However, we still have a poor understanding of how environmental factors and spatiotemporal variability shape these communities. Here, we investigate what drives structure and beta diversity in a plant-pollinator metacommunity from the Australian alpine region using two approaches: pollen DNA metabarcoding (MB) and observations. Individual pollinators often carry pollen from multiple plant species, and therefore we expected MB to reveal a more diverse and complex network structure. We used two gene regions (ITS2 and trnL) to identify plant species present in the pollen loads of 154 insect pollinator specimens from three alpine habitats and construct MB networks, and compared them to networks based on observations alone. We compared species and interaction turnover across space for both types of networks, and evaluated their differences for plant phylogenetic diversity and beta diversity. We found significant structural differences between the two types of networks; notably, MB networks were much less specialized but more diverse than observation networks, with MB detecting many cryptic plant species. Both approaches revealed that alpine pollination networks are very generalized, but we estimated a high spatial turnover of plant species (0.79) and interaction rewiring (0.6) as well as high plant phylogenetic diversity (0.68) driven by habitat differences based on the larger diversity of plant species and species interactions detected with MB. Overall, our findings show that habitat and microclimatic heterogeneity drives diversity and fine-scale spatial turnover of alpine plant-pollinator networks., (© 2022 John Wiley & Sons Ltd.)

'Shuijingmiyou' pummelo (SJ), one of the most popular fruits in Yunnan province of China, is of relatively low fruit shape (FS) quality. In this study, we compared the FS promoting effects of cross pollinations using pollens from seven pummelo varieties, and found that 'Guanximiyou' pummelo (GX) cross-pollination showed the best FS promoting effects on SJ fruits by shortening its fruit neck. To explore the underlying mechanism, physiochemical and transcriptomic differences between self- and cross-pollinated SJ ovaries (SJO and GXO) were investigated. Higher salicylic acid, gibberellin and indole acetic acid contents and superoxide dismutase, peroxidase and catalase activities, and lower polyphenol oxidase activity were determined in GXO compared with SJO. Enrichment analysis of the identified 578 differentially expressed genes (123 up-regulated and 455 down-regulated) in GXO showed that genes involved in solute transport, RNA biosynthesis, phytohormone action and cell wall organization were significantly enriched. The results obtained in this study will be helpful in understanding the influences of cross-pollination on pummelo ovary and fruit development, and can provide the basis for clarifying the underlying mechanism of cross-pollination improved fruit quality., (© 2023. The Author(s).)

Epiphytic microbes frequently affect plant phenotype and fitness, but their effects depend on microbe abundance and community composition. Filtering by plant traits and deterministic dispersal-mediated processes can affect microbiome assembly, yet their relative contribution to predictable variation in microbiome is poorly understood. We compared the effects of host-plant filtering and dispersal on nectar microbiome presence, abundance, and composition. We inoculated representative bacteria and yeast into 30 plants across four phenotypically distinct cultivars of Epilobium canum. We compared the growth of inoculated communities to openly visited flowers from a subset of the same plants. There was clear evidence of host selection when we inoculated flowers with synthetic communities. However, plants with the highest microbial densities when inoculated did not have the highest microbial densities when openly visited. Instead, plants predictably varied in the presence of bacteria, which was correlated with pollen receipt and floral traits, suggesting a role for deterministic dispersal. These findings suggest that host filtering could drive plant microbiome assembly in tissues where species pools are large and dispersal is high. However, deterministic differences in microbial dispersal to hosts may be equally or more important when microbes rely on an animal vector, dispersal is low, or arrival order is important., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Insect pollination is fundamental for natural ecosystems and agricultural crops. The bumblebee species Bombus terrestris has become a popular choice for commercial crop pollination worldwide due to its effectiveness and ease of mass rearing. Bumblebee colonies are mass produced for the pollination of more than 20 crops and imported into over 50 countries including countries outside their native ranges, and the risk of invasion by commercial non-native bumblebees is considered an emerging issue for global conservation and biological diversity. Here, we use genome-wide data from seven wild populations close to and far from farms using commercial colonies, as well as commercial populations, to investigate the implications of utilizing commercial bumblebee subspecies in the UK. We find evidence for generally low levels of introgression between commercial and wild bees, with higher admixture proportions in the bees occurring close to farms. We identify genomic regions putatively involved in local and global adaptation, and genes in locally adaptive regions were found to be enriched for functions related to taste receptor activity, oxidoreductase activity, fatty acid and lipid biosynthetic processes. Despite more than 30 years of bumblebee colony importation into the UK, we observe low impact on the genetic integrity of local B. terrestris populations, but we highlight that even limited introgression might negatively affect locally adapted populations., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Persimmon fruit has a high nutritional value and significantly varies between pollination-constant astringent (PCA) and pollination-constant non-astringent (PCNA) persimmons. The astringency type affects sugar, flavonoids, and tannin accumulation and is well known in persimmon fruit. However, the impact of the fruit astringency type on ascorbic acid (AsA) accumulation is limited. In this study, typical PCA varieties ('Huojing' and 'Zhongshi5') and PCNA varieties ('Yohou' and 'Jiro') of persimmon fruit were sampled at four developing stages (S1-S4) to provide valuable information on AsA content variation in PCA and PCNA persimmon. Persimmon fruit is rich in ascorbic acid; the AsA content of the four varieties 'Zhongshi5', 'Huojing', 'Jiro', and 'Youhou' mature fruit reached 104.49, 48.69, 69.69, and 47.48 mg/100 g. Fruit of the same astringency type persimmon showed a similar AsA accumulation pattern. AsA content was significantly higher in PCA than PCNA fruit at S1-S3. The initial KEGG analysis of metabolites showed that galactose metabolism is the major biosynthetic pathway of AsA in persimmon fruit. There were significant differences in galactose pathway-related metabolite content in developing PCA and PCNA fruit, such as Lactose, D-Tagatose, and D-Sorbitol content in PCA being higher than that of PCNA. Combined gene expression and WGCNA analyses showed that the expression of the GME ( evm.TU.contig4144.37 ) gene was higher in PCA-type than in PCNA-type fruit in S1-S3 and exhibited the highest correlation with AsA content (r = 690 **, p < 0.01). Four hub genes, including the DNA methylation gene , methyltransferase gene , F-box , and Actin-like Protein , were identified as potential regulators of the GME gene. These results provide basic information on how astringency types affect AsA accumulation and will provide valuable information for further investigation on AsA content variation in persimmon fruit.

In the formation of species, adaptation by natural selection generates distinct combinations of traits that function well together. The maintenance of adaptive trait combinations in the face of gene flow depends on the strength and nature of selection acting on the underlying genetic loci. Floral pollination syndromes exemplify the evolution of trait combinations adaptive for particular pollinators. The North American wildflower genus Penstemon displays remarkable floral syndrome convergence, with at least 20 separate lineages that have evolved from ancestral bee pollination syndrome (wide blue-purple flowers that present a landing platform for bees and small amounts of nectar) to hummingbird pollination syndrome (bright red narrowly tubular flowers offering copious nectar). Related taxa that differ in floral syndrome offer an attractive opportunity to examine the genomic basis of complex trait divergence. In this study, we characterized genomic divergence among 229 individuals from a Penstemon species complex that includes both bee and hummingbird floral syndromes. Field plants are easily classified into species based on phenotypic differences and hybrids displaying intermediate floral syndromes are rare. Despite unambiguous phenotypic differences, genome-wide differentiation between species is minimal. Hummingbird-adapted populations are more genetically similar to nearby bee-adapted populations than to geographically distant hummingbird-adapted populations, in terms of genome-wide dXY. However, a small number of genetic loci are strongly differentiated between species. These approximately 20 "species-diagnostic loci," which appear to have nearly fixed differences between pollination syndromes, are sprinkled throughout the genome in high recombination regions. Several map closely to previously established floral trait quantitative trait loci (QTLs). The striking difference between the diagnostic loci and the genome as whole suggests strong selection to maintain distinct combinations of traits, but with sufficient gene flow to homogenize the genomic background. A surprisingly small number of alleles confer phenotypic differences that form the basis of species identity in this species complex., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wessinger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Mating system is one of the major determinants of intra- and interspecific genetic structure, but may vary within and between plant populations. Our study model included all known populations of Moehringia tommasinii (Caryophyllaceae), a narrow endemic plant inhabiting rock crevices in the northwestern Adriatic, and some populations of co-occurring and widespread M. muscosa, an ecologically divergent relative with an overlapping flowering period. We performed reciprocal crosses within and between taxa and used molecular markers to assess the extent of gene flow within and between populations and taxa. Using coefficient of inbreeding, population size, seed weight, pollen-to-ovule ratio, and flower display size, we also looked for evidence of a selfing syndrome., Results: A surprisingly high variation in mating systems was observed among populations of M. tommasinii. These populations exhibited genetic structuring, with their size positively correlated with both seed weight and pollen production. Although a selfing syndrome could not be confirmed as the majority of selfing resulted from allogamous treatments, the occurrence of selfing was notable. In the presence of M. muscosa, at a site where both species coexist closely, a distinct pattern of fruit production was observed in M. tommasinii following various pollination treatments. Molecular and morphometric data provided evidence of hybridization followed by local extinction at this site., Conclusions: Population size proved to be the most important factor affecting the mating system in genetically structured populations of M. tommasinii. Lighter seeds and lower pollen production observed in populations with pronounced selfing do not provide enough evidence for the selfing syndrome. Detected gene flow between M. tommasinii and the sympatric M. muscosa suggested weak reproductive barriers between the taxa, which could pose a conservation problems for the former species. Hybridization leading to local extinction may also resulted in floral polymorphism and disruption of mating patterns of M. tommasinii., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Self-incompatible pitaya varieties have low fruit-setting rates under natural conditions, leading to higher production costs and hindering industrial prosperity. Through transcriptome sequencing, we obtained the 36,900 longest transcripts (including 9167 new transcripts) from 60 samples of flowers. Samples were collected pre- and post-pollination (at 0 h, 0.5 h, 2 h, 4 h, and 12 h) from two varieties of pitaya (self-compatible Jindu No. 1 and self-incompatible Cu Sha). Using the RNA-Seq data and comparison of reference genomes, we annotated 28,817 genes in various databases, and 1740 genes were optimized in their structure for annotation. There were significant differences in the expression of differentially expressed genes (DEGs) in the pitaya stigmas under different pollination types, especially at the late post-pollination stage, where the expression of protease genes increasedal significantly under cross-pollination. We identified DEGs involved in the ribosomal, ubiquitination-mediated, and phyto-signaling pathways that may be involved in pitaya SI regulation. Based on the available transcriptome data and bioinformatics analysis, we tentatively identified HuS-RNase2 as a candidate gynogenetic S gene in the pitaya GSI system.

Studies investigating the evolution of flowering plants have long focused on isolating mechanisms such as pollinator specificity. Some recent studies have proposed a role for introgressive hybridization between species, recognizing that isolating processes such as pollinator specialization may not be complete barriers to hybridization. Occasional hybridization may therefore lead to distinct yet reproductively connected lineages. We investigate the balance between introgression and reproductive isolation in a diverse clade using a densely sampled phylogenomic study of fig trees ( Ficus , Moraceae). Codiversification with specialized pollinating wasps (Agaonidae) is recognized as a major engine of fig diversity, leading to about 850 species. Nevertheless, some studies have focused on the importance of hybridization in Ficus , highlighting the consequences of pollinator sharing. Here, we employ dense taxon sampling (520 species) throughout Moraceae and 1,751 loci to investigate phylogenetic relationships and the prevalence of introgression among species throughout the history of Ficus . We present a well-resolved phylogenomic backbone for Ficus , providing a solid foundation for an updated classification. Our results paint a picture of phylogenetically stable evolution within lineages punctuated by occasional local introgression events likely mediated by local pollinator sharing, illustrated by clear cases of cytoplasmic introgression that have been nearly drowned out of the nuclear genome through subsequent lineage fidelity. The phylogenetic history of figs thus highlights that while hybridization is an important process in plant evolution, the mere ability of species to hybridize locally does not necessarily translate into ongoing introgression between distant lineages, particularly in the presence of obligate plant-pollinator relationships.

Persimmon ( Diospyros kaki ) fruit have significant variation between pollination-constant non-astringent (PCNA) and pollination-constant astringent (PCA) persimmons. The astringency type affects not only the soluble tannin concentration but also the accumulation of individual sugars. Thus, we comprehensively investigate the gene expression and metabolite profiles of individual sugars to resolve the formation of flavor differences in PCNA and PCA persimmon fruit. The results showed that soluble sugar, starch content, sucrose synthase, and sucrose invertase were significantly different between PCNA and PCA persimmon fruit. The sucrose and starch metabolism pathway was considerably enriched, and six sugar metabolites involving this pathway were significantly differentially accumulated. In addition, the expression patterns of diferentially expressed genes (such as bglX , eglC , Cel , TPS , SUS , and TREH genes) were significantly correlated with the content of deferentially accumulated metabolites (such as starch, sucrose, and trehalose) in the sucrose and starch metabolism pathway. These results indicated that the sucrose and starch metabolism pathway maintained a central position of sugar metabolism between PCNA and PCA persimmon fruit. Our results provide a theoretical basis for exploring functional genes related to sugar metabolism and provide useful resources for future studies on the flavor differences between PCNA and PCA persimmon fruit.

Since the release of genetically engineered (GE) crops, there has been increased concern about the introduction of GE genes into non-GE fields of a crop and their spread to feral or wild cross-compatible relatives. More recently, attention has been given to the differential impact of distinct pollinators on gene flow, with the goal of developing isolation distances associated with specific managed pollinators. To examine the differential impact of bee species on gene movement, we quantified the relationship between the probability of getting a GE seed in a pod, and the order in which a flower was visited, or the cumulative distance traveled by a bee in a foraging bout. We refer to these relationships as 'seed curves' and compare these seeds curves among three bee species. The experiments used Medicago sativa L. plants carrying three copies of the glyphosate resistance (GR) allele as pollen donors (M. sativa is a tetraploid), such that each pollen grain carried the GR allele, and conventional plants as pollen recipients. Different foraging metrics, including the number of GR seeds produced over a foraging bout, were also quantified and contrasted among bee species. The lowest number of GR seeds set per foraging bout, and the GR seeds set at the shortest distances, were produced following leafcutting bee visits. In contrast, GR seeds were found at the longest distances following bumble bee visits. Values for honey bees were intermediate. The ranking of bee species based on seed curves correlated well with field-based gene flow estimates. Thus, differential seed curves of bee species, which describe patterns of seed production within foraging bouts, translated into distinct abilities of bee species to move genes at a landscape level. Bee behavior at a local scale (foraging bout) helps predict gene flow and the spread of GE genes at the landscape scale., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: The flowering biology of wheat plants favours self-pollination which causes obstacles in wheat hybrid breeding. Wheat flowers can be divided into two groups, the first one is characterized by flowering and pollination within closed flowers (cleistogamy), while the second one possesses the ability to open flowers during processes mentioned above (chasmogamy). The swelling of lodicules is involved in the flowering of cereals and among others their morphology, calcium and potassium content differentiate between cleistogamic and non-cleistogamous flowers. A better understanding of the chasmogamy mechanism can lead to the development of tools for selection of plants with the desired outcrossing rate. To learn more, the sequencing of transcriptomes (RNA-Seq) and Representational Difference Analysis products (RDA-Seq) were performed to investigate the global transcriptomes of wheat lodicules in two highly chasmogamous (HCH, Piko and Poezja) and two low chasmogamous (LCH, Euforia and KWS Dacanto) varieties at two developmental stages-pre-flowering and early flowering., Results: The differentially expressed genes were enriched in five, main pathways: "metabolism", "organismal systems", "genetic information processing", "cellular processes" and "environmental information processing", respectively. Important genes with opposite patterns of regulation between the HCH and LCH lines have been associated with the lodicule development i.e. expression levels of MADS16 and MADS58 genes may be responsible for quantitative differences in chasmogamy level in wheat., Conclusions: We conclude that the results provide a new insight into lodicules involvement in the wheat flowering process. This study generated important genomic information to support the exploitation of the chasmogamy in wheat hybrid breeding programs., (© 2023. The Author(s).)

The evolutionary transition from self-incompatible distyly to self-compatible homostyly frequently occurs in heterostylous taxa. Although the inheritance of distyly and homostyly has been deeply studied, our understanding on modifications of the classical simple Mendelian model is still lacking. Primula forbesii, a biennial herb native to southwest China, is a typical distylous species, but after about 20 years of cultivation with open pollination, self-compatible homostyly appeared, providing ideal material for the study of the inheritance of distyly and homostyly. In this study, exogenous homobrassinolide was used to break the heteromorphic incompatibility of P. forbesii. Furthermore, we performed artificial pollination and open-pollination experiments to observe the distribution of floral morphs in progeny produced by different crosses. The viability of seeds from self-pollination was always the lowest among all crosses, and the homozygous S-morph plants (S/S) occurred in artificial pollination experiments but may experience viability selection. The distyly of P. forbesii is governed by a single S-locus, with S-morph dominant hemizygotes (S/-) and L-morph recessive homozygotes (-/-). Homostylous plants have a genotype similar to L-morph plants, and homostyly may be caused by one or more unlinked modifier genes outside the S-locus. Open pollinations confirm that autonomous self-pollination occurs frequently in L-morphs and homostylous plants. This study deepens the understanding of the inheritance of distyly and details a case of homostyly that likely originated from one or more modifier genes., (© 2023. The Author(s), under exclusive licence to The Genetics Society.)

Background: Theory suggests that the genetic architecture of traits under divergent natural selection influences how easily reproductive barriers evolve and are maintained between species. Divergently selected traits with a simple genetic architecture (few loci with major phenotypic effects) should facilitate the establishment and maintenance of reproductive isolation between species that are still connected by some gene flow. While empirical support for this idea appears to be mixed, most studies test the influence of trait architectures on reproductive isolation only indirectly. Petunia plant species are, in part, reproductively isolated by their different pollinators. To investigate the genetic causes and consequences of this ecological isolation, we deciphered the genetic architecture of three floral pollination syndrome traits in naturally occurring hybrids between the widespread Petunia axillaris and the highly endemic and endangered P. exserta., Results: Using population genetics, Bayesian linear mixed modelling and genome-wide association studies, we found that the three pollination syndrome traits vary in genetic architecture. Few genome regions explain a majority of the variation in flavonol content (defining UV floral colour) and strongly predict the trait value in hybrids irrespective of interspecific admixture in the rest of their genomes. In contrast, variation in pistil exsertion and anthocyanin content (defining visible floral colour) is controlled by many genome-wide loci. Opposite to flavonol content, the genome-wide proportion of admixture between the two species predicts trait values in their hybrids. Finally, the genome regions strongly associated with the traits do not show extreme divergence between individuals representing the two species, suggesting that divergent selection on these genome regions is relatively weak within their contact zones., Conclusions: Among the traits analysed, those with a more complex genetic architecture are best maintained in association with the species upon their secondary contact. We propose that this maintained genotype-phenotype association is a coincidental consequence of the complex genetic architectures of these traits: some of their many underlying small-effect loci are likely to be coincidentally linked with the actual barrier loci keeping these species partially isolated upon secondary contact. Hence, the genetic architecture of a trait seems to matter for the outcome of hybridization not only then when the trait itself is under selection., (© 2023. The Author(s).)

In a recent issue of Nature, Huang et al. identify and show how to overcome the barriers to successful pollen germination after interspecific crosses. 1 Their findings answer a long-standing question about reproductive barriers in flowering plants and open the door to harnessing genetic diversity of distant relatives for crop improvement., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Saururus chinensis, an herbaceous magnoliid without perianth, represents a clade of early-diverging angiosperms that have gone through woodiness-herbaceousness transition and pollination obstacles: the characteristic white leaves underneath inflorescence during flowering time are considered a substitute for perianth to attract insect pollinators. Here, using the newly sequenced S. chinensis genome, we revisited the phylogenetic position of magnoliids within mesangiosperms, and recovered a sister relationship for magnoliids and Chloranthales. By considering differentially expressed genes, we identified candidate genes that are involved in the morphogenesis of the white leaves in S. chinensis. Among those genes, we verified - in a transgenic experiment with Arabidopsis - that increasing the expression of the "pseudo-etiolation in light" gene (ScPEL) can inhibit the biosynthesis of chlorophyll. ScPEL is thus likely responsible for the switches between green and white leaves, suggesting that changes in gene expression may underlie the evolution of pollination strategies. Despite being an herbaceous plant, S. chinensis still has vascular cambium and maintains the potential for secondary growth as a woody plant, because the necessary machinery, i.e., the entire gene set involved in lignin biosynthesis, is well preserved. However, similar expression levels of two key genes (CCR and CAD) between the stem and other tissues in the lignin biosynthesis pathway are possibly associated with the herbaceous nature of S. chinensis. In conclusion, the S. chinensis genome provides valuable insights into the adaptive evolution of pollination in Saururaceae and reveals a possible mechanism for the evolution of herbaceousness in magnoliids., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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.

Floral syndromes are known by the conserved morphological traits in flowers associated with pollinator attraction, such as corolla shape and color, aroma emission and composition, and rewards, especially the nectar volume and sugar concentration. Here, we employed a phylogenetic approach to investigate sequences of genes enrolled in the biosynthetic pathways responsible for some phenotypes that are attractive to pollinators in Solanaceae genomes. We included genes involved in visible color, UV-light response, scent emission, and nectar production to test the hypothesis that these essential genes have evolved by convergence under pollinator selection. Our results refuted this hypothesis as all four studied genes recovered the species' phylogenetic relationships, even though some sites were positively selected. We found differences in protein motifs among genera in Solanaceae that were not necessarily associated with the same floral syndrome. Although it has had a crucial role in plant diversification, the plant-pollinator interaction is complex and still needs further investigation, with genes evolving not only under the influence of pollinators, but by the sum of several evolutionary forces along the speciation process in Solanaceae.

Because corn pollen can be carried great distances by wind, maintaining genetic purity of corn grain is challenging. The challenge is substantially reduced in popcorn, which carries the Ga1-s allele preventing pollination by ga1 plants, which include the vast majority of non-popcorn commercial maize varieties in the U.S.. Ga1-s can be transferred into dent corn but the effectiveness of the Ga1-s allele in popcorn and dent corn has never been compared, which is important because each are regulated differently regarding GMO contamination. We compared pollen exclusion of commercial popcorn hybrids, Ga1-s dent corn hybrids and normal dent corn hybrids for their ability to exclude ga1 pollen using a sensitive field-based assay. While both popcorn and Ga1-s dent corn had significantly better pollen exclusion than normal dent corn, popcorn was significantly better than Ga1-s dent corn on average. Some Ga1-s dent hybrids excluded as well or better than some popcorn lines suggesting that identification of hybrids comparable to popcorn is possible. The information in this study will support revised gene purity regulations potentially decreasing costs and increasing genetic purity of organic corn., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Plants have evolved various strategies to avoid inbreeding, but the mass flowering displayed by many plants predisposes them to within-plant pollen movements and self-pollination. Mistletoes often aggregate at multiple spatial scales. Their bird pollinators often visit several flowers of the same individual and of others on the same host tree. We hypothesized that hermaphroditic mistletoes have self-incompatibility mechanisms that reduce or prevent selfing. Whether their spatial distribution, affected by host specificity, host distribution, and the behaviour of seed dispersers, influences their mating system and population genetic structure remains unclear. We studied how mating system and spatial distribution affect genetic structure in four populations of the host-generalist mistletoe Dendrophthoe pentandra in southwestern China using microsatellite markers and progeny arrays. We also characterized the fine-scale spatial genetic structure among 166 mistletoes from four host trees in one population. Prevalence and intensity of infection both appeared to vary among host species, strongly affecting the degree of aggregation. Host tree size had a strong effect on infection intensity. Surprisingly, manual pollination experiments indicated that D. pentandra is self-compatible, but genetic analyses revealed that outcrossing rates were higher than expected in all four populations (MLTR t m 0.83-1.20, Bayesian t m 0.772-0.952). Spatial genetic structure was associated with distance between host trees but not at shorter scales (within hosts). Our results demonstrate that the combination of bird pollination, bird-mediated seed dispersal, and post-dispersal processes result in outcrossing and maintain relatively high diversity in the presence of biparental inbreeding, despite very high local densities and possible self-compatibility., (© 2022 John Wiley & Sons Ltd.)

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., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Self-incompatibility (SI) is a major issue in dragon fruit ( Selenicereus spp. ) breeding and production. Therefore, a better understanding of the dragon fruit SI mechanism is needed to improve breeding efficiency and ultimate production costs. To reveal the underlying mechanisms of SI in dragon fruit, plant anatomy, de novo RNA sequencing-based transcriptomic analysis, and multiple bioinformatic approaches were used to analyze gene expression in the pistils of the self-pollinated and cross-pollinated dragon fruit flowers at different intervals of time after pollination. Using fluorescence microscopy, we observed that the pollen of 'Hongshuijing', a self-incompatible dragon fruit variety ( S. monacanthus ), germinated on its own stigma. However, the pollen tube elongation has ceased at 1/2 of the style, confirming that dragon fruit experiences gametophyte self-incompatibility (GSI). We found that the pollen tube elongation in vitro was inhibited by self-style glycoproteins in the SI variety, indicating that glycoproteins were involved in SI. That is to say the female S factor should be homologous of S-RNase or PrsS ( P. rhoeas stigma S factor), both of which are glycoproteins and are the female S factors of the two known GSI mechanism respectively. Bioinformatics analyses indicated that among the 43,954 assembled unigenes from pistil, there were six S-RNase genes, while 158 F-box genes were identified from a pollen transcriptomic dataset. There were no P. rhoeas type S genes discovered. Thus, the identified S-RNase and F-box represent the candidate female and male S genes, respectively. Analysis of differentially expressed genes (DEGs) between the self and cross-pollinated pistils at different time intervals led to the identification of 6,353 genes. We then used a weighted gene co-expression network analysis (WGCNA) to find some non-S locus genes in SI responses in dragon fruit. Additionally, 13 transcription factors (TFs) ( YABBY4, ANL2, ERF43, ARF2, BLH7, KNAT6, PIF3 , two OBF1 , two HY5 and two LHY/CCA ) were identified to be involved in dragon fruit GSI. Thus, we uncovered candidate S and non-S genes and predicted more SI-related genes for a more detailed investigation of the molecular mechanism of dragon fruit SI. Our findings suggest that dragon fruit possesses a GSI system and involves some unique regulators. This study lays the groundwork for future research into SI mechanisms in dragon fruit and other plant species., Competing Interests: The authors declare there are no competing interests., (©2022 Li et al.)

In Brassicaceae, the papillary cells of the stigma are the primary site of the self-incompatibility (SI) responses. SI preserves the genetic diversity by selectively rejecting irrelevant or incompatible pollen, thus promoting cross fertilization and species fitness. Mechanisms that regulate SI responses in Brassica have been studied mainly on the mature stigma that often undermines how stigma papillary cells attain the state of SI during development. To understand this, we integrated PacBio SMRT-seq with Illumina RNA-seq to construct a de novo full-length transcriptomic database for different stages of stigma development in ornamental kale. A total of 48,800 non-redundant transcripts, 31,269 novel transcripts, 24,015 genes, 13,390 alternative splicing, 22,389 simple sequence repeats, 21,816 complete ORF sequences, and 4591 lncRNAs were identified and analyzed using PacBio SMRT-seq. The Illumina RNA-seq revealed 15,712 differentially expressed genes (DEGs) and 8619 transcription factors. The KEGG enrichment analysis of 4038 DEGs in the "incompatibility" group revealed that the flavonoid and fatty acid biosynthesis pathways were significantly enriched. The cluster and qRT-PCR analysis indicated that 11 and 14 candidate genes for the flavonoid and fatty acid biosynthesis pathways have the lowest expression levels at stigma maturation, respectively. To understand the physiological relevance of the downregulation of fatty acid biosynthesis pathways, we performed inhibitor feeding assays on the mature stigma. The compatible pollination response was drastically reduced when mature stigmas were pre-treated with a fatty acid synthase inhibitor. This finding suggested that fatty acid accumulation in the stigmas may be essential for compatible pollination and its downregulation during maturity must have evolved as a support module to discourage the mounting of self-incompatible pollen.

Deceptive pollination often involves volatile organic compound emissions that mislead insects into performing nonrewarding pollination. Among deceptively pollinated plants, Arum maculatum is particularly well-known for its potent dung-like volatile organic compound emissions and specialized floral chamber, which traps pollinators-mainly Psychoda phalaenoides and Psychoda grisescens-overnight. However, little is known about the genes underlying the production of many Arum maculatum volatile organic compounds, and their influence on variation in pollinator attraction rates. Therefore, we performed de novo transcriptome sequencing of Arum maculatum appendix and male floret tissue collected during anthesis and postanthesis, from 10 natural populations across Europe. These RNA-seq data were paired with gas chromatography-mass spectrometry analyses of floral scent composition and pollinator data collected from the same inflorescences. Differential expression analyses revealed candidate transcripts in appendix tissue linked to malodourous volatile organic compounds including indole, p-cresol, and 2-heptanone. In addition, we found that terpene synthase expression in male floret tissue during anthesis significantly covaried with sex- and species-specific attraction of Psychoda phalaenoides and Psychoda grisescens. Taken together, our results provide the first insights into molecular mechanisms underlying pollinator attraction patterns in Arum maculatum and highlight floral chamber sesquiterpene (e.g. bicyclogermacrene) synthases as interesting candidate genes for further study., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Floral divergence can contribute to reproductive isolation among plant lineages, and thus provides an opportunity to study the genetics of speciation, including the number, effect size, mode of action and interactions of quantitative trait loci (QTL). Moreover, flowers represent suites of functionally interrelated traits, but it is unclear to what extent the phenotypic integration of the flower is underlain by a shared genetic architecture, which could facilitate or constrain correlated evolution of floral traits. Here, we examine the genetic architecture of floral morphological traits involved in an evolutionary switch from bill to forehead pollen placement between two species of hummingbird-pollinated Neotropical understorey herbs that are reproductively isolated by these floral differences. For the majority of traits, we find multiple QTL of relatively small effect spread throughout the genome. We also find substantial colocalization and alignment of effects of QTL underlying different floral traits that function together to promote outcrossing and reduce heterospecific pollen transfer. Our results are consistent with adaptive pleiotropy or linkage of many co-adapted genes, either of which could have facilitated a response to correlated selection and helped to stabilize divergent phenotypes in the face of low levels of hybridization. Moreover, our results indicate that floral mechanical isolation can be consistent with an infinitesimal model of adaptation., (© 2021 John Wiley & Sons Ltd.)

Interactions with pollinators are a potent source of natural selection driving the spectacular array of flowering plant diversity on Earth (Kay & Sargent, 2009; Van der Niet et al., 2014). Floral traits play a central role in this process: reliable and effective pollination by animal pollinators depends on complex floral features, including traits that determine pollinator attraction and reward, as well as the mechanics of pollen transfer. Pollinators specify mating events between individuals, and thus differences in flowers have the potential to generate reproductive isolating barriers (floral isolation). A compelling case of floral isolation comes from spiral gingers (Costus), where hummingbird-adapted species have evolved distinct pollen placement strategies (on the bills vs. foreheads of pollinators) due to differences in flower shape and the arrangements of flower parts. This difference in pollen placement causes a mechanical barrier to cross-pollination. In this issue of Molecular Ecology, Kay and Surget-Groba (2022) dissect the genetic basis of these floral differences using a quantitative trait locus (QTL) mapping approach. They find small-effect QTLs that influence multiple correlated traits and allelic effects that suggest a history of directional selection. Their results indicate mechanical isolation reflects adaptive divergence that has built up piecemeal over time., (© 2022 John Wiley & Sons Ltd.)

Floral adaptations to specific pollinators like corolla shape variation often result in reproductive isolation and thus speciation. But despite their ecological importance, the genetic bases of corolla shape transitions are still poorly understood, especially outside model species. Hence, our goal was to identify candidate genes potentially involved in corolla shape variation between two closely related species of the Rhytidophyllum genus (Gesneriaceae family) from the Antilles with contrasting pollination strategies. Rhytidophyllum rupincola has a tubular corolla and is strictly pollinated by hummingbirds, whereas R. auriculatum has more open flowers and is pollinated by hummingbirds, bats, and insects. We surveyed the literature and used a comparative transcriptome sequence analysis of synonymous and non-synonymous nucleotide substitutions to obtain a list of genes that could explain floral variation between R. auriculatum and R. rupincola. We then tested their association with corolla shape variation using QTL mapping in a F2 hybrid population. Out of 28 genes tested, three were found to be good candidates because of a strong association with corolla shape: RADIALIS, GLOBOSA, and JAGGED. Although the role of these genes in Rhytidophyllum corolla shape variation remains to be confirmed, these findings are a first step towards identifying the genes that have been under selection by pollinators and thus involved in reproductive isolation and speciation in this genus., Competing Interests: The authors have declared that no competing interests exist.

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'.

Background: Keel flowers are bilaterally symmetrical, pentamerous flowers with three different petal types and reproductive organs enclosed by keel petals; generally there is also connation of floral parts such as stamens and keel petals. In this study, the evolution of keel flowers within the order Fabales is explored to investigate whether the establishment of this flower type within one of the species-rich families, the Fabaceae (Leguminosae), preceded and could have influenced the evolution of keel flowers in the Polygalaceae. We conducted molecular dating, and ancestral area and ancestral state analyses for a phylogeny constructed for 678 taxa using published matK, rbcL and trnL plastid gene regions., Results: We reveal the temporal and spatial origins of keel flowers and traits associated with pollinators, specifically floral symmetry, the presence or absence of a pentamerous corolla and three distinct petal types, the presence or absence of enclosed reproductive organs, androecium types, inflorescence types, inflorescence size, flower size, plant height and habit. Ancestral area reconstructions show that at the time keel flowers appeared in the Polygaleae, subfamily Papilionoideae of the Fabaceae was already distributed almost globally; at least eight clades of the Papilionoideae had keel flowers with a functional morphology broadly similar to the morphology of the first evolving Polygaleae flowers., Conclusions: The multiple origins of keel flowers within angiosperms likely represent convergence due to bee specialization, and therefore pollinator pressure. In the case of the Fabales, the first evolving keel flowers of Polygaleae have a functional morphology that corresponds with keel flowers of species of the Papilionoideae already present in the environment. These findings are consistent with the keel-flowered Polygaleae exploiting pollinators of keel-flowered Papilionoideae. The current study is the first to use ancestral reconstructions of traits associated with pollination to demonstrate that the multiple evolutionary origins of the keel flower pollinator syndrome in Fabales are consistent with, though do not prove, mimicry., (© 2022. The Author(s).)

The specificity of pollinator host choice influences opportunities for reproductive isolation in their host plants. Similarly, host plants can influence opportunities for reproductive isolation in their pollinators. For example, in the fig and fig wasp mutualism, offspring of fig pollinator wasps mate inside the inflorescence that the mothers pollinate. Although often host specific, multiple fig pollinator species are sometimes associated with the same fig species, potentially enabling hybridization between wasp species. Here, we study the 19 pollinator species (Pegoscapus spp.) associated with an entire community of 16 Panamanian strangler fig species (Ficus subgenus Urostigma, section Americanae) to determine whether the previously documented history of pollinator host switching and current host sharing predicts genetic admixture among the pollinator species, as has been observed in their host figs. Specifically, we use genome-wide ultraconserved element (UCE) loci to estimate phylogenetic relationships and test for hybridization and introgression among the pollinator species. In all cases, we recover well-delimited pollinator species that contain high interspecific divergence. Even among pairs of pollinator species that currently reproduce within syconia of shared host fig species, we found no evidence of hybridization or introgression. This is in contrast to their host figs, where hybridization and introgression have been detected within this community, and more generally, within figs worldwide. Consistent with general patterns recovered among other obligate pollination mutualisms (e.g. yucca moths and yuccas), our results suggest that while hybridization and introgression are processes operating within the host plants, these processes are relatively unimportant within their associated insect pollinators., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Animal pollinators mediate gene flow among plant populations, but in contrast to well-studied topographic and (Pleistocene) environmental isolating barriers, their impact on population genetic differentiation remains largely unexplored. Comparing how these multifarious factors drive microevolutionary histories is, however, crucial for better resolving macroevolutionary patterns of plant diversification. Here we combined genomic analyses with landscape genetics and niche modelling across six related Neotropical plant species (424 individuals across 33 localities) differing in pollination strategy to test the hypothesis that highly mobile (vertebrate) pollinators more effectively link isolated localities than less mobile (bee) pollinators. We found consistently higher genetic differentiation (F ST ) among localities of bee- than vertebrate-pollinated species with increasing geographical distance, topographic barriers and historical climatic instability. High admixture among montane populations further suggested relative climatic stability of Neotropical montane forests during the Pleistocene. Overall, our results indicate that pollinators may differentially impact the potential for allopatric speciation, thereby critically influencing diversification histories at macroevolutionary scales., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Successful reproduction in the Brassicaceae is mediated by a complex series of interactions between the pollen and the pistil, and some species have an additional layer of regulation with the self-incompatibility trait. While the initial activation of the self-incompatibility pathway by the pollen S-locus protein 11/S locus cysteine-rich protein and the stigma S Receptor Kinase is well characterized, the downstream mechanisms causing self-pollen rejection are still not fully understood. In previous studies, we detected the presence of autophagic bodies with self-incompatible (SI) pollinations in Arabidopsis lyrata and transgenic Arabidopsis thaliana lines, but whether autophagy was essential for self-pollen rejection was unknown. Here, we investigated the requirement of autophagy in this response by crossing mutations in the essential AUTOPHAGY7 (ATG7) and ATG5 genes into two different transgenic SI A. thaliana lines in the Col-0 and C24 accessions. By using these previously characterized transgenic lines that express A. lyrata and Arabidopsis halleri self-incompatibility genes, we demonstrated that disrupting autophagy weakened their SI responses in the stigma. When the atg7 or atg5 mutations were present, an increased number of SI pollen was found to hydrate and form pollen tubes that successfully fertilized the SI pistils. Additionally, we confirmed the presence of GFP-ATG8a-labeled autophagosomes in the stigmatic papillae following SI pollinations. Together, these findings support the requirement of autophagy in the self-incompatibility response and add to the growing understanding of the intracellular mechanisms employed in the transgenic A. thaliana stigmas to reject self-pollen., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Yam (Dioscorea spp.) is a staple crop for millions of people in the tropics and subtropics. Its genetic improvement through breeding is being challenged by pre-zygotic and post-zygotic cross-compatibility barriers within and among species. Studies dissecting hybridization barriers on yam for improving the crossability rates are limited. This study aimed to assess the cross-compatibility, which yielded fruit set, viable seeds and progeny plants in an extensive intraspecific and interspecific crossing combinations in a yam genetic improvement effort to understand the internal and exogenous factors influencing pollination success. Cross-compatability was analyzed at the individual genotype or family level using historical data from crossing blocks and seedling nurseries from 2010 to 2020 at the International Institute of Tropical Agriculture (IITA). The average crossability rate (ACR) was lower in interspecific crossing combinations (6.1%) than intraspecific ones (27.6%). The seed production efficiency (SPE) values were 1.1 and 9.3% for interspecific and intraspecific crosses, respectively. Weather conditions and pollinator's skills are the main contributors to the low success rate in the intraspecific cross combinations in yam breeding. At the same time, genetic distance and heterozygosity played little role. Interspecific cross barriers were both pre-zygotic and post-zygotic, resulting from the evolutionary divergence among the yam species. Dioscorea rotundata had higher interspecific cross-compatibility indices than D. alata. Distant parents produced intraspecific crossbred seeds with higher germination rates compared to closest parents (r = 0.21, p = 0.033). This work provided important insights into interspecific and intraspecific cross-compatibility in yam and suggested actions for improving hybridization practices in yam breeding programs., (© 2022. The Author(s).)

Plants living at the edge of their range boundary tend to suffer an overall decline in their fitness, including growth and reproduction. However, the reproductive performance of plants in artificially expanded habitats is rarely investigated, although this type of study would provide a better understanding of range limitations and improved conservation of ex situ plants. In the current study, we transplanted a narrowly dispersed species of Gentiana officinalis H. Smith (Gentianaceae) from its natural area of distribution to two different elevations and natural elevation to comprehensively study its pollination biology, including flowering phenology and duration, floral display, reproductive allocation, pollinator activity, and seed production. The findings indicated that the starting point and endpoint of the flowering phenology of G. officinalis were earlier at the low elevation, but the peak flowering periods did not differ significantly between any of the experimental plots. When transplanted, the flowering duration, especially the female phase, was reduced; the floral display, including spray numbers, flower numbers, and flower size (length and width), decreased, especially at high elevations. Moreover, the pollen numbers and pollen-ovule ratio were decreased at both high and low elevations, although the ovule numbers showed no change, and aboveground reproductive allocation was decreased. Furthermore, pollinator richness and activity were significantly decreased, and the seed-set ratio decreased under both natural conditions and with supplemental pollination. Finally, more severe pollen limitation was found in transplanted individuals. These results indicated an overall decrease in reproductive fitness in plants living outside their original area of distribution when the geographical range of G. officinalis was expanded., (© 2022. The Author(s).)

Sex form is one of the most important characteristics in papaya cultivation in which hermaphrodite is the preferable form. Self-pollination of H*-TSS No.7, an inbred line derived from a rare X chromosome mutant SR*, produced all-hermaphrodite progeny. The recessive lethal allele controlling the all-hermaphrodite phenomenon was proposed to be the recessive Germination suppressor (gs) locus. This study employed next-generation sequencing technology and genome comparison to identify the candidate Gs gene. One specific gene, monodehydroascorbate reductase 4 (MDAR4) harboring a unique polymorphic 3 bp deletion in H*-TSS No.7 was identified. The function of MDAR4 is known to be involved in the hydrogen peroxide (H 2 O 2 ) scavenging pathway and is associated with seed germination. Furthermore, MDAR4 showed higher expression in the imbibed seeds than that in the dry seeds indicating its potential role in the seed germination. Perhaps this is the very first report providing the evidences that MDAR4 is the candidate of Gs locus in H*-TSS No.7. In addition, Gs allele-specific markers were developed which would be facilitated for breeding all-hermaphrodite lines., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The degree of stigma exsertion has a major influence on self-pollination efficiency in tomato, and its improvement is essential for raising productivity and for fixing advantageous traits in cultivated tomato. To study the evolution of stigma exsertion degree in tomato, we searched for genes associated with this trait and other aspects of flower morphology, including the lengths of anthers, styles, and ovaries. We performed a genome-wide association on 277 tomato accessions and discovered a novel stigma exsertion gene (SE3.1). We reannotated the structure of the gene, which encodes a C2H2-type zinc finger transcription factor. A mutation of the lead single nucleotide polymorphism creates a premature termination codon in SE3.1 and an inserted stigma in cultivated tomatoes. SE3.1 is essential for the conversion of flush stigmas to inserted stigmas. This conversion has a major impact on the rate of self-fertilization. Intriguingly, we found that both SE3.1 and Style2.1 contribute to the transition from stigma exsertion to insertion during the domestication and improvement of tomato. Style2.1 controls the first step of exserted stigmas to flush stigmas, and SE3.1 controls the second step of flush stigmas to inserted stigmas. We provide molecular details for the two-step process that controls the transition from stigma exsertion to insertion, which is of great agronomic importance in tomato., (� American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Many pollinator populations are experiencing declines, emphasizing the need for a better understanding of the complex relationship between bees and flowering plants. Using DNA metabarcoding to describe plant-pollinator interactions eliminates many challenges associated with traditional methods and has the potential to reveal a more comprehensive understanding of foraging behaviour and pollinator life history. Here we use DNA metabarcoding of ITS2 and rbcL gene regions to identify plant species present in pollen loads of 404 bees from three habitats in eastern Oregon. Our specific objectives were to (i) determine whether plant species identified using DNA metabarcoding are consistent with plant species identified using observations, (ii) compare characterizations of diet breadth derived from foraging observations to those based on plant species assignments obtained using DNA metabarcoding, and (iii) compare plant species assignments produced by DNA metabarcoding using a "regional" reference database to those produced using a "local" database. At the three locations, 31%-86% of foraging observations were consistent with DNA metabarcoding data, 8%-50% of diet breadth characterizations based on observations differed from those based on DNA metabarcoding data, and 22%-25% of plant species detected using the regional database were not known to occur in the study area in question. Plant-pollinator networks produced from DNA metabarcoding data had higher sampling completeness and significantly lower specialization than networks based on observations. Here, we examine some strengths and limitations of using DNA metabarcoding to identify plant species present in bee pollen loads, make ecological inferences about foraging behaviour and provide guidance for future research., (© 2021 John Wiley & Sons Ltd.)

Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S -locus containing just three genes. We report that the S -locus gene, BAHD , is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii &nbsp; BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen.