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PREMISE The evolution of sex chromosomes is driven by sexual dimorphism, yet it can be challenging to document sexually dimorphic traits in dioecious plant species. At the genetic level, sexual dimorphism can be identified through sequence variation between females and males associated with sexually antagonistic traits and different fitness optima. This study aims to examine sexual dimorphism for 26 traits in three populations of Salix purpurea (a diversity panel and F1 and F2 populations) and determine the effect of the traits on biomass yield, a key trait in Salix bioenergy crops across multiple years, locations, and under manipulated growth conditions. METHODS Sexual dimorphism was evaluated for morphological, phenological, physiological, and wood composition traits in a diversity panel of unrelated S. purpurea accessions and in full-sib F1 and F2 families produced through controlled cross pollinations and grown in replicated field trials. RESULTS We observed sexual dimorphism in the timing of development for several traits that were highly predictive of biomass yield across three populations of S. purpurea. Across all populations and years surveyed, males had significantly shallower branching angle. Male plants highly predictive of biomass yield across three populations of S. purpurea also accumulated more nitrogen under fertilizer amendment as measured by SPAD in the diversity panel and had greater susceptibility to the rust fungus Melampsora americana in the F2 family. Allometric modelling of biomass yield showed an effect of sex and of location on the interaction between yield and stem height. CONCLUSIONS These results provide evidence of sexual dimorphism for certain traits in S. purpurea that may be involved in sex chromosome evolution.

Yam (Dioscorea spp.) anthracnose, caused by Colletotrichum alatae, is the most devastating fungal disease of yam in West Africa, leading to 50%–90% of tuber yield losses in severe cases. In some instances, plants die without producing any tubers or each shoot may produce several small tubers before it dies if the disease strikes early. C. alatae affects all parts of the yam plant at all stages of development, including leaves, stems, tubers, and seeds of yams, and it is highly prevalent in the yam belt region and other yam‐producing countries in the world. Traditional methods adopted by farmers to control the disease have not been very successful. Fungicides have also failed to provide long‐lasting control. Although conventional breeding and genomics‐assisted breeding have been used to develop some level of resistance to anthracnose in Dioscorea alata, the appearance of new and more virulent strains makes the development of improved varieties with broad‐spectrum and durable resistance critical. These shortcomings, coupled with interspecific incompatibility, dioecy, polyploidy, poor flowering, and the long breeding cycle of the crop, have prompted researchers to explore biotechnological techniques to complement conventional breeding to speed up crop improvement. Modern biotechnological tools have the potential of producing fungus‐resistant cultivars, thereby bypassing the natural bottlenecks of traditional breeding. This article reviews the existing biotechnological strategies and proposes several approaches that could be adopted to develop anthracnose‐resistant yam varieties for improved food security in West Africa., Resistance to yam anthracnose, the most devastating fungal disease of yam (Dioscorea spp.), can be produced by genomics‐assisted breeding, high‐throughput phenotyping, genetic engineering, and CRISPR/Cas9 genome editing.

Dioecy, the presence of male and female individual, has evolved independently from hermaphroditism in multiple flowering plant lineages. Spinach, an important leafy vegetable crop worldwide, is a dioecious species with an XY sex-determination system. Although some markers that are fully linked to male-determination locus were identified, the male-specific region (MSR) remained unclear. In this research, five male individuals and five female lines were resequenced to identify the male-specific region. We identified a region (∼21 kb) on chromosome 4 (putative sex chromosome) where the five females had a low reads coverage, while the five males had high coverage. A KASP marker, SponR, developed from a single nucleotide polymorphism (SNP) closely linked to the MSR was co-segregating with the sex determination gene in the population of 958 individuals, suggesting that the MSR might be specifically present in male spinach plants.

Background and Aims Dioecy has evolved up to 5000 times in angiosperms, despite the potentially high intrinsic costs to unisexuality. Dioecy prevents inbreeding, which is especially relevant on isolated islands when gene pools are small. Dioecy is also associated with certain dispersal traits, such as fruit size and type. However, the influence of dioecy on other life history traits and island distribution remains poorly understood. Here, we test the effect of dioecy on palm (Arecaceae) speciation rates, fruit size and frequency on islands. Methods We used phylogenetic comparative methods to estimate the ancestral state of the sexual system and its impact on speciation rates and fruit size. Frequency of sexual systems, effect of insularity on the probability of being dioecious, and phylogenetic clustering of island dioecious vs. mainland species were inferred. Lastly, we determined the interplay of insularity and sexual system on speciation rates. Key Results Palms repeatedly evolved different sexual systems (dioecy, monoecy and polygamy) from a hermaphrodite origin. Differences in speciation rates and fruit size among the different sexual systems were not identified. An effect of islands on the probability of the palms being dioecious was also not found. However, we found a high frequency and phylogenetic clustering of dioecious palms on islands, which were not correlated with higher speciation rates. Conclusions The high frequency and phylogenetic clustering may be the result of in situ radiation and suggest an ‘island effect’ for dioecious palms, which was not explained by differential speciation rates. This island effect also cannot be attributed to long-distance dispersal due to the lack of fruit size difference among sexual systems, and particularly because palm dispersal to islands is highly constrained by the interaction between the sizes of fruit and frugivores. Taken together, we suggest that trait flexibility in sexual system evolution and the in situ radiation of dioecious lineages are the underlying causes of the outstanding distribution of palms on islands.

Salix nigra (black willow) is a widespread tree that hosts many species of polylectic hymenopterans and oligolectic bees of the genus Andrena. The early flowering of S. nigra makes it an important nutritive resource for arthropods emerging from hibernation. However, since S. nigra is dioecious, not all insect visits will lead to successful pollination. Using both visual observation and pan‐trapping, we characterized the community of arthropods that visited S. nigra flowers and assessed differences among male and female trees as well as the chemical and visual drivers that influenced community composition across 3 years. We found that male trees consistently supported higher diversity of insects than female trees and only three insect species, all Andrena spp., consistently visited both sexes. Additionally, Andrena nigrae, which was the only insect that occurred more on female than male flowers, correlated strongly to volatile cues. This suggests that cross‐pollinators cue into specific aspects of floral scent, but diversity of floral visitors is driven strongly by visual cues of yellow male pollen. Through time, the floral activity of two Andrena species remained stable, but A. nigrae visited less in 2017 when flowers bloomed earlier than other years. When native bee emergence does not synchronize with bloom, activity appears to be diminished which could threaten species that subsist on a single host. Despite the community diversity of S. nigra flowers, its productivity depends on a small fraction of species that are not threatened by competition, but rather rapidly changing conditions that lead to host‐insect asynchrony., The widespread, dioecious tree Salix nigra (black willow) hosts many species of arthropods and bees of the genus Andrena on early emerging, nutritive floral resources. Despite the diversity of arthropods that interact with S. nigra flowers, successful pollination is dependent on a small fraction of Andrena native bees that are driven by both visual and volatile cues. Activity of the native bee most active on female flowers, Andrena nigrae, was decoupled from host flowers in 2017 due to an early bloom, demonstrating how rapidly changing climate conditions could threaten plant and pollinator population success in the future.

Fuchsia parviflora is a dioecious shrub that depends on biotic pollination for reproduction. Previous studies suggest that the male plants produce more flowers, and male-biased sex ratios have been found in some natural populations. To assess whether the biased sex ratios found between genders in natural populations are present at the point at which plants reach sexual maturity, and to identify possible trade-offs between growth and reproduction, we performed a common garden experiment. Finally, to complement the information of the common garden experiment, we estimated the reproductive biomass allocation between genders in one natural population. Sex ratios at reaching sexual maturity in F. parviflora did not differ from 0.5, except in one population, which was the smallest seedling population. We found no differences between genders in terms of the probability of germination or flowering. When flowering began, female plants were taller than males and the tallest plants of both genders required more time to reach sexual maturity. Males produced significantly more flowers than females, and the number of flowers increased with plant height in both genders. Finally, in the natural population studied, the investment in reproductive biomass was seven-fold greater in female plants than in male plants. Our results showed no evidence of possible trade-offs between growth and reproduction. Despite the fact that female plants invest more in reproductive biomass, they were taller than the males after flowering, possibly at the expense of herbivory defence.

PREMISE We tested 25 classic and novel hypotheses regarding trait-origin, trait-trait, and trait-environment relationships to account for flora-wide variation in life history, habit, and especially reproductive traits using a plastid DNA phylogeny of most native (96.6%, or 1494/1547 species) and introduced (87.5%, or 690/789 species) angiosperms in Wisconsin, USA. METHODS We assembled data on life history, habit, flowering, dispersal, mating system, and occurrence across open/closed/mixed habitats across species in the state phylogeny. We used phylogenetically structured analyses to assess the strength and statistical significance of associations predicted by our models. RESULTS Introduced species are more likely to be annual herbs, occupy open habitats, have large, visually conspicuous, hermaphroditic flowers, and bear passively dispersed seeds. Among native species, hermaphroditism is associated with larger, more conspicuous flowers; monoecy is associated with small, inconspicuous flowers and passive seed dispersal; and dioecy is associated with small, inconspicuous flowers and fleshy fruits. Larger flowers with more conspicuous colors are more common in open habitats, and in understory species flowering under open (spring) canopies; fleshy fruits are more common in closed habitats. Wind pollination may help favor dioecy in open habitats. CONCLUSIONS These findings support predictions regarding how breeding systems depend on flower size, flower color, and fruit type, and how those traits depend on habitat. This study is the first to combine flora-wide phylogenies with complete trait databases and phylogenetically structured analyses to provide powerful tests of evolutionary hypotheses about reproductive traits and their variation with geographic source, each other, and environmental conditions.

Almost all plants in the genus Populus are dioecious (i.e. trees are either male or female), but it is unknown whether dioecy evolved in a common ancestor or independently in different subgenera. Here, we sequence the small peritelomeric X- and Y-linked regions of P. deltoides chromosome XIX. Two genes are present only in the Y-linked region. One is a duplication of a non-Y-linked, female-specifically expressed response regulator, which produces siRNAs that block this gene’s expression, repressing femaleness. The other is an LTR/Gypsy transposable element family member, which generates long non-coding RNAs. Overexpression of this gene in A. thaliana promotes androecium development. We also find both genes in the sex-determining region of P. simonii, a different poplar subgenus, which suggests that they are both stable components of poplar sex-determining systems. By contrast, only the duplicated response regulator gene is present in the sex-linked regions of P. davidiana and P. tremula. Therefore, findings in our study suggest dioecy may have evolved independently in different poplar subgenera., Dioecy has evolved independently from hermaphroditic ancestors in different plant lineages. Here, the authors assemble Populus deltoides male and female genomes, and show the putative roles of a femaleness gene and a maleness gene in sex determination, which suggests independent evolution in different poplar species.

Premise Variation in local density and sex ratio in dioecious plants can affect mating success through the actions of pollen vectors, principally generalist insects or wind. Increased density and male-biased sex ratios should promote pollen transfer and seed production, but their combined effects have not been investigated for ambophilous species, which exhibit both insect and wind pollination. Methods We manipulated density (low vs. high) and sex ratio (1:1 vs. 3:1 male-biased) in arrays of dioecious ambophilous Thalictrum pubescens. We quantified visitation rates and foraging times to examine whether pollinators exhibited sex-specific preferences and determined the seed set of arrays. Results Pollinators visited more plants per foraging bout at high than low density. Visitation rates and foraging times of visitors were greater for male than for female plants but did not depend on the density or sex ratio of arrays. However, whereas solitary bees displayed a strong preference for males, hover flies were indifferent to plant sex phenotype. Solitary bees also visited significantly more plants per foraging bout than hover flies. There was a significant interaction between density and sex ratio on seed set. At low density, seed set was greater for 3:1 than for 1:1 arrays, but at high density the opposite pattern occurred. Conclusions The demographic factors we investigated had complex influences on pollinator foraging behavior and patterns of seed set. Several factors may explain our results, including the influence of density and sex ratio on pollen export from arrays, grooming by pollinators, and the contribution of wind pollination.

Several possible correlations between dioecy and eco-morphological features have been suggested to infer the conditions that would favor this sexual system. Dioecy has been associated either with specialized or generalized pollination systems. The genus Simarouba has six dioecious species that seem to have generalized pollinators. In this study, we examined the reproductive biology aspects of two Simarouba species to evaluate which of their eco-morphological attributes may favor dioecy. Our data suggest that S. amara and S. versicolor have small, inconspicuous, and abundant flowers of nocturnal anthesis and are only pollinated by nocturnal moths. They have a stable sexual system with flower, inflorescence, and flowering phenology dimorphism that is part of their pollination system strategies. Male plants of both species produce more flowers per individual than do female plants. A larger floral display may have an essential role in increasing male reproductive success. However, both species showed low reproductive efficacy, especially S. versicolor. The low reproductive efficacy may be due to the lack of pollinators or to the inefficacy of the pollinators available locally. Resumo Várias correlações entre dioicia e características ecomorfológicas têm sido sugeridas para inferir as condições que favorecem este sistema sexual. A dioicia tem sido associada a sistemas de polinização especializados ou generalistas. O gênero Simarouba possui seis espécies dióicas que parecem ter polinizadores generalistas. Neste trabalho, examinamos aspectos da biologia reprodutiva de duas espécies desse gênero para investigar quais de seus atributos ecomorfológicos reprodutivos podem favorecer a dioicia. Nossos dados sugerem que S. amara e S. versicolor têm flores pequenas, abertas, abundantes, com antese noturna que são polinizadas apenas por mariposas também noturnas. Elas possuem um sistema sexual estável com dimorfismo na flor, inflorescência e fenologia de floração, que faz parte de suas estratégias de polinização. As plantas masculinas de ambas as espécies produzem mais flores por indivíduo do que as femininas. Esta alta produção de flores tem um papel importante no aumento do sucesso reprodutivo masculino. Ambas as espécies apresentaram baixa eficácia reprodutiva, especialmente S. versicolor. A baixa eficácia reprodutiva pode ser devido à falta de polinizadores ou à ineficácia dos polinizadores disponíveis localmente.

Plant genetic sex determinants that mediate the transition to dioecy are predicted to be diverse, as this type of mating system independently evolved multiple times in angiosperms. Wild Vitis species are dioecious with individuals producing morphologically distinct female or male flowers; whereas, modern domesticated Vitis vinifera cultivars form hermaphrodite flowers capable of self-pollination. Here, we identify the VviPLATZ1 transcription factor as a key candidate female flower morphology factor that localizes to the Vitis SEX-DETERMINING REGION. The expression pattern of this gene correlates with the formation reflex stamens, a prominent morphological phenotype of female flowers. After generating CRISPR/Cas9 gene-edited alleles in a hermaphrodite genotype, phenotype analysis shows that individual homozygous lines produce flowers with reflex stamens. Taken together, our results demonstrate that loss of VviPLATZ1 function is a major factor that controls female flower morphology in Vitis., Unlike wild Vitis species, which produce either female or male flowers, modern grapevine cultivars form hermaphrodite flowers for self-pollination. Here, the authors report that the VviPLATZ1 (plant AT-rich sequence-and zinc-binding protein1) transcription factor functions in controlling female flower morphology determination.

Premise Five to six percent of angiosperm species exhibit a dioecious sexual system, with unisexual “male” or “female” flowers borne on separate plants. The consequent need for inter-individual pollen exchange is a special challenge for taxa where pollen is the sole pollinator reward. Dioecious Australian Solanum assure visits from pollen-foraging bees via production of inaperturate pollen in functionally female (morphologically bisexual) flowers. Biochemical composition of pollen from Australian Solanum has not been assessed nor compared to porate pollen from staminate flowers to reveal whether these flowers differ in their pollinator reward potential. Methods Porate pollen from male flowers and inaperturate pollen from functionally female flowers of two functionally dioecious Australian species were compared for protein and amino acid content. We also assessed pollen from bisexual and staminate flowers of a closely related andromonoecious species, in which all pollen is porate, as a comparison across co-occurring sexual systems. Results In both functionally dioecious species, porate pollen grains from staminate flowers had significantly higher levels of proteins and amino acids than inaperturate pollen grains from functionally female flowers. Levels of proteins and amino acids were highest in bisexual and staminate flowers of the andromonoecious species. Conclusions Higher levels of proteins and amino acids in porate pollen of “male” flowers in our functionally dioecious Solanum species suggests a greater nutritive reward for bees foraging on “male” plants than for those foraging on functionally “female” plants. Greater reward in porate pollen (including andromonoecious species) may be connected to the potential to generate a pollen tube., American Journal of Botany, 108 (11), ISSN:1914-2016, ISSN:0002-9122

Dioecious plants are obligate outcrossers with separate male and female individuals, which can result in decreased seed set with increasing distance between the sexes. Wind pollination is a common correlate of dioecy, yet combined wind and insect pollination (ambophily) could be advantageous in compensating for decreased pollen flow to isolated females. Dioecious, ambophilous gymnosperms Ephedra (Gnetales) secrete pollination drops (PDs) in female cones that capture airborne pollen and attract ants that feed on them. Plant sugary secretions commonly reward ants in exchange for indirect plant defense against herbivores, and more rarely for pollination. We conducted field experiments to investigate whether ants are pollinators and/or plant defenders of South American Ephedra triandra, and whether their contribution to seed set and seed cone protection varies with distance between female and male plants. We quantified pollen flow in the wind and assessed the effectiveness of ants as pollinators by investigating their relative contribution to seed set, and their visitation rate in female plants at increasing distance from the nearest male. Ants accounted for most insect visits to female cones of E. triandra, where they consumed PDs, and pollen load was larger on bigger ants without reduction in pollen viability. While wind pollination was the main contributor to seed set overall, the relative contribution of ants was distance dependent. Ant contribution to seed set was not significant at shorter distances, yet at the farthest distance from the nearest male (23 m), where 20 times less pollen reached females, ants enhanced seed set by 30% compared to plants depending solely on wind pollination. We found no evidence that ants contribute to plant defense by preventing seed cone damage. Our results suggest that, despite their short-range movements, ants can offset pollen limitation in isolated females of wind-pollinated plants with separate sexes. We propose that ants enhance plant reproductive success via targeted delivery of airborne pollen, through frequent contact with ovule tips while consuming PDs. Our study constitutes the first experimental quantification of distance-dependent contribution of ants to pollination and provides a working hypothesis for ambophily in other dioecious plants lacking pollinator reward in male plants.

PREMISE Five to six percent of angiosperm species exhibit a dioecious sexual system, with unisexual "male" or "female" flowers borne on separate plants. The consequent need for inter-individual pollen exchange is a special challenge for taxa where pollen is the sole pollinator reward. Dioecious Australian Solanum assure visits from pollen-foraging bees via production of inaperturate pollen in functionally female (morphologically bisexual) flowers. Biochemical composition of pollen from Australian Solanum has not been assessed nor compared to porate pollen from staminate flowers to reveal whether these flowers differ in their pollinator reward potential. METHODS Porate pollen from male flowers and inaperturate pollen from functionally female flowers of two functionally dioecious Australian species were compared for protein and amino acid content. We also assessed pollen from bisexual and staminate flowers of a closely related andromonoecious species, in which all pollen is porate, as a comparison across co-occurring sexual systems. RESULTS In both functionally dioecious species, porate pollen grains from staminate flowers had significantly higher levels of proteins and amino acids than inaperturate pollen grains from functionally female flowers. Levels of proteins and amino acids were highest in bisexual and staminate flowers of the andromonoecious species. CONCLUSIONS Higher levels of proteins and amino acids in porate pollen of "male" flowers in our functionally dioecious Solanum species suggests a greater nutritive reward for bees foraging on "male" plants than for those foraging on functionally "female" plants. Greater reward in porate pollen (including andromonoecious species) may be connected to the potential to generate a pollen tube.

Dioecy, the separation of sex at the individual level, evolved in angiosperms from hermaphroditic ancestors. One of the postulated evolutionary pathways is by way of the gynodioecious stage, i.e., the coexistence of female and hermaphroditic individuals. In a morphologically gynodioecious species, Daphne jezoensis., seed fertility in hermaphrodites is limited compared to that of females, indicating that its sexual system is close to dioecy. The aim of this study was to clarify whether the gynodioecy in this species is a stable sexual system or a transitional state leading to dioecy. We evaluated variations in the degree of pollen limitation, sexual and mating systems, floral morphology, sex ratio, and the genetic structure of 14 populations across the species’ distribution range in Japan. Sexual systems were similar among populations throughout the distribution range although the southernmost populations had unique genetic structures to some extent. Hermaphrodites were commonly self-compatible, but their fruiting capacity was low in every population. This suggests that gynodioecy composed of females and hermaphrodites having low seed fertility is a consistent sexual system in D. jezoensis. Low seed fertility in hermaphrodites throughout the distribution range suggests that this sexual system is not necessarily a transitional state from gynodioecy to dioecy if occasional seed production in hermaphrodites has any advantage. One possible ecological advantage for the maintenance of hermaphrodites is that seed production by autonomous self-pollination may be beneficial in the colonization process after long-distance seed dispersal.

The increased frequency and intensity of drought pose great threats to the survival of trees, especially in dioecious tree species with sexual differences in mortality and biased sex ratios. The sex-specific mechanisms underlying stem xylem anatomy and function and carbon metabolism in drought resistance and recovery were investigated in dioecious Populus cathayana Rehder. The sex-specific drought resistance and subsequent recovery were linked to the xylem anatomy and carbon metabolism. Females had a greater xylem vessel area per vessel, biomass and theoretically hydraulic efficiency under well-watered conditions. Conversely, males had a lower xylem lumen area, but greater vessel numbers, and a higher cell wall thickness, suggesting a theoretically conservative water-use strategy and drought resistance. The recovery of photosynthetic ability after drought in males was largely dependent on the recovery of xylem function and the regulation of the xylem carbohydrate metabolism. Additionally, the number of upregulated genes related to xylem cell wall biogenesis was greater in males relative to females under drought stress and subsequent rewatering, which facilitated drought resistance and xylem function restoration in males. These results suggested that sex-specific drought resistance and restoration were related to xylem anatomy and function, carbohydrate metabolism and cell turgor maintenance.

Background The fragrant flower plant Osmanthus fragrans has an extremely rare androdioecious breeding system displaying the occurrence of males and hermaphrodites in a single population, which occupies a crucial intermediate stage in the evolutionary transition between hermaphroditism and dioecy. However, the molecular mechanism of androdioecy plant is very limited and still largely unknown. Results Here, we used SWATH-MS-based quantitative approach to study the proteome changes between male and hermaphroditic O. fragrans pistils. A total of 428 proteins of diverse functions were determined to show significant abundance changes including 210 up-regulated and 218 down-regulated proteins in male compared to hermaphroditic pistils. Functional categorization revealed that the differentially expressed proteins (DEPs) primarily distributed in the carbohydrate metabolism, secondary metabolism as well as signaling cascades. Further experimental analysis showed the substantial carbohydrates accumulation associated with promoted net photosynthetic rate and water use efficiency were observed in purplish red pedicel of hermaphroditic flower compared with green pedicel of male flower, implicating glucose metabolism serves as nutritional modulator for the differentiation of male and hermaphroditic flower. Meanwhile, the entire upregulation of secondary metabolism including flavonoids, isoprenoids and lignins seem to protect and maintain the male function in male flowers, well explaining important feature of androdioecy that aborted pistil of a male flower still has a male function. Furthermore, nine selected DEPs were validated via gene expression analysis, suggesting an extra layer of post-transcriptional regulation occurs during O. fragrans floral development. Conclusion Taken together, our findings represent the first SWATH-MS-based proteomic report in androdioecy plant O. fragrans, which reveal carbohydrate metabolism, secondary metabolism and post-transcriptional regulation contributing to the androdioecy breeding system and ultimately extend our understanding on genetic basis as well as the industrialization development of O. fragrans.

Dasiphora fruticosa comprises male, female and hermaphrodite plants, which are distributed sympatrically in some populations on the Qinghai-Tibet Plateau. To explore what governs the coexistence of these three sexual phenotypes, we investigated the DNA contents, pollen and ovule production, pollen deposition, and performed hand-pollination in both hermaphroditic and dioecious individuals of D. fruticosa. Flow cytometry confirmed that the DNA content of males and females were almost twice as much as that of the hermaphrodites. Male and female flowers produced more pollen grains and ovules than hermaphroditic flowers. Hand-pollinated treatments showed that unisexual flowers were sterile in one sexual function and bisexual flowers were fertile for both functions, but no sterile seeds were produced between unisexual and bisexual flowers. Our findings imply that polyploidy is related to gender dimorphism, and both are likely to play a strong role in the coexistence of two cryptic biological species of D. fruticosa (low ploidy hermaphroditic species and high ploidy dioecious species) in the Qinghai-Tibet Plateau. Keywords: Gender polymorphism, Dioecy, Dasiphora fruticosa, Polyploidy, Reproductive isolation

Sex-determination in Mercurialis annua is not related to chromatin conformation or DNA methylation of floral homeotic genes but might be regulated upstream of these genes by one or more unknown gender-specific factors that affect hormonal homeostasis., In plants, dioecy characterizes species that carry male and female flowers on separate plants and it occurs in about 6% of angiosperms; however, the molecular mechanisms that underlie dioecy are essentially unknown. The ability for sex-reversal by hormone application raises the hypothesis that the genes required for the expression of both sexes are potentially functional but are regulated by epigenetic means. In this study, proteomic analysis of nuclear proteins isolated from flower buds of females, males, and feminized males of the dioecious plant Mercurialis annua revealed differential expression of nuclear proteins that are implicated in chromatin structure and function, including floral homeotic proteins. Focusing on floral genes, we found that class B genes were mainly expressed in male flowers, while class D genes, as well as SUPERMAN-like genes, were mainly expressed in female flowers. Cytokinin-induced feminization of male plants was associated with down-regulation of male-specific genes concomitantly with up-regulation of female-specific genes. No correlation was found between the expression of class B and D genes and the changes in DNA methylation or chromatin conformation of these genes. Thus, we could not confirm DNA methylation or chromatin conformation of floral genes to be the major determinant regulating sexual dimorphisms. Instead, determination of sex in M. annua might be controlled upstream of floral genes by one or more sex-specific factors that affect hormonal homeostasis. A comprehensive model is proposed for sex-determination in M. annua.

Salicaceae plants are dioecious woody plants. Previous studies have shown that male individuals are more tolerant to water deficiency than females for male‐biased poplars. However, Salix paraplesia is a female‐biased species in nature. It is still unknown whether female willows are more tolerant to drought stress than males. To better understand the sexually different tolerance to water deficiency in willows, a greenhouse experiment combined with a field investigation was conducted, and physiological traits were tested in male and female S. paraplesia under a drought‐stressed condition (50% of soil water capacity). Our field investigation showed that S. paraplesia was a species with female‐biased sex ratio along altitude gradients (2,400 m, 2,600 m and 2,800 m) in their natural habitats. Our results showed that the height growth, biomass accumulation, total chlorophyll pigment content (TChl), and the net photosynthetic rate were higher in female willows than in males at the low and middle altitudes (2,400 m and 2,600 m) rather than at a high altitude (2,800 m) under well‐watered conditions. Under drought‐stressed conditions, the growth, biomass, and photosynthesis were greatly inhibited in both sexes, while females showed higher biomass and TChl content and suffered less negative effects than did males. Particularly, females that originated from a high altitude showed lower leaf relative electrolyte leakage, malondialdehyde content, and less disorder of chloroplast ultrastructures but a higher peroxidase activity (POD) than that of males. Therefore, S. paraplesia females exhibited a better drought tolerance and self‐protective ability than males from high altitude. There is a reason to speculate that the population structure of S. paraplesia at a high altitude would be likely to further female biases with the increased drought intensity in the alpine regions.

Dioecious woody species often exhibit male-biased sex ratios under drought habitat, which may result in vulnerability in drying soils. However, whether biased sex ratios are associated with sex-specific responses in growth and defence against insect herbivory under drought stress conditions is unknown. We investigated the sexual responses in terms of growth and defence in Populus yunnanensis Dode in response to two water treatments (40% and 100% field capacity). Drought stress reduced the plant growth (e.g., height increment, gas exchange, photosynthetic nitrogen use efficiency, and water potential) of both sexes and reduced the leaf water content and defensive performance (increased damaged area by leaf-chewing generalists and decreased leaf contents of total phenolics, condensed tannins, and flavonoids) of the females but not the males. Moreover, female defensive performance was lower than that of the males under drought conditions. Additionally, the plant mortality rate increased for both sexes, and it was higher for females than for males during drought stress in response to all leaf defoliation treatments. Our results suggest that sexual responses in terms of growth and defence to drought are significantly different and that P. yunnanensis females have a lower defence capability than males, which may explain the male-biased sex ratio that exists in the natural population. Hence, this study provides new insight into forecasting population dynamics for dioecious plant species in drying habitats.

PCD role in unisexual flowers. The developmental processes underlying the transition from hermaphroditism to unisexuality are key to understanding variation and evolution of floral structure and function. A detailed examination of the cytological and histological patterns involved in pollen and ovule development of staminate and pistillate flowers in the dioecious Opuntia robusta was undertaken, and the potential involvement of programmed cell death in the abortion of the sex whorls was explored. Flowers initiated development as hermaphrodites and became functionally unisexual by anthesis. Female individuals have pistillate flowers with a conspicuous stigma, functional ovary, collapsed stamens and no pollen grains. Male individuals have staminate flowers, with large yellow anthers, abundant pollen grains, underdeveloped stigma, style and an ovary that rarely produced ovules. In pistillate flowers, anther abortion resulted from the premature degradation of the tapetum by PCD, followed by irregular deposition of callose wall around the microsporocytes, and finally by microspore degradation. In staminate flowers, the stigma could support pollen germination; however, the ovaries were reduced, with evidence of placental arrest and ovule abortion through PCD, when ovules were present. We demonstrate that PCD is recruited in both pistillate and staminate flower development; however, it occurs at different times of floral development. This study contributes to the understanding of the nature of the O. robusta breeding system and identifies developmental landmarks that contribute to sexual determination in Cactaceae.

In clonal dioecious plants, the frequency and spatial distribution of flowering ramets contains information on the underlying genet sex ratio. These measures can also provide insight on potential ecological mechanisms causing variation and bias in sex ratios among populations. We used a novel likelihood‐based approach and spatial clustering model to estimate the genet sex ratios from flowering ramet data collected from 32 populations of dioecious Thalictrum pubescens, a clonal species from eastern N. America that occupies moist wetland and forested environments. We investigated sex ratios of seed families, clone size, patterns of flowering and plant height to determine potential causes of sex‐ratio bias. Flowering ramet sex ratios varied considerably among populations but were significantly male‐biased. Seed families grown to flowering also exhibited the same degree of male bias. Both models predicted close correspondence between ramet and genet sex ratios. The likelihood model revealed that sex differences in ramet production could not account for biased ramet sex ratios. The spatial clustering model indicated that ramets were significantly clustered at two spatial scales and estimated similar cluster sizes and densities for both sexes. There was no evidence for spatial segregation of the sexes. Both sexes were equally likely to flower in consecutive years and repeated bouts of flowering had no effect on ramet height. Synthesis. Our analyses suggest that the widespread occurrence of male‐biased sex ratios in Thalictrum pubescens is unlikely to result from sexual differences in clonal growth or habitat preferences. The bias appears to become established early in the life cycle, perhaps at the seed stage as a consequence of local resource competition.

Dioecy has often broken down in flowering plants, yielding functional hermaphroditism. We reasoned that evolutionary transitions from dioecy to functional hermaphroditism must overcome an inertia of sexual dimorphism, because modified males or females will express the opposite sexual function for which their phenotypes have been optimised. We tested this prediction by assessing the siring success of monoecious individuals of the plant Mercurialis annua with an acquired male function but that are phenotypically still female‐like. We found that pollen dispersed by female‐like monoecious individuals was ~ 1/3 poorer at siring outcrossed offspring than pollen from monoecious individuals with an alternative male‐like inflorescence. We conclude that whereas dioecy might evolve from functional hermaphroditism by conferring upon individuals certain benefits of sexual specialisation, reversion from a strategy of separate sexes to one of combined sexes must overcome constraints imposed by the advantages of sexual dimorphism. The breakdown of dioecy must therefore often be limited to situations in which outcrossing cannot be maintained and where selection favours a capacity for inbreeding by functional hermaphrodites.

SUMMARY Brandies of male and female plants of the dioecious Leucadendron rubrum Burm. f. were collected on the site of the Weikamp weather-station in the Jonkershoek State Forest, Stellenbosch, from July 1990 to September 1991. Xylem sap was extracted from the branches by vacuum extraction. HPLC was employed to purify the xylem sap and to separate the cytokinins prior to quantitation by ELISA. The cytokinin levels in the purified plant samples were determined using rabbit antiserum-based ELISAs for the determination of the levels of the cytokinins DHZR and IPA, and a trans-ZR specific monoclonal antibody-based ELISA for the determination of the levels of the cytokinin ZR. It was observed that the levels of the cytokinins DHZR and ZR did not differ significantly between male and female plants but did correlate with the seasonal growth patterns. The levels of the cytokinin IPA, in male and female plants of L. rubrum, not only correlated specifically with the different seasonal growth patterns observed but differed significantly between male and female plants. Peaks in the levels of all three cytokinins. i.e. ZR, DHZR and IPA, occurred during the study period. These peaks corresponded with the phenological development of L. rubrum, specifically with vegetative bud break and inflorescence differentiation and appeared to be synchronized with the growth and development. An attempt was also made to correlate cytokinin levels with the extreme differences in morphological characters exhibited by male and female plants, and possibly to explain to what extent these phytohormones influence sex expression in L. rubrum

The stages of floral development in staminate and pistillate plants of hop (Humulus lupulus) were defined using scanning electron microscopy and light microscopy. Vegetative meristems of male and female plants are morphologically indistinguishable. On transition to the reproductive phase, inflorescence apices reduce greatly in size and striking developmental sex differences become apparent. The first sex-specific differences occur extremely early in floral ontogeny. Both male and female plants initiate inflorescence meristems at each leaf node, each meristem being enclosed within a bract. Male secondary inflorescence meristems give rise to clusters of asynchronously developing flowers. Female inflorescence meristems produce flowers arranged in 'cones'. Each male floral meristem initiates a whorl of five sepal primordia, followed by an inner whorl of five stamen primordia. There is no sign of carpel development at any stage. In females, two carpel primordia are initiated, surrounded at their base by a vestigial perianth whorl. No stamen development is observed. Several monoecious lines carry bisexual flowers, either within cymose panicles or within the basal bracts of terminal female inflorescences. Bisexual flowers usually possess perianth, stamen and carpel whorls. The central whorls are often highly variable, and range from a pair of stigmas fused to a thin central filament to a well developed gynoecium. Chimaeric central whorls consisting of fused staminoid-carpelloid structures also occur. Sex differences in unisexual hop flowers are determined at an extremely early stage in ontogeny. The inappropriate set of sex organs is suppressed before it becomes visible or, more probably, it is not initiated at all. Genes directing the development of sex are likely to act at an extremely early stage, well in advance of floral organogenesis. The sex chromosomes of dioecious hop plants are described, as well as the chromosome constitutions of monoecious plants and those carrying bisexual flowers.

Significance We studied the grape sex-determining region (SDR) in 12 Vitis genomes and demonstrated its conservation across 556 genotypes including 193 accessions from 47 world-wide wild grapevine species and 363 accessions of cultivated grapevine. Although the grape SDR is recombination free in all wild species, we found two distinct hermaphrodite (H) haplotypes (H1 and H2) among the cultivated grapevines, both chimeras of male (M) and female (f) haplotypes. The two independent recombinations carry different genetic signatures which long predate the domestication of grapevine, suggesting independent evolutions of this trait in wild European grapevine gene pools prior to human domestication., Hermaphroditic (perfect) flowers were a key trait in grapevine domestication, enabling a drastic increase in yields due to the efficiency of self-pollination in the domesticated grapevine (Vitis vinifera L. ssp. vinifera). In contrast, all extant wild Vitis species are dioecious, each plant having only male or female flowers. In this study, we identified the male (M) and female (f) haplotypes of the sex-determining region (SDR) in the wild grapevine species V. cinerea and confirmed the boundaries of the SDR. We also demonstrated that the SDR and its boundaries are precisely conserved across the Vitis genus using shotgun resequencing data of 556 wild and domesticated accessions from North America, East Asia, and Europe. A high linkage disequilibrium was found at the SDR in all wild grape species, while different recombination signatures were observed along the hermaphrodite (H) haplotype of 363 cultivated accessions, revealing two distinct H haplotypes, named H1 and H2. To further examine the H2 haplotype, we sequenced the genome of two grapevine cultivars, 'Riesling' and 'Chardonnay'. By reconstructing the first two H2 haplotypes, we estimated the divergence time between H1 and H2 haplotypes at ∼6 million years ago, which predates the domestication of grapevine (∼8,000 y ago). Our findings emphasize the important role of recombination suppression in maintaining dioecy in wild grape species and lend additional support to the hypothesis that at least two independent recombination events led to the reversion to hermaphroditism in grapevine.

BackgroundPersimmon (Diospyros kakiThunb.) has various labile sex types, and studying its sex differentiation can improve breeding efficiency. However, studies on sexual regulation patterns in persimmon have focused mainly on monoecy and dioecy, whereas little research has been published on andromonoecy. In order to reveal the sex differentiation regulation mechanism of andromonoecious persimmon, we performed histological and cytological observations, evaluatedOGIandMeGIexpression and conducted phytohormones assays and mRNA and small RNA transcriptome analyses of the male and hermaphroditic floral buds of the andromonoecious persimmon ‘Longyanyeshi 1’.ResultsStages 2 and 4 were identified as the critical morphological periods for sex differentiation of ‘Longyanyeshi 1’ by histological and cytological observation. At both stages,OGIwas differentially expressed in male and hermaphroditic buds, butMeGIwas not. This was different from their expressions in dioecious and monoecious persimmons. Meantime, the results of phytohormones assays showed that high IAA, ABA, GA3, and JA levels at stage 2 may have promoted male floral bud differentiation. However, high JA levels at stage 4 and high ZT levels at stages 2 and 4 may have promoted hermaphroditic floral bud differentiation. In these phytohormone biosynthesis and signaling pathways, 52 and 54 differential expression genes (includingAux/IAA,ARFs,DELLA,AHP,A-ARR,B-ARR,CYP735A,CRE1,PP2C,JAZ,MYC2,COI1,CTR1,SIMKK,ACO,andMPK6) were identified, respectively. During the development of male floral buds, five metacaspases genes may have been involved in pistil abortion. In addition,MYB,FAR1,bHLH, WRKY,andMADStranscription factors might play important roles in persimmon floral bud sex differentiation. Noteworthy, miR169v_1, miR169e_3, miR319_1, and miR319 were predicted to contribute to phytohormone biosynthesis and signaling pathways and floral organogenesis and may also regulate floral bud sex differentiation.ConclusionThe present study revealed the differences in morphology and phytohormones content between male and hermaphroditic floral buds of ‘Longyanyeshi 1’ during the process of sex differentiation, and identified a subset of candidate genes and miRNAs putatively associated with its sex differentiation. These findings can provide a foundation for molecular regulatory mechanism researching on andromonoecious persimmon.

Dioecious plants possess diverse sex determination systems and unique mechanisms of reproductive organ development; however, little is known about how sex-linked genes shape the expression of regulatory cascades that lead to developmental differences between sexes. InSilene latifolia, a dioecious plant with stable dimorphism in floral traits, early experiments suggested that female-regulator genes act on the factors that determine the boundaries of the flower whorls. To identify these regulators, we sequenced the transcriptome of male flowers with fully developed gynoecia induced by rapid demethylation in the parental generation. As the hermaphrodite flower trait is holandric (transmitted only from male to male, inherited on the Y chromosome), we screened for genes that are differentially expressed between male, female, and hermaphrodite flowers. Dozens of candidate genes that are upregulated in hermaphrodite flowers compared to male and female flowers were detected and found to have putative roles in floral organization, affecting the expression of floral MADS-box and other genes. Amongst these genes, eight candidates were found to promote gynoecium formation in female and hermaphrodite flowers, affecting organ size, whorl boundary, and the expression of mainly B class flower genes. To complement our transcriptome analysis, we closely examined the floral organs in their native state using a field emission environmental scanning electron microscope. Our results reveal the principal regulatory pathways involved in sex-specific flower development in the classical model of dioecy,S. latifolia.

The diversity of inflorescences among flowering plants is captivating. Such charm is not only due to the variety of sizes, shapes, colors, and flowers displayed, but also to the range of reproductive systems. For instance, hermaphrodites occur abundantly throughout the plant kingdom with both stamens and carpels within the same flower. Nevertheless, 10% of flowering plants have separate unisexual flowers, either in different locations of the same individual (monoecy) or on different individuals (dioecy). Despite their rarity, dioecious plants provide an excellent opportunity to investigate the mechanisms involved in sex expression and the evolution of sex-determining regions (SDRs) and sex chromosomes. The SDRs and the evolution of dioecy have been studied in many species ranging from Ginkgo to important fruit crops. Some of these studies, for example in asparagus or kiwifruit, identified two sex-determining genes within the non-recombining SDR and may thus be consistent with the classical model for the evolution of dioecy from hermaphroditism via gynodioecy, that predicts two successive mutations, the first one affecting male and the second one female function, becoming linked in a region of suppressed recombination. On the other hand, aided by genome sequencing and gene editing, single factor sex determination has emerged in other species, such as persimmon or poplar. Despite the diversity of sex-determining mechanisms, a tentative comparative analysis of the known sex-determining genes and candidates in different species suggests that similar genes and pathways may be employed repeatedly for the evolution of dioecy. The cytokinin signaling pathway appears important for sex determination in several species regardless of the underlying genetic system. Additionally, tapetum-related genes often seem to act as male-promoting factors when sex is determined via two genes. We present a unified model that synthesizes the genetic networks of sex determination in monoecious and dioecious plants and will support the generation of hypothesis regarding candidate sex determinants in future studies.

Research We recently described, in Cannabis sativa, the oldest sex chromosome system documented so far in plants (12–28 Myr old). Based on the estimated age, we predicted that it should be shared by its sister genus Humulus, which is known also to possess XY chromosomes. Here, we used transcriptome sequencing of an F1 family of H. lupulus to identify and study the sex chromosomes in this species using the probabilistic method SEX-DETECTOR. We identified 265 sex-linked genes in H. lupulus, which preferentially mapped to the C. sativa X chromosome. Using phylogenies of sex-linked genes, we showed that a region of the sex chromosomes had already stopped recombining in an ancestor of both species. Furthermore, as in C. sativa, Y-linked gene expression reduction is correlated to the position on the X chromosome, and highly Y degenerated genes showed dosage compensation. We report, for the first time in Angiosperms, a sex chromosome system that is shared by two different genera. Thus, recombination suppression started at least 21–25 Myr ago, and then (either gradually or step-wise) spread to a large part of the sex chromosomes (c. 70%), leading to a degenerated Y chromosome info:eu-repo/semantics/publishedVersion

The recovery of inaperturate pollen from functionally female flowers in archaeological layers opens the question of a possible pollen-based discrimination between wild and domesticated Vitis vinifera in prehistoric times. Pollen analysis applied to archaeology has not routinely considered the existence of pollen dimorphism in Vitis, a well-known trait in the field of agrarian studies. Therefore, the inaperturate shape of grapevine pollen is ignored by studies on the archaeobotanical history of viticulture. In this paper we investigate pollen morphology of the domesticated and wild subspecies of V. vinifera, and report the first evidence of inaperturate Vitis pollen from an archaeological site. We studied exemplar cases of plants with hermaphroditic flowers, belonging to the subspecies vinifera with fully developed male and female organs, cases of dioecious plants with male or female flowers, belonging to the wild subspecies sylvestris and cases of V. vinifera subsp. vinifera with morphologically hermaphroditic but functionally female flowers. The pollen produced by hermaphroditic and male flowers is usually trizonocolporate, the pollen produced by female flowers is inaperturate. This paper reports on the inaperturate pollen of Vitis found in an archeological site of the Po Plain, Northern Italy. The site dated to the Bronze Age, which is known to have been a critical age for the use of this plant with a transition from wild to domesticated Vitis in central Mediterranean. Can the inaperturate Vitis pollen be a marker of wild Vitis vinifera in prehistoric times? Palynology suggests a possible new investigation strategy on the ancient history of the wild and cultivated grapevine. The pollen dimorphism also implies a different production and dispersal of pollen of the wild and the domesticated subspecies. Grapevine plants are palynologically different from the other Mediterranean "cultural trees". In fact, Olea, Juglans and Castanea which are included in the OJC index, have the same pollen morphology and the same pollen dispersal, in wild and domesticated plants. In contrast, the signal of Vitis pollen in past records may be different depending on the hermaphroditic or dioecious subspecies.

Date palm is dioecious, characterized by unisexual flowers located on separate male or female trees. Dioecy has always been an issue in date palm breeding because the sex of the plant cannot be known until it reaches its reproductive stage, which takes 5‒9 years to first flowering. Hence, early sex identification can enhance breeding and genetic improvement success in date palm. The breeding programs help in the selection and identification of superior seedlings of important traits. However, the sex of dioecious plants is usually tedious to identify. Sex determination may not only enhance genetic resources but also gear up breeding programs. No doubt, substantial progress has been made in understanding mechanisms of sex identification in date palm using morphological, physiological, biochemical, and cytological procedures but the discrimination in male and female plants by these techniques is not reliable. Sexuality in plants is regulated by genetic, epigenetic, and physiological mechanisms, and the most specialized mechanism of sex determination is the sex chromosomes. Molecular markers have been applied accurately in date palm breeding programs to differentiate male and female seedlings, but the development of more reliable molecular markers is needed. Hence, this chapter is focused on an understanding of floral biology, genetics of sex, and methods applied for sex determination in date palm.

International audience; The genetic basis and evolution of sex determination in dioecious plants is emerging as an active area of research with exciting advances in genome sequencing and analysis technologies. As the sole species within the sister lineage to all other extant flowering plants, Amborella trichopoda is an important model for understanding the evolution and development of flowers. Plants typically produce only male or female flowers, but sex determination mechanisms are unknown for the species. Sequence data derived from plants of natural origin and an F1 mapping population were used to identify sex-linked genes and the nonrecombining region. Amborella trichopoda has a ZW sex determination system. Analysis of genes in a 4 Mb nonrecombining sex-determination region reveals recent divergence of Z and W gametologs, and few Z- and W-specific genes. The sex chromosomes of A. trichopoda evolved less than 16.5 Myr ago, long after the divergence of the extant angiosperms.

Background A key step in domestication of the grapevine was the transition from separate sexes (dioecy) in wild Vitis vinifera ssp. sylvestris (V. sylvestris) to hermaphroditism in cultivated Vitis vinifera ssp. sativa (V. vinifera). It is known that V. sylvestris has an XY system and V. vinifera a modified Y haplotype (Yh) and that the sex locus is small, but it has not previously been precisely characterized. Results We generate a high-quality de novo reference genome for V. sylvestris, onto which we map whole-genome re-sequencing data of a cross to locate the sex locus. Assembly of the full X, Y, and Yh haplotypes of V. sylvestris and V. vinifera sex locus and examining their gene content and expression profiles during flower development in wild and cultivated accessions show that truncation and deletion of tapetum and pollen development genes on the X haplotype likely causes male sterility, while the upregulation of a Y allele of a cytokinin regulator (APRT3) may cause female sterility. The downregulation of this cytokinin regulator in the Yh haplotype may be sufficient to trigger reversal to hermaphroditism. Molecular dating of X and Y haplotypes is consistent with the sex locus being as old as the Vitis genus, but the mechanism by which recombination was suppressed remains undetermined. Conclusions We describe the genomic and evolutionary characterization of the sex locus of cultivated and wild grapevine, providing a coherent model of sex determination in the latter and for transition from dioecy to hermaphroditism during domestication.

Androdioecy is a rare reproductive system. Fraxinus platypoda, a woody canopy species in Japan’s mountainous riparian zones, is described as a morphologically androdioecious species. In this study, we tried to detect whether F. platypoda is also functionally androdioecious. We analyzed its sexual expression, seed development, pollen morphology and germination ability, pollination systems, and mast flowering behavior. We found that the hermaphrodite trees are andromonoecious, with inflorescences bearing male and hermaphroditic flowers, whereas male individuals had only male flowers. Pollen morphology was identical in male flowers, in hermaphrodite flowers of an andromonoecious individual, and in male flowers of male individuals. Pollen from both types of individuals was capable of germination both ex vivo (on nutrient medium) and in vivo in pollination experiments. However, compared with pollen from andromonoecious trees, pollen from male trees showed a higher germination rate. The self-pollination rate of bagged hermaphroditic flowers was almost zero. The fruit set rate following cross-pollination with male pollen from a male tree was higher than that following natural pollination, whereas the rate with hermaphroditic pollen was the same. The flowering and fruiting of F. platypoda have fluctuated over 17 years; the flowering of the two types of sexual individuals exhibited clear synchronization during this period. The frequency of male individuals within the populations is 50%. The maintenance of such a proportion of males in populations of the self-incompatible F. platypoda is either indicative of a true androdioecious species with a diallelic self-incompatibility system or a cryptic-dioecious species. This alternative is discussed here.

Kiwifruit (Actinidia spp.) is a significant plantation crop belonging to family Actinidiaceae, having deciduous, dioecious, and scrambling vines with chromosome number 2n=58. Dioecy in kiwifruit forms the basis for several breeding programs. The present study was carried out for diversity analysis in kiwifruit genotypes using RAPD markers. 7 kiwifruit genotypes (2 males viz. Allision & Tomuri and 5 females viz. Hayward, Bruno, Allision, Monty & Abbott) were analysed for molecular polymorphism using 94 RAPD primers, out of which 23 primers amplified the genomic DNA in all the genotypes. RAPD data was analysed using NTSYS-pc software and dendrogram construction was done using UPGMA method. Two separate clusters of male and female genotypes were formed. Similarity matrix indices showed maximum similarity between Tomuri (M) and Allision (M) with a similarity coefficient of 0.719 while Abbott (F) and Allision (M) were found to have least similarity having a similarity coefficient of 0.521. Four RAPD primers amplified unique amplicons in Monty, Hayward, Bruno, Allision (M) and Abbott and two primers amplified unique amplicons in Allision (M) & Tomuri (M) along with the male and female plants of Allision genotype respectively. Therefore, these primers can help in distinguishing the genotypes of kiwifruit and can also be validated as putative markers for the sex identification in kiwifruit.

Within the Cucurbitaceae family, most of its species develop unisexual female and male flowers, either on the same plant (monoecy) or on different plants (dioecy). As in other plant families, these two sex morphotypes have evolved from hermaphrodite species; however, many evolutionary events have occurred in cucurbits allowing easy conversion from dioecy to monoecy and vice versa. The variability in sex morphotypes is higher in the domesticated species of the family, which together with recent advances in genomics, make cucurbits an ideal model to study the genetic and molecular mechanisms that control sex determination in plants. Conventional studies demonstrated that ethylene was the master regulator of sex determination in cucurbits, although some cultivated species may respond differently to ethylene action. In this article, we survey the new advances in hormonal and genetic control of sex determination in cucurbit species, control which establishes the ethylene biosynthesis and signaling genes as being those that determine the floral meristem towards a male, female or hermaphrodite flower. The interactions between these genes are integrated into a model that explains the occurrence and distribution of unisexal and hermaphrodite flowers within the different sex morphotypes. We underline the significance of this scientific progress with regard to breeding programs for agronomically-important sex-associated traits.

Secondary (flowering) sex ratio of a wild population of the dioecious woody shrub Lindera benzoin (spicebush) in Maryland has been persistently female biased over a time span of ∼ 17 years. Female-biased secondary sex ratios are relatively uncommon in dioecious woody plants, and are not readily explained by differential costs of reproduction, which are generally higher in females. In fact, prior studies of reproductive adults in L. benzoin populations have shown higher growth rates in males. This leads to questions concerning the source of female-biased sex ratios in some populations. Using seeds collected from 15 maternal plants at the Maryland site in 2013, a potted plant study was conducted from 2014–2020 in which periodic flowering censuses were taken, and plant size parameters were recorded. This study improves upon an analogous common garden study conducted under highly competitive conditions that induced considerable preflowering mortality. In the current study, plants were grown in pots to eliminate root competition and in a loose array to minimize shoot competition; mortality was thus kept to ∼ 1% after seed germination. Like the source population, genet sex ratios in the current study were significantly female-biased, partly due to a trend toward female-biased sex ratios in the offspring of a number of maternal plants. Estimates of plant size were generally higher for females in 2018, just prior to the onset of significant fruit production, supporting earlier results suggesting that females may compensate for differential costs of reproduction that can manifest after fruiting begins. Even in the absence of biased primary sex ratios, formation of female bias in wild populations could involve development of a relatively larger size in the pre-fruiting stage that gives females a temporary advantage over males. This could lead to relatively greater survival and/or earlier flowering of female plants in the early stages of population establishment, and could thus explain observed patterns of sex ratio bias.

The high occurrence of dioecy on oceanic islands has attracted much attention from plant evolutionary biologists. Tournefortia argentea (Boraginaceae), an Indo-Pacific strand plant, has an unusual sexual system which remains poorly understood. Here, we investigated the sexual system of T. argentea based on morphological and micromorphological studies, as well as manipulated pollination treatments. Two morphs of plants were found in the natural populations on Xisha Islands: hermaphrodite and female plants. Each sexual morph exhibited different floral traits, showing a modest form of sexual dimorphism. Flowers of hermaphrodites display significantly larger size in corolla and anther than those of females. Fruit sets under natural condition and hand pollination in hermaphroditic individuals were both far lower than that of female ones, which shows characteristics of both leaky dioecy and gynodioecy. We investigated how these differences in floral traits between females and hermaphrodites affect the choice of pollinators. Although females and hermaphroditic flowers in T. argentea attracted the same assemblage of pollinator species, including Apidae, Syrphidae and Utetheisa lotrix (Arctiidae), the reduction in allocation to pollinator-attracting structures (corolla) and rewarding products caused a significant reduction in Apidae visitation to female flowers. Accordingly, T. argentea is another example of a gynodioecious species of flowering plants in Boraginaceae with clear sexual dimorphism occurring in oceanic islands. The sexual system in T. argentea may imply a transitional stage in the evolution to dioecy and may have been favored during colonization to newly reclaimed habitats, where mating partners may be limited.

A well-established hypothesis for the evolution of dioecy involves two genes linked at a sex-determining region (SDR). Recently there has been increased interest in possible single gene sex determination. Work in Populus has finally provided direct experimental evidence for single gene sex determination in plants using CRISPR-Cas9 to knock out a single gene and convert individuals from female to male. In poplar, the feminizing factor popARR17 acts as a "master regulator", analogous to the mammalian masculinizing factor SRY. The production of fully functional males from females by a simple single gene knockout is experimental evidence that an antagonistic male-determining factor does not exist in Populus. Mammals have a "default sex" (female), as do poplar trees (Populus), although the default sex in poplars is male. The occurrence of single gene sex determination with a default sex may be much commoner in plants than hitherto expected, especially when dioecy evolved via monoecy. The master regulator does not even need to be at the SDR (although it may be). In most poplars the feminizing factor popARR17 is not at the SDR, but instead a negative regulator of it. So far there is little information on how high-level regulators are connected to floral phenotype. A model is presented of how sex-determining genes could lead to different floral morphologies via MADS-box floral developmental genes.

The origin and early evolution of sex chromosomes have been hypothesized to involve the linkage of factors with antagonistic effects on male and female function. Garden asparagus (Asparagus officinalis) is an ideal species to investigate this hypothesis, as the X and Y chromosomes are cytologically homomorphic and evolved from an ancestral autosome pair in association with a shift from hermaphroditism to dioecy. Mutagenesis screens paired with single-molecule fluorescence in situ hybridization directly implicate Y-specific genes that respectively suppress female (pistil) development and are necessary for male (anther) development. Comparison of contiguous X and Y chromosome assemblies shows that hemizygosity underlies the loss of recombination between the genes suppressing female organogenesis (SUPPRESSOR OF FEMALE FUNCTION) and promoting male function (TAPETAL DEVELOPMENT AND FUNCTION1 [aspTDF1]). We also experimentally demonstrate the function of aspTDF1. These findings provide direct evidence that sex chromosomes can function through linkage of two sex determination genes.

Plant sex determining systems and sex chromosomes are often evolutionarily young. Here, we present the early stage of sex chromosome in a fully dioecious plant,P. deltoides, by determining separate sequences of the physically small X- and Y-linked regions. Intriguingly, two Y genes are absent from the X counterpart. One gene represses female structures by producing siRNAs that block expression of a gene necessary for development of female structures, via RNA-directed DNA methylation and siRNA-guided mRNA cleavage. The other gene generates long non-coding RNA transcripts that, in males, soak up miRNAs that specifically inhibit androecium development. Transformation experiments inArabidopsis thalianashow that the two genes affect gynoecium and androecium development independently and antagonistically. Sex determination in the poplar therefore has the properties proposed for the first steps in the evolution of dioecy in flowering plants, with two genes whose joint effects favor close linkage, as is observed in poplar.

Sex expression in the bitter gourd is mainly categorized into monoecious—staminate and pistillate flowers are produced separately in the same plant. Genes for its sex determination mechanism are quite important for genetic improvement through breeding, including the production of hybrid cultivars. In Cucurbitaceae, the mechanism of physiology and genetics of sex determination and expression in cucumber and melon have been extensively studied. For elucidating on sex determination in bitter gourd, a few gynoecious bitter gourd lines, showing only the female flowers, are found and employed as the maternal parent of F1 hybrid cultivars. Each of these gynoecious lines was predicted to be determined by a single recessive locus, which was genetically mapped. In monoecious bitter gourd, various ratios between female and male flowers per a plant (sex ratio) were observed and frequency of female flowers is an influential trait for the yield of fruits. By genetic mapping approaches, quantitative trait loci for female flower frequency were found. It was already well known that ethylene signaling is a key of sex determination in melon and cucumber, and causal genes for their sex determination were identified. According to bitter gourd draft genome sequences, putative orthologs of these sex determination genes could be identified. In other Momordica species, both monoecious and dioecious species were diverged. Genetic and genomic studies of sex determination in bitter gourd will greatly contribute to elucidate the evolution of monoecy and dioecy.

Aside from selection, controlled mating is another powerful tool available to the plant breeder for making desirable genetic changes to plant populations. Unlike animals, plants do not select mating partners through behavior. Natural mating in plants is accomplished by altering the morphology and chemistry of flowers and flower parts to increase the probability of targeted mating alternatives. Certain floral modifications promote self-pollination while others favor cross-pollination. An overlay of mating type on parsimony analysis of plant taxa reveals that reproductive biology is highly mutable over short periods of evolutionary time. Surveys of floral structure and function within species often reveal diversity, suggesting that floral biology is a highly mutable feature of plants. Over the past 25 years, the implication of the MADS-box family of transcription factors has been discovered to be profoundly implicated in plant flower development. By understanding the molecular bases of floral development, plant breeders will be able to craft methods to manipulate underlying genes to change floral function and mating system in desirable ways. One fascinating strategy that plants have developed to enhance cross-pollination is self-incompatibility (SI). Two different forms of SI have been found in plants based on the determination of pollen SI phenotype by the parental sporophyte genotype or gametophyte haplotype. Methods have been crafted by plant breeders to avert SI during the breeding phase and to utilize SI for large-scale controlled mating. Certain plant taxa have been discovered in which the embryos within seeds are of maternal sporophyte origin, not the sexual fusion of gametes; known as apomixis. This phenomenon may be obligate or reversible either environmentally or according to genotype. By controlling apomixis, plant breeders can reap benefits that are difficult or impossible via obligate sexual reproduction. The genetic control of developmental processes that lead to other floral modifications that promote cross-pollination is also increasingly well understood. For example, the genetic changes that accompany the evolutionary transition from hermaphrodity to dioecy have been elucidated, and the underlying molecular biology of male sterility has been progressively revealed. This knowledge will empower the plant breeder to incorporate specific genes into breeding programs that will culminate in desirable reproductive outcomes. At present, plant breeders relay mostly on relatively mundane strategies to alter mating system. Advances in our understanding of floral development and reproduction at the molecular level will, however, provide more opportunities to use genomics for controlled mating in the future.

Although increasing attention has been paid to the relationships between heavy metal and nitrogen (N) availability, the mechanism underlying adaptation to cadmium (Cd) stress in dioecious plants has been largely overlooked. This study examined Cd accumulation, translocation and allocation among tissues and cellular compartments in Populus cathayana Rehder females and males. Both leaf Cd accumulation and root-to-shoot Cd translocation were significantly greater in females than in males under a normal N supply, but they were reduced in females and enhanced in males under N deficiency. The genes related to Cd uptake and translocation, HMA2, YSL2 and ZIP2, were strongly induced by Cd stress in female roots and in males under a normal N supply. Cadmium largely accumulated in the leaf blades of females and in the leaf veins of males under a normal N supply, while the contrary was true under N deficiency. Furthermore, Cd was mainly distributed in the leaf epidermis and spongy tissues of males, and in the leaf palisade tissues of females. Nitrogen deficiency increased Cd allocation to the spongy tissues of female leaves and to the palisade tissues of males. In roots, Cd was preferentially distributed to the epidermis and cortices in both sexes, and also to the vascular tissues of females under a normal N supply but not under N deficiency. These results suggested that males possess better Cd tolerance compared with females, even under N deficiency, which is associated with their reduced root-to-shoot Cd translocation, specific Cd distribution in organic and/or cellular compartments, and enhanced antioxidation and ion homeostasis. Our study also provides new insights into engineering woody plants for phytoremediation.

It remains a major challenge to identify the genes and mutations that lead to plant sexual differentiation. Here, we study the structure and evolution of the sex-determining region (SDR) in Vitis species. We report an improved, chromosome-scale Cabernet Sauvignon genome sequence and the phased assembly of nine wild and cultivated grape genomes. By resolving twenty Vitis SDR haplotypes, we compare male, female, and hermaphrodite haplotype structures and identify sex-linked regions. Coupled with gene expression data, we identify a candidate male-sterility mutation in the VviINP1 gene and potential female-sterility function associated with the transcription factor VviYABBY3. Our data suggest that dioecy has been lost during domestication through a rare recombination event between male and female haplotypes. This work significantly advances the understanding of the genetic basis of sex determination in Vitis and provides the information necessary to rapidly identify sex types in grape breeding programs., Grapevine is one of a few ancestrally dioecious crops that are reverted to hermaphroditism during domestication. Here, the authors identify candidate genes related to male- and female-sterility in grapes and describe the genetic process that led to hermaphroditism during domestication.

Sexuality in flowering plants is distinctly correlated with mating systems. Maximum numbers of flowering plants are outbreeders, and cross-pollination is the predominant type of pollen transfer to increase genetic variation. Morphological traits that promote outbreeding include temporal (dichogamy) and spatial separation (herkogamy) of male and female functions of flowers, monoecy and dioecy. Monoecy permits both inbreeding and outbreeding, while outbreeding is the only means of reproduction in dioecious species. Outbreeding confers on plant an opportunity to ‘move’ its genes amongst conspecific populations and is largely influenced by the availability of pollinators. Though pollinator-driven mating is favoured by most plant species, certain populations may occasionally maximize their reproductive fitness under adverse conditions by adopting multiple strategies such as autonomous selfing, dichogamy, late and early selfing, prepotency and outbreeding. Sexual plasticity manifested through mixed-mating, multiple mating, biparental inbreeding and sex switching has helped plants not only reproduce sexually but also ‘move on’. In addition, pollination environment also alters the outcrossing rates in a population emphasizing the fact that mating systems are labile. How various populations maintain potential for such shifts needs to be investigated. This chapter focuses on the outbreeding strategies in plants and also discusses how mating systems may evolve over the next few decades in response to challenges presented by changing environment.

Dioecious plants constitute 7% of all angiosperm species, yet they occur in many habitat types, partially through the deployment of sexual dimorphisms that assist in reproduction. In the present work, the dioecious monocot Lomandra leucocephala ssp. robusta (Asparagaceae: Lomandroideae) was studied to understand how sexual dimorphisms can assist species conservation and inform us of a species’ potential significance in an ecosystem. Floral display was sexually dimorphic, as male inflorescences were displayed more prominently and more conspicuously in UV range. Male nectar analysed by thin-layer chromatography contained a higher glucose content than female nectar. However, both sexes contained hexose-rich nectar, a common indicator of generalist pollination, which was supported by observations of floral visitors. Floral extract comparison conducted via gas chromatography-mass spectrometry showed that male extracts contained more compounds that potentially convey greater resistance to biotic and abiotic threats. Chemical comparison of leaves by high performance liquid chromatography with peak area ratio analysis revealed this technique could be used as a tool for gender identification of individuals during non-flowering periods. Due to the generalist pollination mechanisms of L. leucocephala ssp. robusta, may have an important role in the conservation and support of local insect populations. The presence of chemical biotic and abiotic resistance may also make L. leucocephala ssp. robusta a significant contributor to the ongoing stabilisation of the sand dunes. Conservation efforts required for L. leucocephala ssp. robusta are likely to be minimal, as pollination services are provided by a diversity of pollinating taxa, including introduced species, which will be abundant regardless of variable flowering periods. Further observational study of L. leucocephala ssp. robusta pollinators and differences in pollinator visitation behaviours between sexes is recommended to better understand efficient pollination for the species, and potentially reveal a greater extent of ecosystem benefit for this species.

Background The evolution of dioecious plants is occasionally accompanied by the establishment of sex chromosomes: both XY and ZW systems have been found in plants. Structural studies of sex chromosomes are now being followed up by functional studies that are gradually shedding light on the specific genetic and epigenetic processes that shape the development of separate sexes in plants. Scope This review describes sex determination diversity in plants and the genetic background of dioecy, summarizes recent progress in the investigation of both classical and emerging model dioecious plants and discusses novel findings. The advantages of interspecies hybrids in studies focused on sex determination and the role of epigenetic processes in sexual development are also overviewed. Conclusions We integrate the genic, genomic and epigenetic levels of sex determination and stress the impact of sex chromosome evolution on structural and functional aspects of plant sexual development. We also discuss the impact of dioecy and sex chromosomes on genome structure and expression.