Anatomy and ultrastructure of the floral nectary in Peganum harmala L. (Nitrariaceae)

Anatomy and ultrastructure of the floral nectary of Peganum harmala L. were studied using light and transmission electron microscopy. The floral nectary was visible as a glabrous, regularly five‐lobed circular disc encircling the base of the ovary. Anatomically, it comprised a single layered epidermis and 15–20 layers of small, subepidermal secretory cells overlying several layers of large, ground parenchyma cells. The floral nectary was supplied by phloem and both sieve tubes and companion cells were found adjacent to the ground parenchyma. Based on our ultrastructural observations, plastids of secretory cells during the early stages of development were rich in starch grains and/or osmiophilic plastoglobuli, but these disappeared as nectar secretion progressed. The nectar appeared to exude through the modified stomata along symplastic and apoplastic routes. The abundant plastids and mitochondria suggest an eccrine mechanism of nectar secretion in P. harmala.     

Biosystematic studies inErythronium (Liliaceae-Tulipeae)

The floral biology ofErythronium japonicum has been studied from two approaches: a reinvestigation of its floral morphology and a pollinator case history. The perianth, differentiated into a sepal and petal cycle, has a tubular, but free arrangement basally around a slightly stipitate ovary. The two cycles of stamens with dimorphic filaments are positioned by the differently lobed auricles of the mature sepals and petals. These auricles also form a trap-lid mechanism for the inverted nectary which also has passageways. The perianth parts are highly UV absorbant due to the presence of flavonoids. This pattern contrasts strikingly with the purple trident basal guide lines so prominent in the visible spectrum. The weakly protandrous flowers also have exserted styles, thus functioning to exclude its own pollen and insure outbreeding. These floral adaptations are related specifically to the pollination behavior ofXylocopa appendiculata, and in general to the floral evolution within the genusErythronium. This work was supported in part by the U.S.-Japan Cooperative Science Program Grant GF-41367 and Grant-in-Aid No. 934053 from the Ministry of Education, Japan.     

Androecium and floral nectaries ofHarungana madagascariensis (Clusiaceae)

The mature flower ofHarungana madagascariensis (Choisy)Poir. has an androecium of five antipetalous fascicles, consisting of four stamens each. The stamen fascicles alternate with five indented nectary scales. A SEM-study of the floral development, as well as a study of the floral anatomy was carried out to understand whether the nectariferous scales represent staminodia or are receptacular in nature and consequently whether or not the androecium ofHarungana, and theClusiaceae in general, is originally diplostemonous. The five petals originate by the splitting of petal-stamen complexes. Next the upper part of each complex differentiates basipetally in four stamens. The stamens remain fascicled and are lifted on a long stalk at maturity. Five carpel primordia are initiated united in a low ringwall. The five nectary scales appear after carpel inception and develop an external morphology reminiscent of anthers. The floral anatomy reveals an independent origin of sepal median traces and common sepal lateral traces, free petal traces, stamen fascicle traces and alternating vascular tissue which supplies the nectaries. The petal-stamen complexes are the result of a retardation in petal inception, linked with the absorption of petal tissue into the stamen primordia. The development of the stamen fascicles is discussed; it is suggested that they are of a secondary nature and do not appear as a reduction from a multistaminate androecium. The external morphology and vascular anatomy of the scales speaks in favour of a staminodial nature. The comparison with some other species of theClusiaceae gives evidence of a diplostemonous ancestry of the androecium.     

Floral nectary morphology and evolution in Pedicularis (Orobanchaceae)

Intricate associations between floral morphology and pollinator foraging behaviour are common. In this context, the presence and form of floral nectaries can play a crucial role in driving floral evolution and diversity in flowering plants. However, the reconstruction of the ancestral state of nectary form is often hampered by a lack of anatomical studies and well‐resolved phylogenetic trees. Here, we studied 39 differentially pollinated Pedicularis spp., a genus with pronounced interspecific variation in colour, shape and size of the corolla. Anatomical and scanning electron microscopy observations revealed two nectary forms [bulged (N = 27) or elongated (N = 5)] or the absence of nectaries (N = 7). In a phylogenetic context, our data suggest that: (1) the bulged nectary should be the ancestral state; (2) nectaries were independently lost in some beaked species; and (3) elongated nectaries evolved independently in some clades of beakless species. Phylogenetic path analysis showed that nectary presence is indirectly correlated with beak length/pollinator behaviour through an intermediate factor, nectar production. No significant correlation was found between nectary type and nectar production, beak length or pollinator behaviour. Some beaked species had nectary structures, although they did not produce nectar. The nectary in beaked species may be a vestigial structure retained during a recent rapid radiation of Pedicularis, especially in the Himalaya–Hengduan Mountains of south‐western China. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 592–607.     

Is nectar reabsorption restricted by the stalk cells of floral and extrafloral nectary trichomes?

Reabsorption is a phase of nectar dynamics that occurs concurrently with secretion; it has been described in floral nectaries that exude nectar through stomata or unicellular trichomes, but has not yet been recorded in extrafloral glands. Apparently, nectar reabsorption does not occur in multicellular secretory trichomes (MST) due to the presence of lipophilic impregnations – which resemble Casparian strips – in the anticlinal walls of the stalk cells. It has been assumed that these impregnations restrict solute movement within MST to occur unidirectionally and exclusively by the symplast, thereby preventing nectar reflux toward the underlying nectary tissues. We hypothesised that reabsorption is absent in nectaries possessing MST. The fluorochrome lucifer yellow (LYCH) was applied to standing nectar of two floral and extrafloral glands of distantly related species, and then emission spectra from nectary sections were systematically analysed using confocal microscopy. Passive uptake of LYCH via the stalk cells to the nectary tissues occurred in all MST examined. Moreover, we present evidence of nectar reabsorption in extrafloral nectaries, demonstrating that LYCH passed the stalk cells of MST, although it did not reach the deepest nectary tissues. Identical (control) experiments performed with neutral red (NR) demonstrated no uptake of this stain by actively secreting MST, whereas diffusion of NR did occur in plasmolysed MST of floral nectaries at the post‐secretory phase, indicating that nectar reabsorption by MST is governed by stalk cell physiology. Interestingly, non‐secretory trichomes failed to reabsorb nectar. The role of various nectary components is discussed in relation to the control of nectar reabsorption by secretory trichomes.     

Genetic and evolution analysis of extrafloral nectary in cotton

Extrafloral nectaries are a defence trait that plays important roles in plant–animal interactions. Gossypium species are characterized by cellular grooves in leaf midribs that secret large amounts of nectar. Here, with a panel of 215 G. arboreum accessions, we compared extrafloral nectaries to nectariless accessions to identify a region of Chr12 that showed strong differentiation and overlapped with signals from GWAS of nectaries. Fine mapping of an F₂ population identified GaNEC1, encoding a PB1 domain‐containing protein, as a positive regulator of nectary formation. An InDel, encoding a five amino acid deletion, together with a nonsynonymous substitution, was predicted to cause 3D structural changes in GaNEC1 protein that could confer the nectariless phenotype. mRNA‐Seq analysis showed that JA‐related genes are up‐regulated and cell wall‐related genes are down‐regulated in the nectary. Silencing of GaNEC1 led to a smaller size of foliar nectary phenotype. Metabolomics analysis identified more than 400 metabolites in nectar, including expected saccharides and amino acids. The identification of GaNEC1 helps establish the network regulating nectary formation and nectar secretion, and has implications for understanding the production of secondary metabolites in nectar. Our results will deepen our understanding of plant–mutualism co‐evolution and interactions, and will enable utilization of a plant defence trait in cotton breeding efforts.     

FLORAL DEVELOPMENT IN CAESALPINIA (LEGUMINOSAE)

Utilizing scanning electron microscopy, we studied the early floral ontogeny of three species of Caesalpinia (Leguminosae: Caesalpinioideae): C. cassioides, C. pulcherrima, and C. vesicaria. Interspecific differences among the three are minor at early and middle stages of floral development. Members of the calyx, corolla, first stamen whorl, and second stamen whorl appear in acropetal order, except that the carpel is present before appearance of the last three inner stamens. Sepals are formed in generally unidirectional succession, beginning with one on the abaxial side next to the subtending bracts, followed by the two lateral sepals and adaxial sepal, then lastly the other adaxial sepal. In one flower of C. vesicaria, sepals were helically initiated. In the calyx, the first-initiated sepal maintains a size advantage over the other four sepals and eventually becomes cucullate, enveloping the remaining parts of the flower. The cucullate abaxial sepal is found in the majority of species of the genus Caesalpinia. Petals, outer stamens, and inner stamens are formed unidirectionally in each whorl from the abaxial to the adaxial sides of the flower. Abaxial stamens are present before the last petals are visible as mounds on the adaxial side, so that the floral apex is engaged in initiation of different categories of floral organs at the same time.     

Structure of the receptacular nectary and circadian metabolism of starch in the ant‐guarded plant Ipomoea cairica (Convolvulaceae)

Nectaries occur widely in Convolvulaceae. These structures remain little studied despite their possible importance in plant–animal interactions. In this paper, we sought to describe the structure and ultrastructure of the receptacular nectaries (RNs) of Ipomoea cairica, together with the dynamics of nectar secretion. Samples of floral buds, flowers at anthesis and immature fruits were collected, fixed and processed using routine methods for light, scanning and transmission electron microscopy. Circadian starch dynamics were determined through starch measurements on nectary sections. The secretion samples were subjected to thin layer chromatography. RNs of I. cairica were cryptic, having patches of nectar‐secreting trichomes, subglandular parenchyma cells and thick‐walled cells delimiting the nectary aperture. The glandular trichomes were peltate type and had typical ultrastructural features related to nectar secretion. The nectar is composed of sucrose, fructose and glucose. Nectar secretion was observed in young floral buds and continued as the flower developed, lasting until the fruit matured. The starch content of the subglandular tissue showed circadian variation, increasing during the day and decreasing at night. The plastids were distinct in different portions of the nectary. The continuous day–night secretory pattern of the RNs of I. cairica is associated with pre‐nectar source circadian changes in which the starch acts as a buffer, ensuring uninterrupted nectar secretion. This circadian variation may be present in other extrafloral nectaries and be responsible for full daytime secretion. We conclude that sampling time is relevant in ultrastructural studies of dynamic extranuptial nectaries that undergo various changes throughout the day.     

An APETALA3 homolog controls both petal identity and floral meristem patterning in Nigella damascena L. (Ranunculaceae)

Flower architecture mutants provide a unique opportunity to address the genetic origin of flower diversity. Here we study a naturally occurring floral dimorphism in Nigella damascena (Ranunculaceae), involving replacement of the petals by numerous sepal‐like and chimeric sepal/stamen organs. We performed a comparative study of floral morphology and floral development, and characterized the expression of APETALA3 and PISTILLATA homologs in both morphs. Segregation analyses and gene silencing were used to determine the involvement of an APETALA3 paralog (NdAP3–3) in the floral dimorphism. We demonstrate that the complex floral dimorphism is controlled by a single locus, which perfectly co‐segregates with the NdAP3–3 gene. This gene is not expressed in the apetalous morph and exhibits a particular expression dynamic during early floral development in the petalous morph. NdAP3–3 silencing in petalous plants perfectly phenocopies the apetalous morph. Our results show that NdAP3–3 is fully responsible for the complex N. damascena floral dimorphism, suggesting that it plays a role not only in petal identity but also in meristem patterning, possibly through regulation of perianth organ number and the perianth/stamen boundary.     

Functional decoupling between flowers and leaves in the Ameroglossum pernambucense complex can facilitate local adaptation across a pollinator and climatic heterogeneous landscape

Decoupling between floral and leaf traits is expected in plants with specialized pollination systems to assure a precise flower–pollinator fit, irrespective of leaf variation associated with environmental heterogeneity (functional modularity). Nonetheless, developmental interactions among floral traits also decouple flowers from leaves regardless of selection pressures (developmental modularity). We tested functional modularity in the hummingbird‐pollinated flowers of the Ameroglossum pernambucense complex while controlling for developmental modularity. Using two functional traits responsible for flower–pollinator fit [floral tube length (TL) and anther–nectary distance (AN)], one floral trait not linked to pollination [sepal length (SL), control for developmental modularity] and one leaf trait [leaf length (LL)], we found evidence of flower functional modularity. Covariation between TL and AN was ca. two‐fold higher than the covariation of either of these traits with sepal and leaf lengths, and variations in TL and AN, important for a precise flower–pollinator fit, were smaller than SL and LL variations. Furthermore, we show that previously reported among‐population variation of flowers associated with local pollinator phenotypes was independent from SL and LL variations. These results suggest that TL and AN are functionally linked to fit pollinators and sufficiently decoupled from developmentally related floral traits (SL) and vegetative traits (LL). These results support previous evidences of population differentiation due to local adaptation in the A. pernambucense complex and shed light on the role of flower–leaf decoupling for local adaptation in species distributed across biotic and abiotic heterogeneous landscapes.     

Floral morphology and structure of Phyllonoma (Phyllonomaceae): systematic and evolutionary implications

Phyllonoma, a small tree genus of four species distributed from Mexico to Peru, has been placed in various families (mainly in Saxifragaceae), but now, based on molecular evidence, is placed in a distinct family Phyllonomaceae in Aquifoliales. To better understand the morphological relationships of the genus and family, I studied its floral morphology, anatomy, and vasculature using P. tenuidens. Most of the external and internal floral characteristics were described more than 120 years ago. Although some of them were confirmed, some were substantially revised, mainly those concerning the gynoecial structure. Flowers are small and basically pentamerous, consisting of five sepals, five petals, five stamens, and a gynoecium composed of two carpels usually in transversal position. Comparisons with other Aquifoliales show that Phyllonomaceae share the inferior ovary, epiphyllous inflorescence and epigynous disc nectary with East-Asian Helwingiaceae (Helwingia only), but clearly differ from Helwingiaceae in having glandular trichomes on the sepal margins and a bicarpellate, unilocular gynoecium bearing many ovules on the parietal placentae. Evidence from floral morphology and structure supports the distinctness of Phyllonomaceae and its sister-group relationship with Helwingiaceae. Its floral characteristics suggest that Phyllonomaceae have evolved by adapting to distinct biological habitats in relation to pollination and seed dispersal.     

GoNe encoding a class VIIIb AP2/ERF is required for both extrafloral and floral nectary development in Gossypium

The floral nectary, first recognized and described by Carl Linnaeus, is a remarkable organ that serves to provide carbohydrate-rich nectar to visiting pollinators in return for gamete transfer between flowers. Therefore, the nectary has indispensable biological significance in plant reproduction and even in evolution. Only two genes, CRC and STY, have been reported to regulate floral nectary development. However, it is still unknown what genes contribute to extrafloral nectary development. Here, we report that a nectary development gene in Gossypium (GoNe), annotated as an APETALA 2/ethylene-responsive factor (AP2/ERF), is responsible for the formation of both floral and extrafloral nectaries. GoNe plants that are silenced via virus-induced gene silencing technology and/or knocked out by Cas9 produce a nectariless phenotype. Point mutation and gene truncation simultaneously in duplicated genes Ne₁Ne₂ lead to impaired nectary development in tetraploid cotton. There is no difference in the expression of the CRC and STY genes between the nectary TM-1 and the nectariless MD90ne in cotton. Therefore, the GoNe gene responsible for the formation of floral and extrafloral nectaries may be independent of CRC and STY. A complex mechanism might exist that restricts the nectary to a specific position with different genetic factors. Characterization of these target genes regulating nectary production has provided insights into the development, evolution, and function of nectaries and insect-resistant breeding.     

Cunoniaceae in the Cretaceous of Europe: Evidence from Fossil Flowers

Fossil flowers of the Cunoniaceae from Late Cretaceous sedimentsof southern Sweden are described in detail. The flowers aresmall, bisexual, actinomorphic, tetramerous with broadly attachedvalvate sepals; they have narrowly attached petals; eight stamensin two whorls; a massive, lobed nectary; a semi-inferior, syncarpousgynoecium with axile placentation; numerous ovules; separatestyles; and peltate, probably secretory, trichomes. They sharemany features with extant representatives of both the Cunoniaceaeand Anisophylleaceae. However, the gynoecium structure in particularindicates a closer relationship to the Cunoniaceae. The floralcharacters are not specific for any extant genus of the familyand therefore a new genus and species, Platydiscus peltatusgen. et sp. nov., is formally described. This is the first recordof cunoniaceous floral structures from the Northern Hemisphereand the oldest record of Cunoniaceae flowers worldwide. Copyright2001 Annals of Botany Company Anisophylleaceae, Cunoniaceae, fossil flowers, Late Cretaceous, Oxalidales, Platydiscus peltatus gen. et sp. nov., Santonian-Campanian, southern Sweden     

Influence of elevated CO2 and ultraviolet-B radiation levels on floral nectar production: a nectary-morphological perspective

 Investigations of the effects of two global events – elevated CO₂ levels and enhanced ultraviolet-B (UV-B) radiation – on floral nectar production are reviewed from twelve dicotyledonous families. Furthermore, to allow comparisons between nectary morphology and nectar production in treated plants of these fifteen species, new data on floral nectary structure are provided for Malcolmia maritima (L.) R. Br. (Brassicaceae) and Scabiosa columbaria L. (Dipsacaceae). All but the last taxon possessed mesenchymatic floral nectaries with surface stomata. Few clear relationships existed between nectary morphology and various physiological responses to CO₂ or UV-B enrichment, indicating that species responded notwithstanding nectary structure itself. Overall, nectar-solute concentration was least affected by elevated CO₂ or UV-B radiation; consequently, changes in nectar volume were responsible for differences in nectar-sugar production per flower. Three species of Fabaceae experienced no change in floral nectar production upon exposure to elevated CO₂. To date, no study of enhanced UV-B radiation reported a consistent reduction in floral nectar production; three species of Brassicaceae responded differently, but various levels of ozone depletion were simulated. Experimentation with more taxa – including those possessing nectary types such as septal (gynopleural) nectaries (e.g. many monocotyledons) or aggregations of glandular trichomes – and expanding such physiological studies to species possessing extrafloral nectaries, are recommended. Received August 8, 2002; accepted November 23, 2002 Published online: June 2, 2003     

DISTRIBUTION OF DEFENSE NECTARIES IN IPOMOEA (CONVOLVULACEAE)

The structure and distribution of defense nectaries in the genus Ipomoea (Convolvulaceae) were investigated. These nectaries do not reward pollinators and probably contribute to antiherbivore defense. Of 22 species sectioned, 15 had defense nectaries on the sepals. Of 12 other species observed, ten had sepal nectaries and two did not. Structurally, 14 of the species sectioned had crypt sepal nectaries and one had a basin nectary. Of the 14 species with crypt nectaries, two had invaginated spaces adding greatly to the internal area of these nectaries, and forming the most complex nectaries that have been reported. We term these labyrinthine crypt nectaries. All three types of nectaries are lined with secretory trichomes along the proximal surfaces of the crypts. Species with defense nectaries on the sepals tend to have petiolar defense nectaries as well, but the two locations may have different nectary types; e.g., basins on the petiole and crypts on the sepals. Since most reports of the function of these nectaries have shown antiherbivore defense by nectar feeders, the distributions of defense nectaries with respect to region and life history of the species were sought. Plants without sepal nectaries were found to have significantly smaller seeds than plants with sepal nectaries; they were also more frequently annuals. No significant relationship was found between region or breeding system and defense nectaries.     

How do plants balance multiple mutualists? Correlations among traits for attracting protective bodyguards and pollinators in cotton (Gossypium)

Many species, both plants and animals, are simultaneously engaged in interactions with multiple mutualists. However, the extent to which separate traits that attract different mutualist guilds display negative or positive relationships remains largely unstudied. We asked whether correlations exist among extrafloral nectary traits to attract arthropod bodyguards and floral traits to attract pollinator mutualists. For 37 species in the cotton genus (Gossypium), we evaluated correlations among six extrafloral nectary traits and four floral traits in a common greenhouse environment, with and without correction for phylogenetic non-independence. Across Gossypium species, greater investment in extrafloral nectary traits was positively correlated with greater investment in floral traits. Positive correlations remained after accounting for the evolutionary history of the clade. Our results demonstrate that traits to maintain multiple mutualist guilds can be positively correlated across related species and build a more general understanding of the constraints on trait evolution in plants.     

Fruit production and floral traits: correlated evolution in <Emphasis Type="Italic">Govenia</Emphasis> (Orchidaceae)

The most common estimate of reproductive success in orchids is usually fruit set. Factors such as resource limitation and certain floral traits may influence reproductive success in animal-pollinated plants. Correlated evolution of reproductive success vs. seven floral traits (inflorescence length, flower number, flower distribution along inflorescence, dorsal sepal length, lateral sepal length, flower color, and column position) was studied in eight species of Govenia. Taxa represented three lineages in the genus. Independent contrasts were calculated on a phylogeny inferred from chloroplast (trnL-F IGS) DNA sequences, and a correlation test and multiple regression were then performed. Two data sets were evaluated, one including all eight species and another excluding G. utriculata, which is autogamous. The historical analyses showed that there is a correlation between reproductive success and dorsal sepal length, column position, and flower number, these correlations suggest that changes in these floral traits usually accompany evolutionary shifts in reproductive success. Multiple regression tests suggest that changes in reproductive success can be explained by shifts in flower number, inflorescence length, column position and by dorsal sepal length. When phylogeny is taken into account, our analyses showed that evolutionary shifts in these floral traits were correlated with changes in reproductive success. Evolutionary correlation between reproductive success and floral traits might be explained by the natural selection of certain floral phenotypes by pollinators.     

蓝堇草属(毛茛科)花形态发生的扫描电子显微镜观察

花的发生和发育过程研究可以发现早期进化的轨迹,为系统发育的研究提供重要线索。蓝堇草属(Leptopyrum)为毛茛科唐松草亚科一单种属,仅包含蓝堇草一种,其花的发生和发育过程仍为空白。为了深入理解唐松草亚科乃至毛茛科花发育多样性和演化规律,该文运用扫描电子显微镜(SEM)观察了蓝堇草各轮花器官的形态发生和发育过程。结果表明:该属植物所有的萼片、花瓣、雄蕊和雌蕊均为螺旋状发生,花器官排列式样也为螺旋状; 5枚萼片原基宽阔,5枚花瓣原基圆球形、位于萼片原基的间隔,且在后期表现为延迟发育现象,雄蕊原基较小、为圆球形;花瓣原基和雄蕊原基连续发生,无明显的时空间隔,但与萼片原基有时空间隔;心皮原基为马蹄形对折,柱头组织由单细胞乳突组成;胚珠倒生、具单珠被。该属花器官螺旋状排列、胚珠具单珠被在唐松草亚科中是独有的性状,花发育形态学证据支持了该属的特殊性。     

Anatomy and ultrastructure of the floral nectary of Tontelea micrantha (Celastraceae: Salacioideae)

Among several native species of the Brazilian cerrado, a shrub, Tontelea micrantha, is exploited by traditional communities for the valuable oil extracted from its seeds, which has anti‐inflammatory properties. There have been no studies on the anatomy of its flower, and so the aim of this study is to describe the anatomy and ultrastructure of its floral nectary. Flower buds and flowers in anthesis were collected, fixed and processed for light and electron microscopy. The discoid floral nectary is composed of epidermis and a secretory parenchyma. Secretory cells are rich in plastids with starch grains and mitochondria. The nectar, sucrose dominant, is just sufficient to form a thin film on the nectary. The secretory cells show starch and oil droplets; however, during nectar production there is no evidence of hydrolysis of starch and some lipid reserves remain unchanged. Our results suggest a reduction in the amount of oil in the secretory cells during the secretory phase but this does not appear to imply a release of oil as a nectar component. In addition to maintaining part of the reserves, the lower frequency of organelles involved in nectar synthesis reinforces the hypothesis that phloem sap is the origin of nectar sugars. The tiny nectar film, released through modified stomata, is attractive to small insects such as flies. Considering the importance and intensity of use of T. micrantha in the Brazilian cerrado, we think that these data about its floral nectary can help to better explain its reproductive biology with positive impacts on its management and conservation.