Floral glands in myophilous and sapromyophilous species of Pleurothallidinae (Epidendroideae, Orchidaceae)-osmophores, nectaries, and a unique sticky gland.

Pleurothallidinae orchids have been the focus of many multidisciplinary studies due to their challenging systematics and taxonomy. The synapomorphies already recognized in the group are mostly related to floral characters, the last proposed being the occurrence of alkanes in the floral fragrance. The composition of the floral bouquet varied significantly among the studied species, leading us to hypothesize that the variations in volatiles emitted could be linked to the structure of osmophores, especially when comparing the myophilous and sapromyophilous pollination syndromes. Sepals and labellum at different developmental stages of seven Brazilian Pleurothallidinae species were examined using light, scanning, and transmission electron microscopy. Nectar reabsorption was assessed by Lucifer Yellow CH tracer and imaged under confocal microscopy. Nectaries were restricted to the labellum of the myophilous species, whereas osmophores occurred in the dorsal and/or lateral sepals, varying according to species. In the sapromyophilous species, floral nectaries were not detected and osmophores were restricted to the labellum. Osmophore structure was correlated with the volatiles emitted, being the trichome osmophores notably present on the sepals of both myophilous species that possess nectaries. For the first time, we demonstrated reabsorption of the released nectar in Pleurothallidinae and the occurrence of a unique gland named sticky-exudate glands, which occurred in the lateral sepals and labellum of Echinosepala aspasicensis, a sapromyophilous species, that released a heterogeneous exudate composed of polysaccharides and lipids. Similar glands have been reported in Bulbophyllum, highlighting the convergence between both groups.

Molecular identification of Stylops advarians (Strepsiptera: Stylopidae) in western Canada.

Strepsiptera are an enigmatic order of insects with extreme sexual dimorphism which makes it difficult to "match-up" free-living adult males with parasitic conspecific females of the Stylopidia, and free-living females of the Mengenillidae using morphological characters. Species identification is further complicated for the Stylopidia because adult females are endoparasitic and neotenic. Therefore, we used DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) to confirm the species identity of adult strepsipterans that were morphologically identified as Stylops advarians. These specimens, collected from Saskatoon (Saskatchewan, Canada), included one adult male, and eight females, the latter of which had been collected from solitary bees (Andrena milwaukeensis). Also included in the analyses were three pools of first-instar larvae that had emerged from three of the females. The results of the molecular analyses revealed that all specimens had an identical cox1 sequence, and belonged to a clade, with total statistical support (bootstrap value of 100%), that contained specimens of S. advarians from New York and Maine (USA). Hence, the results were consistent with the morphological identification of S. advarians. This study demonstrates the usefulness of a molecular approach for the identification of endoparasitic adult female and larval strepsipterans, life cycle stages that lack significant morphological characters for species identification.

Description of the Adult Male of Stylops nubeculae Pierce (Strepsiptera: Stylopidae), a New Record for Canada.

The morphology of the adult male of Stylops nubeculae Pierce, encountered in stylopized gasters of two adult bees of Andrena peckhami, is described for the first time. This species was previously known only from the endoparasitic adult female found in Colorado, USA. We report a new locality for this species in Alberta, Canada.

Life history traits and interactions of Stylops advarians (Strepsiptera) with its bee host, Andrena milwaukeensis.

Specimens of Stylops advarians were sampled by collecting foraging bees of Andrena milwaukeensis along the South Saskatchewan River within Saskatoon, Saskatchewan. As the foraging season progressed from early May till late June over three consecutive years (2016-2018), most stylopized bees possessed endoparasitic adult (neotenic) females of S. advarians protruding from the bee gaster's dorsum. In contrast, very few adult bees stylopized by male puparia, and no free-living males, were encountered. Over the sampling period, prevalence remained around 22% each year; mean intensity was 1.2 (range of 1-3 female parasites per bee); and parasite abundance was 0.27. Also newly reported for Stylops is the occurrence of one bee bearing four Stylops (two neotenic females and two males with puparia), plus another bee with a male puparium extruded from its gaster's sternites. Around 2 May each year, a high proportion of the earliest captured female bees were stylopized. However, non-stylopized female bees typically were not encountered until about 10 days later, suggesting the parasites manipulate female bee hosts to emerge earlier, in close synchrony to male bee emergence. First-instar larvae of S. advarians appeared from 22-25 May, indicating that adults of S. advarians matured and mated at similar times each season.

Description of the Adult Male of Stylops advarians Pierce (Strepsiptera: Stylopidae).

The morphology of the adult male of Stylops advarians Pierce is described for the first time. This species was previously known only from the endoparasitic adult female and the host-seeking, first-instar larva. Members of Stylops are cosmopolitan, and Stylops advarians can be found parasitizing Andrena milwaukeensis Graenicher in western Canada.

Adaptive morphology of the host-seeking first-instar larva of Stylops advarians Pierce (Strepsiptera, Stylopidae), a parasite of Andrena milwaukeensis Graenicher (Hymenoptera, Andrenidae).

The morphology of the prognathous, host-seeking first-instar larvae of Stylops advarians was examined to advance our understanding of their adaptations to reach immature bee hosts, a process requiring temporal phoresy on an adult bee. Sensory structures on the larval head, including eye spots and two pairs of olfactory pits, evidently assist recognition of an adult bee and eventual detection of a permanent host within a nest cell. First-instar larvae utilize various features of their appendages to travel securely on their phoretic host. Flexible adhesive tarsi of the pro- and mesothoracic legs allow them to embark and be retained on a flying bee. The tips of the pair of caudal filaments appear modified for a similar purpose. Spinulae of two lengths, and arranged in distinct patterns, cover the posterior edges of the thoracic and abdominal segments both dorsally and ventrally. These projections can cause lodging of larvae in the plumose hairs of the phoretic host, and may lock into the exine of pollen collected by the foraging bee. Discovery of a first-instar larva partially packed into a pollen load and in the crop of Andrena milwaukeensis demonstrates that Stylops is adapted to travel with a phoretic host both externally and internally.

Pollen, ovules, and pollination in pea: Success, failure, and resilience in heat.

Field pea (Pisum sativum), a major grain legume crop, is autogamous and adapted to temperate climates. The objectives of this study were to investigate effects of high temperature stress on stamen chemical composition, anther dehiscence, pollen viability, pollen interactions with pistil and ovules, and ovule growth and viability. Two cultivars ("CDC Golden" and "CDC Sage") were exposed to 24/18°C (day/night) continually or to 35/18°C for 4 or 7 days. Heat stress altered stamen chemical composition, with lipid composition of "CDC Sage" being more stable compared with "CDC Golden." Heat stress reduced pollen viability and the proportion of ovules that received a pollen tube. After 4 days at 35°C, pollen viability in flower buds decreased in "CDC Golden," but not in "CDC Sage." After 7 days, partial to full failure of anthers to dehisce resulted in subnormal pollen loads on stigmas. Although growth (ovule size) of fertilized ovules was stimulated by 35°C, heat stress tended to decrease ovule viability. Pollen appears susceptible to stress, but not many grains are needed for successful fertilization. Ovule fertilization and embryos are less susceptible to heat, but further research is warranted to link the exact degree of resilience to stress intensity.

Seed set, pollen morphology and pollen surface composition response to heat stress in field pea.

Pea (Pisum sativum L.) is a major legume crop grown in a semi-arid climate in Western Canada, where heat stress affects pollination, seed set and yield. Seed set and pod growth characteristics, along with in vitro percentage pollen germination, pollen tube growth and pollen surface composition, were measured in two pea cultivars (CDC Golden and CDC Sage) subjected to five maximum temperature regimes ranging from 24 to 36 °C. Heat stress reduced percentage pollen germination, pollen tube length, pod length, seed number per pod, and the seed-ovule ratio. Percentage pollen germination of CDC Sage was greater than CDC Golden at 36 °C. No visible morphological differences in pollen grains or the pollen surface were observed between the heat and control-treated pea. However, pollen wall (intine) thickness increased due to heat stress. Mid-infrared attenuated total reflectance (MIR-ATR) spectra revealed that the chemical composition (lipid, proteins and carbohydrates) of each cultivar's pollen grains responded differently to heat stress. The lipid region of the pollen coat and exine of CDC Sage was more stable compared with CDC Golden at 36 °C. Secondary derivatives of ATR spectra indicated the presence of two lipid types, with different amounts present in pollen grains from each cultivar.

The relationship between cell division and elongation during development of the nectar-yielding petal spur in Centranthus ruber (Valerianaceae).

BACKGROUND AND AIMS: Floral spurs are hollow, tubular outgrowths that typically conceal nectar. By their involvement in specialized pollinator interactions, spurs have ecological and evolutionary significance, often leading to speciation. Despite their importance and diversity in shape and size among angiosperm taxa, detailed investigations of the mechanism of spur development have been conducted only recently. METHODS: Initiation and growth of the nectar-yielding petal spur of Centranthus ruber 'Snowcloud' was investigated throughout seven stages, based on bud size and developmental events. The determination of the frequency of cell division, quantified for the first time in spurs, was conducted by confocal microscopy following 4',6-diamidino-2-phenylindole (DAPI) staining of mitotic figures. Moreover, using scanning electron microscospy of the outer petal spur surface unobstructed by trichomes, morphometry of epidermal cells was determined throughout development in order to understand the ontogeny of this elongate, hollow tube. KEY RESULTS: Spur growth from the corolla base initially included diffuse cell divisions identified among epidermal cells as the spur progressed through its early stages. However, cell divisions clearly diminished before a petal spur attained 30 % of its final length of 4·5 mm. Thereafter until anthesis, elongation of individual cells was primarily responsible for the spur's own extension. Consequently, a prolonged period of anisotropy, wherein epidermal cells elongated almost uniformly in all regions along the petal spur's longitudinal axis, contributed principally to the spur's mature length. CONCLUSIONS: This research demonstrates that anisotropic growth of epidermal cells - in the same orientation as spur elongation - chiefly explains petal spur extension in C. ruber. Representing the inaugural investigation of the cellular basis for spur ontogeny within the Euasterids II clade, this study complements the patterns in Aquilegia species (order Ranunculales, Eudicots) and Linaria vulgaris (order Lamiales, Euasterids I), thereby suggesting the existence of a common underlying mechanism for petal spur ontogeny in disparate dicot lineages.

Annular floral nectary with oil-producing trichomes in Salvia farinacea (Lamiaceae): Anatomy, histochemistry, ultrastructure, and significance.

PREMISE OF THE STUDY: Many angiosperms produce nectar that entices pollinator visits. Each floral nectary tends to embody a singular form, such as the receptacular ring arising beneath the ovary in mint flowers (Lamiaceae). Exceptionally, the annular floral nectary in Salvia farinacea possesses modified stomata plus secretory trichomes. This first study of nectary ultrastructure within the largest genus of Lamiaceae examined this unusual condition. METHODS: Nectary anatomy, histochemistry, and ultrastructure were investigated from fresh and fixed material using light microscopy and scanning electron and transmission electron microscopy. KEY RESULTS: The annular nectary encircled the ovary plus extended ventrally as a projection. Modified stomata occurred only in the projection's abaxial epidermis. Conversely, peltate trichomes with a basal cell, a stalk cell, and 4-7 head cells were interspersed among the ovary lobes and covered the projection's adaxial surface. Phloem and xylem supplied the nectary interior, where parenchyma cells had numerous mitochondria and plastids with little starch, but few dictyosomes and little endoplasmic reticulum. Nectar accumulated as a drop opposite the projection's abaxial surface, escaping through stomatal pores and probably the cuticle. However, the annular nectary's glistening trichomes secreted a Sudan-positive product largely retained below the distended cuticle, but not nectar. CONCLUSIONS: This first ultrastructural study of co-occurring secretory trichomes and modified stomata on a mint nectary suggests multiple interactive functions for this atypical structure. These trichomes-possibly generating a substance informative to pollinators or as an ovarian defense against phytophagy-produced oil in an aqueous milieu, rather than contributing fluid to nectar.

Evaluation of inflorescence visitors as pollinators of Echinacea angustifolia (Asteraceae): comparison of techniques.

Inflorescences (heads or capitula) of the putative self-incompatible species, purple coneflower (Echinacea angustifolia (DC) Cronq. (Asteraceae)), were visited by insects representing the Coleoptera, Diptera, Hymenoptera, and Lepidoptera, in accordance with a generalist pollination syndrome. Measurement of the effectiveness of insect species as pollinators was accomplished by permitting solitary visits to receptive, central disc florets of virgin (previously bagged) heads. Four parameters were quantified: total stigmatic pollen load and proportion of pollen grains germinated, numbers of pollen tubes at style bases, and percentages of total receptive florets that had retracted (shrivelled) styles. Quantifying total and germinated pollen grains proved ineffective, partly owing to the tendency of self-pollen to initiate pollen tubes. The most effective pollinators were Apidae, especially bumble bees (Bombus spp.) and the European honey bee (Apis mellifera L.) (mean: 39 - 61% of styles retracted). Other noteworthy pollinators were cloudless sulfur butterflies (Phoebis sennae L.--Pieridae; mean 47% of style bases with pollen tubes), golden blister beetles (Epicauta ferruginea Say--Meloidae; 44%), and grasshopper bee flies (Systoechus vulgaris Loew--Bombyliidae; 22%). Sunflower leafcutter bees (Megachile pugnata Say) were less effective (4% of styles retracted). Promisingly, analysis of the proportion of retracted styles provided similar results to the established technique of pollen-tube quantification, but had the significant advantages of being completed more rapidly, without a microscope, and in the field. The quantitative technique of retracted-style analysis appears well suited for prompt measurement of inflorescence-visiting insects as pollinators of many asteraceans.

Ontogeny of floral organs in flax (Linum usitatissimum; Linaceae).

PREMISE OF THE STUDY: Flax (Linum usitatissimum) is an important crop worldwide; however, a detailed study on flower development of this species is lacking. Here we describe the pattern of initiation and a program of key developmental events in flax flower ontogeny. This study provides important fundamental information for future research in various aspects of flax biology and biotechnology. METHODS: Floral buds and organs were measured throughout development and examined using scanning electron microscopy. KEY RESULTS: Floral organs were initiated in the following sequence: sepals, stamens and petals, gynoecium, and nectaries. The five sepals originated in a helical pattern, followed evidently by simultaneous initiation of five stamens and five petals, the former opposite of the sepals and the latter alternate to them. The gynoecium, with five carpels, was produced from the remaining, central region of the floral apex. Stamens at early stages were dominated by anther growth but filaments elongated rapidly shortly before anthesis. Early gynoecium development occurred predominantly in the ovary, and ovule initiation began prior to enclosure of carpels. A characteristic feature was the twisted growth of styles, accompanied by the differentiation of papillate stigmas. Petal growth lagged behind that of other floral organs, but petals eventually grew rapidly to enclose the inner whorls after style elongation. Flask-shaped nectaries bearing stomata developed on the external surface of the filament bases. CONCLUSIONS: This is the first detailed study on flax floral organ development and has established a key of 12 developmental stages, which should be useful to flax researchers.

Floral nectar production and nectary anatomy and ultrastructure of Echinacea purpurea (Asteraceae).

BACKGROUND AND AIMS: In spite of the impressive species diversity in the Asteraceae and their widespread appeal to many generalist pollinators, floral-nectary ultrastructure in the family has rarely been investigated. To redress this, a study using Echinacea purpurea, a plant of horticultural and nutraceutical value, was undertaken. Nectar secretion of disc florets was compared with floral nectary ultrastructure taking into account nectar's potential impact upon the reproductive success of this outcrossing species. METHODS: Micropipette collections of nectar in conjunction with refractometry were used to determine the volume and nectar-sugar quantities of disc florets throughout their phenology, from commencement of its production to cessation of secretion. Light, scanning-electron and transmission-electron microscopy were utilized to examine morphology, anatomy and ultrastructure of nectaries of the disc florets. KEY RESULTS: Florets were protandrous with nectar being secreted from anthesis until the third day of the pistillate phase. Nectar production per floret peaked on the first day of stigma receptivity, making the two innermost whorls of open florets most attractive to foraging visitors. Modified stomata were situated along the apical rim of the collar-like nectary, which surrounds the style base and sits on top of the inferior ovary. The floral nectary was supplied by phloem only, and both sieve elements and companion cells were found adjacent to the epidermis; the latter participated in the origin of some of the precursor cells that yielded these specialized cells of phloem. Companion cells possessed wall ingrowths (transfer cells). Lobed nuclei were a key feature of secretory parenchyma cells. CONCLUSIONS: The abundance of mitochondria suggests an eccrine mechanism of secretion, although dictyosomal vesicles may contribute to a granulocrine process. Phloem sap evidently is the main contributor of nectar carbohydrates. From the sieve elements and companion cells, an apoplastic route via intercellular spaces and cell walls, leading to the pores of modified stomata, is available. A symplastic pathway, via plasmodesmata connecting sieve elements to companion, parenchyma and epidermal cells, is also feasible. Uncollected nectar was reabsorbed, and the direct innervation of the nectary by sieve tubes potentially serves a second important route for nectar-sugar reclamation. Microchannels in the outer cuticle may facilitate both secretion and reabsorption.

Comparative structure and pollen production of the stamens and pollinator-deceptive staminodes of Commelina coelestis and C. dianthifolia (Commelinaceae).

BACKGROUND AND AIMS: Flowers of Commelina coelestis and C. dianthifolia provide pollen alone as a floral reward, and rely on visual cues to attract pollinators. Three stamen types, all producing pollen, occur in each of these species: two cryptically coloured lateral stamens, a single cryptically coloured central stamen and three bright yellow staminodes that sharply contrast with the blue to purple corolla. The objective was to compare the stamen structure and pollen characteristics of each of the three stamen types, and to test the hypothesis that the staminodes are poor contributors of viable pollen for the siring of seed. The pollination roles of the three stamen types and the breeding systems of both species were also explored. METHODS: Light, fluorescence and scanning electron microscopy were utilized to examine stamen morphology and pollen structure and viability. Controlled hand pollinations were used to explore the breeding system of each species. Filament and style lengths were measured to investigate herkogamy and autogamy. KEY RESULTS: Pollen from all stamen morphs is viable, but staminode pollen has significantly lower viability. Pollen polymorphism exists both (a) between the lateral and central stamens and the staminodes, and (b) within each anther. Lateral and central stamens have thicker endothecia with a greater number of secondary cell wall thickenings than the staminodes. CONCLUSIONS: Both species are entomophilous and facultatively autogamous. Lateral stamen pollen is important for cross-pollination, central stamen pollen is utilized by both species as a pollinator reward and for delayed autogamy in C. dianthifolia, and the staminodes mimic, by means of both colour and epidermal features, large amounts of pollen to attract insects to the flowers. Pollen from all three anther morphs is capable of siring seed, although staminode pollen is inferior. The thin staminode endothecium with fewer secondary thickenings retards staminode dehiscence.

Isolation and characterization of a Brassica napus cDNA corresponding to a B-class floral development gene.

B-class floral homeotic genes are required for the proper formation and identity of petals and stamens in dicot flowers. A partial cDNA clone encoding a B-class gene, BnAP3 (Brassica napus APETALA3), was isolated from a B. napus cDNA library derived from young inflorescence meristems. The 5' region of the cDNA was retrieved by RACE. The deduced amino acid sequence of the full-length clone exhibited high similarity to APETALA3 of Arabidopsis thaliana and functionally homologous proteins from other species. 5' RACE and Southern analysis suggests that BnAP3 has multiple alleles in B. napus. Expression analysis assayed by RT-PCR shows that BnAP3 is expressed in floral tissues, as well as non-floral tissues such as root and bract. Transformation of wild-type A. thaliana and B. napus plants with BnAP3 under the control of a promoter specific to reproductive organs converts carpels to stamens, while the expression of this construct in A. thaliana plants mutant for AP3 restores the development of third-whorl stamens in addition to directing a carpel to stamen conversion in the fourth whorl.

Temporal changes in floral nectar production, reabsorption, and composition associated with dichogamy in annual caraway (Carum carvi; Apiaceae).

The dynamics of nectar production were studied in perfect florets of two varieties (Karzo, Moran) of annual caraway (Carum carvi L., Apiaceae). Florets were protandrous and strongly dichogamous, lasting 7-15 d but producing nectar from the stylopodia for 4-12 d, in an interrupted fashion. Nectar secretion began during a floret's phase of stamen elongation and anther dehiscence. After reabsorption of uncollected nectar, at which point nectary surfaces were completely dry, the two styles elongated and a second bout of secretion commenced during the female phase, up to 5 d later, when a floret became receptive to pollination. During the male and female phases, respectively, 0.392 ± 0.064 µL and 1.083 ± 0.261 µL of nectar of similar solute concentration (844 mg/mL) was produced per ten florets. On a daily basis, florets yielded 1.5-fold more nectar in the female than during the male phase. First-time nectar removal throughout the female phase did not match the sum of nectar quantities from male and female phases combined, suggesting that under natural conditions, any uncollected male-phase nectar, once reabsorbed, is not made available to visitors of the same florets when in the female phase. Nectar-sugar composition differed between bouts of secretion; it was hexose-rich (59.6% fructose, 26.9% glucose, 13.6% sucrose) initially, but hexose-dominant (70.2, 26.8, 3.1) during the female phase. A 5.7-fold difference in mean nectar production per floret occurred among plants.