Pollination and Floral Biology of a Rare Morning Glory Species Endemic to Thailand, Argyreia siamensis

Argyreia siamensis is extremely rare, and very little is known about its reproduction. The species has colorful flowers that seem likely to attract pollinators, but population sizes are typically small (<30 individuals). To determine whether poor reproduction contributes to its rarity, we investigated its mating system and potential pollinators in two populations. We also examined the staminal trichomes and floral nectary to investigate their role in pollinator attraction. The mating system was assessed with a bagging experiment and pollinator visits were recorded with action cameras. Additionally, we tested the staminal trichomes and floral nectary for terpenes and flavonoids and examined floral nectary micromorphology via scanning electron microscope and compound light microscope. Our results reveal that A. siamensis is self-incompatible and dependent on pollinators; the western population was pollinated by bees (Meliponini and Amegilla), while the eastern population was mainly pollinated by skipper butterflies (Hesperiidae). Both staminal trichomes and the floral nectary appear to contribute to pollinator attraction through the presence of terpenes and flavonoids (in both secretory structures) and nectariferous tissue and nectarostomata (in the nectary). Our results indicate that A. siamensis has reliable and effective pollinators and that insufficient pollination is likely not a primary cause of its rarity.

Validation of QuEChERS Method for Estimation of Imidacloprid and its Metabolites in Cotton Flower, Pollen, Nectariferous Tissue, and Honey

Background Exposure of Apis mellifera to neonicotinoid insecticides is one of the factors attributed to the recent decline in A. mellifera populations resulting in economic and ecological losses due to loss of pollination services. Honeybees can get exposed to neonicotinoids like imidacloprid directly in the field at the time of application as well as during consumption of pollen and nectar from treated plants. In addition, some metabolites of imidacloprid are more toxic than the parent compound. So, the fate of imidacloprid and its metabolites in commodities to which honeybees get exposed needs to be overhauled. Objective To validate QuEChERS method for estimation of imidacloprid and its metabolites in cotton flower, pollen, nectariferous tissue, and honey using HPLC. Methods The QuEChERS method was validated in terms of selectivity, linearity, LOD, LOQ, matrix match, accuracy, and precision. The estimation of residues was done by HPLC. Results Recoveries of imidacloprid and its metabolites for cotton flowers, nectariferous tissue, pollen and honey samples were in the range of 80.42–99. 83%. LOQ for imidacloprid and its metabolites was 0.01 µg/g. Acceptable precision (RSD &lt; 20%) was obtained. Conclusion The method allows simple and fast extraction of imidacloprid and its metabolites from cotton flower, pollen, nectariferous tissue, and honey. Highlights An accurate, simple, and sensitive analytical method was validated for imidacloprid and its metabolites. The method was validated according to the SANTE/12682/2019 guidelines.

Floral development of Condylocarpon isthmicum (Apocynaceae)

Apocynaceae is one of the largest families of angiosperms. Its representatives have flowers with relatively simple morphology, ranging from anthers free from the style head to more complex flowers in which the anthers are postgenitally united with the style head, forming a gynostegium, and those with a style head that is vertically differentiated into distinct functional regions. The aim of this study is to understand the morphology and secretory structures of Condylocarpon isthmicum (Vell.) A.DC. at different stages of development. This species, which is in the family Apocynaceae, has morphologically simple flowers. Flowers at four different stages of development were collected and processed for anatomical and histochemical analysis; floral anatomy was examined using light and scanning electron microscopy. The simplicity of the C. isthmicum flower morphology was contrasted with the complexity observed in the secretory structures at different stages of flower development. Four secretory structures were identified in this species: colleters, style head epidermal cells, nectariferous tissue, and an obturator. The colleters were observed in the bracts and bracteoles of the young inflorescences. The style head began the secretory phase in the pre-anthetic stage and remained in this phase until anthesis. The nectariferous tissue was secreted during anthesis, and the obturator was present only in post-anthetic flowers. We identified a nectary in the wall of the ovary, and we verified and described a new structure in the Apocynaceae, the obturator.

The morphology and ultrastructure of the nectaries of marrow (Cucurbita pepo L. convar. giromontiina)

The present study investigated the size and structure of the nectaries in flowers of marrow – <em>Cucurbita pepo </em>convar.<em> giromontiina </em>cv. ‘Weiser Busch’. The diameter and thickness of nectariferous layer were compared in female and male flowers of this taxon. The micromorphology as well as the anatomical and ultrastructural characters of the nectary from the female flower were observed using light, scanning and transmission electron microscopy. The density and size of stomata of the nectary epidermis from both types of flowers were examined using light microscopy. The nectaries in female flowers were found to have a larger size than in male flowers. The stomata occurring in the nectary epidermis of both types of flowers have a similar size and density. We observed that nectar was released onto the surface of the nectary not only via the stomata, but also through the walls of the epidermal cells. In TEM examination, large nuclei, different-shaped plastids, ER tubules, dictyosomes, and ribosomes were observed in the nectariferous tissue cells. A large number of mitochondria accompanying the plastids were found in the parenchyma cells of the nectary. The degradation of the nectary parenchyma cells in the flowers living for about 6 hours was asynchronous.

Characteristics of blooming, floral nectaries and nectar of Malus sargentii Rehd.

In the years 2007-2008, the flowering biology of <i>Malus sargentii</i>, an ornamental apple tree native to Japan, was studied in the conditions of Lublin (Poland). The daily rate of flower opening, flowering duration and flower visitation by insects were determined. The amount of nectar produced per flower and sugar content in the nectar were investigated. The size of nectaries and the micromorphology of their surface were examined using light and scanning electron microscopy. It was found that the greatest amount of flowers opened between 11.00 and 13.00. During this time, the largest number of insects was observed in the flowers. Bees (90%) were predominant among the insects, with a much smaller number of bumblebees (6%) and butterflies (4%). The flower life span was 5 days. Over this period, the flower produced, on the average, 0.71 mg of nectar with an average sugar content of 32%. The nectaries of <i>Malus sargentii</i> are orange-yellow coloured and they represent the hypanthial type. Due to the protrusion of the nectariferous tissue, they are classified as automorphic nectaries. The surface of the epidermal cells of the nectary was distinguished by distinct cuticle folds. A small number of stomata were located only in the basal part of the nectary. At the beginning of flowering, all stomata were closed, but secretion traces were observed near well-developed outer cuticular ledges.

Micromorphology of flowers, anatomy and ultrastructure of Chamomilla recutita (L.) Rausch. (Asteraceae) nectary

Investigations of the micromorphology of flowers and the structure of nectaries in <i>Chamomilla recutita</i> L. (Rausch.) were carried out with the use of stereoscopic, light, scanning and transmission electron microscopy. Biseriate glandular trichomes consisting of 5-6 cell layers were found on the surface of the corollas of ray and disc florets. Accumulation of secretion within the subcuticular space was accompanied by degradation of trichome cells. Secretion release followed rupture of the cuticle in the apical part of the trichome. The ovary of the ray florets exhibited characteristic ribs covered with epidermis composed of radially elongated palisade cells. Nectariferous glands were present only in the disc florets. The ring-like nectary (93 × 163 µm; height × diameter) was located above the inferior ovary. The gland structure was formed by single-layer epidermis and 5-8 layers of specialised nectariferous parenchyma. Nectar was released via modified 15-20 µm wide stomata. The guard cells were slightly elevated above the surface of the other epidermal cells or were located slightly below them. The stomatal cells were characterised by small external and internal cuticular ledges. No vascular bundles were observed in the nectary. The gland was supplied by branches of vascular bundles reaching the style and ending at the nectary base. The nectariferous tissue was formed by isodiametric cells with a diameter of 11-20 µm. The cell interior was filled with electron dense cytoplasm containing a large nucleus, numerous pleomorphic plastids, mitochondria with a distinct system of cristae, Golgi bodies, ER profiles, and ribosomes. The plastid stroma was characterised by presence of pastoglobuli, intraplastid tubules, and lighter zones. Several small vacuoles were found in each cell. Plasmodesmata were visible in the walls of some cells. Lighter periplasmic space in which apoplastic transport of nectar might take place was observed between the plasmalemma and the cell wall. The presence of an osmiophilic substance in the intercellular spaces additionally corroborates this assumption.

Structure of nectaries of Malus sylvestris (L.) Mill.

The structure of floral nectary of <i>Malus sylvestris</i> was examined using light and scanning electron microscopy. Nectaries in <i>M. sylvestris</i> flowers were situated on the adaxial surface of the receptacle, between the style and the base of filaments. The middle part of the nectary was covered epidermal cells with striated cuticle. The remaining part of the nectary was covered with smooth cuticle. Open and modified nectarostomata were situated at the same level as epidermal cells. The nectariferous tissue was formed by densely packed small parenchyma cells (secretory cells) with dark protoplasts.

Anatomy and ultrastructure of floral nectaries of Asphodelus aestivus Brot. (Asphodelaceae)

The structure of septal nectaries in <i>Asphodelus aestivus</i> flowers was investigated by using light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (EM). It was found that the outlets of the three parts of the nectary were situated on the ovary surface at 2/3 of its height and had the shape of elongated openings. The nectariferous tissue was in the septa of the lower part of the ovary. The secretory tissue cells formed 1-3 layers surrounding the nectary slits. They contained thin cell walls with the cuticle layer from the slit side, large cell nuclei, numerous mitochondria and plastids characterised by various shapes. In plastids, small starch grains occurred sporadically. At the beginning of anthesis, the cells were poorly vacuolized. ER cisternae and secretory vesicles were located near the outer cell wall. Fibrous substance was present in the nectary slits. In the subglandular tissue, numerous starch grains occurred at the beginning of anthesis. In this zone, cells containing raphides and xylem elements were observed. Based on the ultrastructure of the nectary it can be stated that granulocrine nectar secretion occurs in <i>A. aestivus</i>.

Comparison of features of the epidermis and the size of the floral nectary in four species of the genus Cotoneaster Med.

The investigations involved four species of the <i>Cotoneaster</i> genus: <i>C. divaricatus</i>, <i>C. horizontalis</i>, <i>C. lucidus</i>, <i>C. praecox</i>, which are commonly grown for decorative purposes. In Poland, these plants bloom in May and June and are a source of abundant spring nectar flow for insects. The floral nectaries of the above-mentioned species were examined using stereoscopic, light, and scanning electron microscopy in order to assess their size and epidermal microstructure. In the plants studied, the upper part of the hypanthium is lined by nectariferous tissue. The nectaries in the four species vary in terms of their sizes. Nectar is secreted onto the surface of the epidermis through anomocytic, slightly elongated or circular stomata. The largest stomata on the nectary epidermis were found in the flowers of <i>C. horizontalis</i>, and the smallest ones in <i>C. divaricatus</i>.Their size and location in relation to other epidermal cells were taxon-specific. The highest density of stomata in the nectary epidermis was found in <i>C. divaricatus</i> (205 per mm²), whereas <i>C. horizontalis</i> flowers exhibited the lowest (98 per mm²) stomatal density. The cuticular ornamentation on the nectary epidermis surface was diverse. The stomatal indices calculated for the nectary epidermis were considerably lower than for the leaves in the particular species.