Micromorphological differentiation of Korean Disporum species using light and scanning electron microscopy.

The genus Disporum Salisb. is widely distributed in East Asia, yet phylogenetically relevant morphological traits useful for differentiating many of the small, perennial, herbaceous species remain poorly described. To address this, leaf, floral, pollen, and orbicule micromorphology of four Korean Disporum species was investigated using light and scanning electron microscopy. All Korean Disporum species examined had hypostomatic leaves, with anomocytic stomatal complexes found only on the abaxial epidermis. Guard cell length varied among species, ranging from 44.30 µm in D. viridescens to 53.49 µm in D. uniflorum. The epidermal cells of the investigated Disporum taxa had sinuate anticlinal cell walls on both adaxial and abaxial surfaces. The surface of the guard and subsidiary cells were either smooth with weak striations or had strongly wrinkled striations. The pollen grains of all Korean Disporum taxa were monads, monosulcate with granular aperture membranes, subprolate to prolate in shape with microreticulate or verrucate exine surfaces. The mean size of pollen grains ranged from 46.38 to 49.92 µm in polar length and from 34.39 to 39.58 µm in equatorial diameter across species. Sexine ornamentation was a taxonomically relevant trait for differentiating Korean Disporum taxa. Additionally, the presence of orbicules as well as the orbicular characters (e.g., size, shape, ornamentation, and association pattern) are described for the first time in species from this genus. The present investigation of leaf and floral micromorphology using light and scanning electron microscopy provides valuable information for the taxonomic differentiation and identification of Disporum species in Korea. RESEARCH HIGHLIGHTS: A detailed micromorphological description of leaf, floral characters (tepal, stigma, style), pollen and orbicule is provided for Korean Disporum species using scanning electron microscopy (SEM) and light microscopy (LM). The presence of orbicules and their taxonomic implications in Korean Disporum species are described for the first time. Phylogenetically informative pollen and orbicule micromorphological characters are described, improving understanding the systematic relationships of Korean species in the genus Disproum.

Developmental and ultrastructural characters of the pollen grains and tapetum in species of Nymphaea subgenus Hydrocallis.

Variations in pollen characters and tapetum behavior were recently acknowledged in the early-divergent family Nymphaeaceae and even within the genus Nymphaea, which probably is not monophyletic; some traits such as infratectum and tapetum type are also a matter of different interpretations. In this study, developmental characters of the pollen grains and tapetum in Nymphaea subgenus Hydrocallis are provided for the first time. Observations were made in N. amazonum, N. gardneriana, and N. prolifera using light, scanning, and transmission electron microscopy. Tapetum is of the secretory type and produces orbicules. At microspore and pollen grain stages, the distal and proximal walls differ considerably. This result supports the operculate condition of the aperture in Hydrocallis, and such aperture might be plesiomorphic for Nymphaeoideae. The infratectum is intermediate, composed of inter-columellae granular elements, robust columellae consisting of agglomerated granules, complete columellae, and fused columellae. Narrow microchannels are present and persist until the mature pollen grain stage. The membranous granular layer is often present in the pollen grains of Nymphaeaceae. In N. gardneriana, this layer is most probably a component of the intine because it is lost after acetolysis. Orbicules in the Nymphaeaceae are characterized as spherical or subspherical, with a smooth sporopolleninic wall that surrounds an electron-lucent core and with individual orbicules that usually merge to give irregular aggregations. The aperture, pollen wall ultrastructure, and the tapetum of the studied species are discussed in an evolutionary and systematic context, and these characters are also compared with those of other angiosperm lineages.

Esporogénesis y esporas de Equisetum bogotense (Equisetaceae) de las áreas montañosas de Colombia / Sporogenesis and spores of Equisetum bogotense (Equisetaceae) from mountain areas of Colombia

Studies on some reproductive traits in Equisetum species are scarce and valuable to understand species distribution. Therefore, a detailed study of the sporogenesis process and spore development in E. bogotense is presented, with an analysis of the main events during meiosis, maturation of spores, spore wall ultrastructure, orbicules and elaters. Specimens were collected from 500 to 4 500m in Cauca, Colombia. Strobili at different maturation stages were fixed, dehydrated, embedded in resin, and ultra-microtome obtained sections were stained with Toluidine blue. Observations were made with optical microscopy with differential interference contrast illumination technique (DIC), transmission and scanning electron microscopy (TEM and SEM). Ultrathin sections (70-80μm) for TEM observations were stained with uranyl acetate and lead citrate; while samples for SEM observations, were fixed, dehydrated in 2.2-dimethoxypropane and dried at critical point as in standard methods. Strobili have numerous mature sporangiophores, each one with a peltate structure, the scutellum, bearing five-six sessile sporangia attached to the axis of strobilus by the manubrium. Immature sporocytes (spore mother cells) are tightly packed within the young sporangia. The sporocytes quickly undergo meiosis, by passing the stage of archesporium and give origin to tetrads of spores. The tapetum loses histological integrity during early stages of sporogenesis, intrudes as a plasmodial mass into the cavity of the sporangium, partially surrounding premeiotic sporocytes, and then, tetrads and adult spores. The tapetum disintegrates towards the end of the sporogenesis, leaving spores free within the sporangial cavity. Spores present several cytological changes that allow them to achieve greater size and increase the number of plastids, before reaching the adult stage. Sporoderm includes three layers external to the cytoplasmic membrane of the spore cell, and they are pseudoendospore, exospore and perispore. Viewed with SEM, the exospore is smooth to rugulate, with micro perforations, while the perispore is muriform, rugate, with narrow, delicate, discontinuous, randomly distributed folds delimiting incomplete, irregular areolae, externally covered by of different size, densely distributed orbicules. These orbicules are also found all over the external face and margins of the elaters, while the internal face is smooth and lack orbicules. Viewed with TEM, the exospore is a thick layer of fine granular material, while perispore is a thinner layer of dense, separate orbicules. The elaters are composed by two layers of fibrillar material: an inner layer with longitudinally oriented fibrils and an outer, thicker and less dense layer with fibrils transversely fibrils and abundant, external orbicules. It is suggested that the processes of ontogeny and characters of the sporoderm are relatively constant in Equisetum; however, sporogenesis in E. bogotense is synchronous and this condition has been observed so far only in E. giganteum, a tropical genus also found in Colombia.

Los estudios sobre aspectos reproductivos son escasos en Equisetum. Por eso, hemos realizado un análisis detallado del proceso de esporogénesis, desarrollo de las esporas, ultraestructura de procesos que tienen lugar durante la meiosis, formación de la pared esporal, orbículas y eláteres de E. bogotense, en especímenes procedentes del Cauca, Colombia. Los estudios se efectuaron mediante microscopía fotónica, electrónica de transmisión (TEM) y de barrido (SEM). Los estróbilos llevan numerosos esporangióforos maduros, cada uno con un escutelo peltado, unido al eje del estróbilo por el manubrio y portador de 5-6 esporangios sésiles. Los esporocitos experimentan meiosis dando origen a tétradas de esporas. El tapete pierde la integridad histológica en las primeras etapas de esporogénesis y rodea los esporocitos premeióticos, posteriormente a las tétradas y finalmente las esporas inmaduras, que experimentan cambios citológicos y de tamaño antes de alcanzar la etapa adulta. El esporodermo de las esporas adultas de E. bogotense consiste de seudoendosporio, exosporio y perisporio. Vistos con MEB, el exosporio de las esporas adultas es liso a rugulado con microperforaciones y el perisporio es muriforme, rugado, con pliegues delicados, estrechos, discontinuos, que se distribuyen al azar y delimitan aréolas incompletas. Externamente el perisporio está cubierto por orbículas, que se forman también en la cara externa y los márgenes de los eláteres. Vistos con TEM, el exosporio es una capa de material granular fino y el perisporio, una capa mucho más delgada con orbículas discretas. Los eláteres están formados por dos capas de naturaleza fibrilar, orientadas longitudinalmente y transversalmente. La esporogénesis en E. bogotense es sincrónica, similar a la de E. giganteum, otra especie de distribución tropical que también crece en Colombia.

THE PROCESSES OF ANTHER DEHISCENCE AND POLLEN DISPERSAL: II. THE FORMATION AND THE TRANSFER MECHANISM OF POLLENKITT, CELL-WALL DEVELOPMENT OF THE LOCULUS TISSUES AND A FUNCTION OF ORBICULES IN POLLEN DISPERSAL.

The development of the locule of Gasteria verrucosa (Mill.) H. Duval and Lilium hybrida cv. Enchantment, especially the border between the sporophyte and the gametophyte, is investigated by means of light and electron microscopy, histochemistry and micromanipulation and related to pollen dispersal. The pecto-cellulosic cell walls of both the middle layer and the tapetum and a part of the endothecium disappear, and the tapetum cells are covered with sporopollenin-containing tapetal membranes and orbicules. The cell contents of the tapetum turn into the hydrophobic pollenkitt. In the locule, similar changes are observed as the pecto-cellulosic and callose walls of the meiocytes disappear and the future pollen grains are covered with the sporopollenin-containing exine. Due to their non-wettability, these hydrophobic substances on both sides of the sporophyte-gametophyte surface are important in pollen dispersal. The transfer of pollenkitt from the tapetum to the locule appears to be due to capillary forces in the locule after the continuous expansion of the pollen, rather than to active flow or to direct attraction by the pollen grains.