Scanning Electron Microscopy of Cookeina sulcipes (Berk.) Kuntze (Pezizales, Ascomycota) From a New Location in Mexico

Abstract

Cookeina sulcipes (Berk.) Kuntze 1891 [1], is a xylophagous fungus. Taxonomically, Cookeina sulcipes is classified within the class Pezizomycetes, the order Pezizales, and the family Sarcoscyphaceae, mainly found in the tropics, predominantly in mountain cloud forest. This fungus grows on both deciduous and perennial trees. It has been described that this species can be found either alongside the road or deep in the forest Cappello, Rosique and Cifuentes (2018) [2], and it thrives in single to cluster. Cookeina sulcipes is commonly recognized for its striking and brightly colored fruiting bodies. It is a saprophytic species that grows on decaying wood, playing an important role in nutrient recycling within forest ecosystems. Due to its distinctive appearance, it is frequently cited in mycological studies and field guides. This species has a wide distribution in tropical and subtropical regions, including Central America and South America, Southeast Asia, the Caribbean, and parts of Africa. It is typically found in humid environments, especially in lowland rainforests where moisture and organic substrates are abundant. Morphologically, it is distinguished by a cup-shaped ascoma (apothecium), which is typically pink to orange-red, and is supported by a slender stipe. The inner surface features the hymenium, where asci and ascospores are generated, while the outer surface is frequently finely textured or hairy. Optical microscopy was employed to examine microscopic structures utilizing visible light and a lens system. The procedure commences with appropriate sample preparation, which includes sectioning, fixation with Potassium Hydroxide (KOH), and staining to maintain structures and enhance contrast. The prepared specimen was placed on a glass slide and covered with a coverslip to safeguard it and improve image clarity. The illumination is subsequently adjusted by aligning the light source, condenser, and diaphragm to achieve uniform lighting and optimal contrast, typically employing Köhler illumination. Observation initiates at low magnification to identify the specimen, followed by higher magnifications for thorough analysis, utilizing coarse and fine focus controls to refine the image. Structural and morphological characteristics are scrutinized, and images are captured with a digital camera for documentation purposes. Ultimately, the gathered observations or images are analyzed to extract data, including measurements of size, shape, and distribution of microscopic components. On the other hand, Scanning Electron Microscopy (SEM) was used to examine the surface morphology and composition of materials at high resolution. Starting with sample preparation, drying and coating the specimen with a thin conductive layer of gold to prevent charging under the electron beam. The prepared sample is placed in a vacuum chamber, where a focused beam of high-energy electrons scans the surface in a raster pattern. As the electrons interact with the sample, they generate signals such as secondary electrons and backscattered electrons. These signals were detected and converted into detailed images that reveal surface topography, texture, and structural features. The results were: Pezizomycotina Pezizales Sarcoscyphaceae Cookeina sulcipes (Berk.) Kuntze 1891. Hyphal hairs arise from cells originating in the ectal excipulum, with thickened, globose walls that elongate until they become prismatic and merge into a single structure that protrudes from the apothecium. Under SEM, elongated fibers without ornamented walls are observed, forming a compact bundle of hyphae. In the medullary excipulum, elongated thin-walled cells with abundant extracellular matrix are observed, corresponding to a intricata imbuta texture, whereas the cells of the ectal excipulum have thin walls, some slightly thickened, with little extracellular matrix, constituting a typical textura globularis. Regarding the spores, they are ornamented, grooved with thin longitudinal striations that are parallel to each other, and are citriform to amygdaloid in shape. In the genus Cookeina, highlights that the use of tools such as scanning electron microscopy is of utmost importance in identifying taxonomic characteristics. In Cookeina sulcipes, it was identified that in the case of hyphal hairs, they emerge from the cells of the ectal excipulum, while the ornamentation of the spores is not very evident under optical microscopy, but stands out clearly in SEM images. Furthermore, this species is reported for the first time in the state of Veracruz, in the Los Tuxtlas Biosphere Reserve. A, Longitudinal section of the apothecium of Cookeina sulcipes (Berk.) Kuntze. a, hymenium, b, medullary excipulum, c, ectal excipulum, d, hyphal hair. B, Detail of the hyphal hair. C, Detail of the medullary excipulum and ectal excipulum. D, Detail of the hymenium showing asci and ascospores. A, Longitudinal section of the apothecium of Cookeina sulcipes (Berk.) Kuntze. B and C, Hyphal hairs. D, Detail of ascospore ornamentation. E, detail of excipular cells.