Botany 201 Laboratory Spring 2007 Ascomycota There has never been uniform agreement on a classification scheme in the Ascomycota. However, over the last few years, new concepts in the phylogeny of the Ascomycota have made classification of this group of fungi even more difficult. In the classification scheme we will be using, three informal groups of Ascomycota are recognized: Archiascomycetes, Saccaromycetes and the Filamentous Ascomycetes. Because of the great diversity in this phylum, there is only one characteristic that is present in all species in this phylum, and that is the production of ascospores, which are borne in an enclosed sac-like cell called the ascus (pl. = asci), during sexual reproduction. Cell wall is primarily composed of chitin as in all of the true fungi. The remaining characteristics that are utilized are more general and are not always present, or present only in specific taxonomic groups. Mycelium is usually produced, and when present is septate, but thalli may also be yeast, and some fungi have both and are said to be dimorphic. Asci and ascospores usually are produced in an ascocarp, but in many taxa they may be borne naked. Asexual reproduction, when present, is by means of conidia (sing. = conidium) that are borne on conidiophores. In order to make it somewhat comprehensible, descriptive headings, i.e., Yeast and yeast- like fungi, cleistothecium forming Ascomycetes, etc., will be utilized, for each order. These descriptive headings actually represent the concept of older, but no longer recognized taxa. Although this system of classification will not be entirely accurate, it should be easier to understand. I. Archiascomycetes Members of this group lack ascocarps and produce a prototunicate ascus during sexual reproduction. This type of ascus is typically globose to elliptical. There is no apparent mechanism of releasing the ascospores other than through the disintegration of the ascus. Yeast and Yeast-like Fungi A. Order: Schizosaccharomycetales This order has commonly been referred to as the fission yeast because of their means of asexual reproduction. During fission, after mitosis occurs, the cell elongates and a wall is formed between the two daughter nuclei. This order has long been classified with the Saccharomycetales. However, recent rDNA sequence analyses indicate that they are distinct groups that are not closely related. Representative Schisosaccharomycetales to be examined: Schizosaccharomyces octosporus 1. Schizosaccharomyces octosporus: This is an example of a fission yeast. 9 Mount this species in 3%KOH and stain in 1% Phloxine. Identify and draw a few yeast cells that are in the process of dividing. How does fission differ from budding? You should also be able to see naked asci with ascospores in your slide. How many ascospores can you see in each ascus? II. Saccharomycetes This group of fungi is commonly referred to as the budding yeast because of their means of asexual reproduction. In Saccharomyces, which is the genus we will be examining, an area of the cell wall, of the yeast cell, becomes enzymatically softened inside of a newly developed chitinous ring. The softened wall inside the ring is pushed outward to produce the "bud". Mitosis of the nucleus occurs and one of the daughter nuclei migrates into the expanding bud. When the expansion of the bud is completed, new wall layer is laid down between the "isthmus" leading to the two cells. Separation of the two cells soon follows. During sexual reproduction, the asci are prototunicate and are not produced in an ascocarp. A. Saccharomyces cerevisiae: This species of yeast is an example of a budding yeast. Mount some yeast cells on a slide with, a toothpick, in 3% KOH and stain with phloxine. Identify and draw yeast cells that are budding and those that contain ascospores. The yeast cells, with ascospores, and budding, diploid spores can readily be recognized by their larger size. However, because there are relatively few cells that contain ascospores, they will be more difficult to find. B. Dipodascopsis uninucleatus: Mount this species as described above. This species was once thought to be homologous to the zygospore characteristic of the Zygomycota. However, recent studies do not support this hypothesis. Identify and draw a multi-ascospored ascus in your slide. Explain why this structure was once believed to be homologous to the sporangium that germinates from the zygospore in the Zygomycota 10 III. Filamentous Ascomycetes The remaining group of the Ascomycetes that we will be studying is mycelial and produce asci and ascospores in an ascocarp. A yeast stage is not produced in these species. There are four types of ascocarps recognized; cleistothecium, perithecium, apothecium and ascostroma. We will examine representative orders producing each type of ascocarp. Asexual reproduction, when present, is by conidia. Asexual spores borne in sporangia do not occur. A. Cleistothecium Forming Ascomycetes (=Plectomycetes) 1. Order: Eurotiales Asci and ascospores are borne in an ascocarp called a cleistothecium. A cleistothecium is an ascocarp that is entirely enclosed, without an opening through which the ascospores may be dispersed. This order has thin walled, globse to sac-shaped asci that have a single layer without any means of dispersing its ascospores. Asci are prototunicate. Dispersal of ascospores occur when asci disintegrate. The asci are scattered, randomly, throughout the interior and are not arranged in a hymenium. A hymenium of asci refers to the asci being arranged in a single basal layer, which does not occur in this order. Representative species to be examined: Emericella variecolor (Telomorph) and Aspergillus variecolor (Anamorph) a. Eurotium variecolor. (=Aspergillus variecolor.): Mount a small piece of the green part of this fungus on a slide with 3%KOH and phloxine. This is the asexual stage (anamorph) of this species. Identify and draw the.this stage and label the foot cell, conidiophore, phialide, and conidia. The sexual stage (telomorph) is the small, roundish, gray structures in this culture. Identify and draw a cleistothecium, and carefully pressed down on the covership in order to squeeze asci and ascospores out. How are the asci arranged in this type of ascocarp? 11 B. Perithecium Forming Species of Ascomycetes (= Pyrenomycetes) 1. Order: Sordariales The order usually produces an ascocarp called a perithecium. The perithecium is dark or pallid and can take various shapes, but globose, urn- or flasked-shaped perithecia are the most common. Regardless of their shape, the perithecium has a small opening, an ostiole, through which the ascospores are released. The asci are arranged in a hymenium or more specifically a fascicle, which do not include sterile filaments called paraphyses. Ascospores are hyaline to dark and usually one or two celled. At maturity, the ascospores are forcibly ejected from their asci into the air where they are dispersed. Representative species to be examined: Sordaria fimicola a. Sordaria fimicola: Mount a few perithecia on a glass slide and examine them under the 10X objective of your compound microscope before covering them with a cover slip. Describe the shape of the ascocarp, in this species. How does it differ from a cleistothecium? Draw the perithecium and the ostiole. Using the same perithecia, add a drop of 3% KOH and press down firmly with a cover slip. Identify and draw an ascus with ascospores. 2. Order: Xylariales Ascocarp produced is usually a peithecium that is dark and is on top of or embedded in a stroma. Asci are said to be unitunicate and release the ascospores by forcibly ejecting them through a pore at the tip of the asci. Asci are produced in a true hymenium with paraphysis, which are thought to form the central cavity where the asci and ascospores are borne. This order is a very diverse and difficult group to comprehend. Representative species to be examined: Penzigia globosum a. Penzigia globosum: This is an example of a species that produces stromata (sing. = stroma). A stroma is a compact mass of tightly interwoven mycelium or host tissue and mycelium, in or on which sporulating structures are formed. Make a thin section 12 through a part of the stroma that includes perithecia and mount in 3% KOH. Examine the part of the section with perithecia. The perithecia are embedded at the periphery of the stroma. Draw the l-sect., of the stroma, which will include a few of the embedded perithecia. Note that the perithecia have a wall that is distinct from the stroma. This is a feature that distinguishes the stromatic species of Xylariales from those producing ascostroma in the Dothideales. b. Xylaria polymorpha: Briefly examine and note the similarities in color and texture of the stroma and perithecia. How does this differ from the above species? C. Apothecium Forming Species of Ascomycetes (=Discomycetes) 1. Order: Pezizales The order produces an ascocarp called an apothecium. Typically an apothecium is cup-shaped which is why members of the Pezizales are sometimes called cup fungi. However, the shape of the apothecium is quite variable. Whatever their shape may be, the asci form a hymenium that is usually entirely exposed to the environment at maturity. The asci are unitunicate and have an operculum that cover the tip of the ascus until ascospores are forcibly ejected. Paraphysis are generally present. Representative Discomycetes to be examined: Sclerotinia sp. (prepared slide) and Morchella esculenta a. Sclerotinia: Examine the prepared slide of a longitudinal section through the apothecium of this species. What is the shape of the ascocarp? Identify the asci, ascospores and paraphyses. b. Morchella esculenta (Morel): Examine the preserved specimens of this species. As you can see, the ascocarp is not always the typical cup- shaped structure you observed in the above species. Where are the asci and ascospores produced in this species? 13 D. Filamentous Ascomycetes with Ascostromata (=Loculoascomycetes) Species in this group produce their asci in ascostroma.