Lab: Exploring the Fungi

Bio 213 Name: ______Lab 2: Exploring the Kingdom Fungi and the Fungus-Like Protists

Objectives:  Identify and classify “molds” of the Kingdom Protista specifically plasmodial and cellular slime molds and water molds.  Describe the structures of the bodies of slime molds.  Examine and identify the life cycle stages of the cellular and plasmodial slime molds  Identify the structural characteristics typical of Oomycota, and distinguish between the structures of asexual and sexual reproductive phases  Identify the structural characteristics typical of zygomycetes, and distinguish between asexual and sexual reproductive structures  Identify the structures typical of ascomycetes, and distinguish between asexual and sexual reproductive structures  Identify the structures typical of basidiomycetes  Describe how a “mushroom” is formed  Explain the basis for the names “Zygomycota”, “Ascomycota”, “Basidiomycota”  Observe life-cycle stages of chytrids  Classify a variety of local fungal specimens by phylum or type

General 1. Work in a group (the size of the groups will be determined by the size of the class and by the amount of equipment available 2. Examine the slides and the mold specimens provided in the lab. 3. Please do not hoard all slides or specimens at your lab station, and allow other students to have access to all slides. 4. Use the pictures in the photo atlas and your textbook to guide you through the slides and specimens. 5. For each major group described below, sketch the slide provided, labeling key structures in your sketch. These sketches should be in your lab notebook.

Note: Some of these slides are difficult to interpret. Do your best and ask me if you have questions.

General Introduction to the Fungus-Like Protists: Before the origins of plants, animals, and fungi, the earliest eukaryotic cells were of the Kingdom Protista. This kingdom is a diverse collection of organisms with a wide variety of appearance, function, and metabolism. This diversity includes the Protozoans (early animals) that we studied in Biology 212, the algae that we will study in a later lab, and the molds. The “molds” we will study in this lab are actually from three phyla: the plasmodial slime molds (Phylum Myxomycota or Myxogastrida), and the cellular slime molds (Phylum Acrasiomycota or Dictyostelida). The slime molds were once classified in the Kingdom Fungi due to their similar appearance and metabolism. They differ with the fungi in their basic body plan and they lack the chitinous cell walls of fungi. We will also look at the water molds or egg molds (Phylum Oomycota)

1) Phylum Myxomycota (Plasmodial Slime Molds) Plasmodial slime molds live along the damp forest floor in a brightly colored plasmodium. The plasmodium is a coenocytic mass of “cells” that forms through mitosis without cytokinesis. Thus, the plasmodium is a multinucleate cytoplasmic mass of nuclei not separated by cell walls. Often these plasmodial slime molds live in fallen bark, decomposing plants, and in the leaf litter and soil of a forest floor. The streaming plasmodium eats through phagocytosis of living cells and organic particles. The life cycle of the plasmodial slime molds is a complicated series of stages. We will examine the stages of the life cycle in lecture, and you should recognize several of these stages in lab.

Terms to know: plasmodium, coenocyte, cytoplasmic streaming, swarm cells, sporangia, sclerotium Structures to Recognize and Identify: plasmodium, sporangiophore, sporangia Slides: Dictydium spp. Live specimens: Physarum spp., (if available) Describe the characteristics of the living specimens including color, size and shape

Optional Exercise: Culturing live Physarum (Instructor’s Option/Availability) To set up the culture plates (using sterile technique): 1. Use innoculating spatulas to cut and remove a 1cm by 1cm cube from the culture 2. Place the cube upside down on a plate of corn meal agar 1 Lab: Exploring the Fungi 3. Place a coverslip on the corn meal agar near the cube 4. Sprinkle oat grains (about 10) on the corn meal agar plate 5. Cover and label the new corn meal agar plate 6. Examine the plates over the next few class periods.

2) Phylum Acrasiomycota (Cellular Slime Molds) The cellular slime molds challenge our definition of “individual organism”. The feeding stages of the cellular slime molds are solitary amoeboid cells that move through pseudopodia and feed through phagocytosis. When food is scarce, the individual cells aggregate to function as a “multicellular” organism. This life cycle stage is slightly different than the plasmodium of the myxomycetes, as the cells retain their individual identity and remain divided by their cell membranes.

We will look at live specimens of this phylum. Specifically we will look at the genus Dictyostelium.

Define: amoebae, pseudoplasmodium, fruiting body Structures to Recognize and Identify: amoebas, pseudoplasmodium, fruiting body (sorocarp) Live specimens: Dictyostelium spp. (Observe and draw the solitary amoebas, the “multicellular” slugs and reproductive sorocarps if available)

3) Phylum Oomycota (Water Molds or Egg Molds) The water molds are currently classified as stramenopiles and are essentially viewed as “algal” protists that have lost their chloroplasts. We will examine them in this lab however as they are quite similar to the molds. Some of the water molds are unicellular, but we will focus on the multicellular species. These “multicellular” forms are composed of thin hyphae that are multi-nucleated (coenocytic) and are quite similar to the coenocytic hyphae of some fungal species. Water molds have cell walls composed of cellulose (as compared to the chitinous cell walls of fungi). [This is a great example of convergent evolution.] The diploid form is predominant in the life cycle of the water molds. Biflagellated cells occur in the life cycle as diploid “zoospores” released from the zoosporangium. The term “egg mold” refers to the presence of haploid egg nuclei that are produced in the sexual life cycle of the water molds. The egg nuclei are fertilized by sperm nuclei to give rise to another diploid organism. Some water molds are saprophytic decomposers that grow as filaments (cottony) off of dead animals and/or algae (mostly in aqueous environments). Other water molds are parasitic and may grow on the skin or gills of fish.

Terms to know: oogonium, antheridia, zoosporangia, zygote, oospores Structures to Recognize and Identify: zoosporangia, oogonium, antheridium Slides: Saprolegnia spp. Live specimens: Saprolegnia spp., Dictyuchus spp. if available

General Introduction to the Fungi: The Kingdom Fungi is composed of heterotrophic eukaryotes (similar to the Kingdom Animalia). As heterotrophs, these organisms must acquire their organic nutrients from their environment. Typically, they digest their food outside their bodies and absorb the digestive products into their cells. The fungi are primarily saprophytic or parasitic. The bodies can be unicellular (yeasts), or multicellular. The fungi often have very complex life cycles with alternating sexual and asexual reproduction. The sexual life cycles consists of both haploid and diploid stages, and spores that are produced asexually or sexually depending on the species. In these life cycles, fertilization is often divided into two processes, plasmogamy (fusion of cytoplasm) and karyogamy (fusion of nuclei), that are separated by a certain length of time. Plasmogamy before karyogamy creates a dikaryotic stage in which the cells contain both unfused, haploid nuclei. Some fungi are quite beneficial to humans as well as their environment. We have used fungi to make food products (bread, wine, and beer. Medically, we use the antibiotic products of some fungi to fight bacterial infections. Ecologically, the fungi are important recyclers of nutrients as they (and the bacteria) are important decomposers. Some fungi are harmful to other organisms as they are parasitic on animal or plant hosts. Fungi may also form important symbiotic relationships with photosynthesizers (e.g. lichens and mycorrhizae). In this lab, we will examine the body structures and the complex life cycles within specific fungi in the major phyla of the kingdom. We will focus on three of the most common phyla, the Zygomycota Ascomycota, and Basidiomycota. We will also view several fungal symbiotic relationships.

Phylum Zygomycota (Zygote Fungi) The zygomycetes are mostly terrestrial organisms living in soil or decaying plant or animal material. Some of the zygomycete species form mycorrhizae, symbiotic associations with plant roots. The name of this phylum refers to the zygosporangia stage unique to the life cycle of this fungus. The hyphae of the zygomycetes are coenocytic and

2 Lab: Exploring the Fungi haploid. Asexual reproduction as haploid spores can be produced by mitosis of the haploid hyphae. Sexually, the hyphae of two different mating strains fuse through plasmogamy to form the zygosporangium. Dividing walls called septa separate the young dikaryotic zygosporangium from the hyphae of the two individuals. Inside the zygosporangium karyogamy occurs fusing the two haploid nuclei. From the now diploid zygosporangium, a sporangium develops and meiosis in this structure produces the haploid sexual spores. Both asexual and sexual spores germinate when conditions are good and hyphae grow from them.

Define and recognize: hypha, coenocytic, rhizoids, zygosporangium, zygospores Slides: Rhizopus spp. (germinating spores, sporangia, zygotes-sporangia) Live specimens: as available

Phylum Ascomycota (Sac Fungi) The largest and most diverse fungal phylum is made up of marine, freshwater and terrestrial organisms. About half of the ascomycetes form symbiotic communities with algae or cyanobacteria called lichens. Similarly to the zygomycetes, some ascomycetes form mycorrhizae (“fungus-roots”) with plant roots. Unique to the life cycle of the organisms of this phylum is the sac-like ascus where sexual spores are produced. Asci are part of the macroscopic fruiting bodies known as ascocarps. The hyphae of the vegetative body are haploid and septate. Asexually ascomycetes can produce spores through mitosis. These asexual spores, conidia, are formed at the ends of the hyphae and are released for dispersal by the wind or water. Sexually, the hyphae af two separater mating strains fuse to form dikaryotic hyphae. The dikaryotic hyphae grow to form the ascocarp. At the ends of the reproductive dikaryotic hyphae, the asci form. Karyogamy occurs in the asci producing a diploid nucleus, and then as a result of meiosis and mitosis typically eight ascospores are produced. The ascospores are released and like the conidia will germinate to form a new haploid organism.

Define and recognize: conidiospores, conidiophores, ascospores, ascocarp Slides: Peziza spp. (apothecium) Live specimens: as available

Phylum Basidiomycota The basidiomycetes include mushrooms, puffballs, and shelf fungi among the numerous species. Basidiomycetes are important decomposers of plant material in the ecosystems that they live in as they can very effectively digest the lignin of wood. Basidiomycetes are commonly seen on the downed woody plants in a forest. Like the other two phyla, several basidiomycete species form mycorrhizal associations with plant roots. The phylum is named for the basidium stage of the life cycle. The basidium is a temporary diploid stage in which the sexual basidiospores are produced. The vegetative hyphae of the basidiomycetes are haploid and septate. Again, sexually, plasmogamy of two separate mating strains creates dikaryotic hyphae that grow to produce the signature basidiocarps (like a mushroom). The basidia that line the basidiocarp undergo karyogamy to become diploid and then through meiosis, produce the haploid basidiospores. The basidiospores are released to hopefully germinate giving rise to the next generation of hyphae.

Define and recognize: basidiospores, basidium, basidiocarp, cap, gills, stalk, Slides: Coprinus spp. (gills, pileus) Live specimens: various mushrooms

Miscellaneous Fungi (including the Deuteromycetes) Specializations have evolved in certain zygomycetes, ascomycetes, and basidiomycetes that make it difficult to classify these species. These fungal forms are common and are often used commercially by humans (e.g. yeasts). Some of these fungal “species” are actually symbiotic with photosynthetic organisms actually living in a community with the photosynthesizers (e.g. lichens and mycorrhizae). Also included in this collection of fungi are the imperfect fungi (Deuteromycetes) that cannot be classified in the phyla described previously as the deuteromycetes do not have sexual stages in their life cycle. We will examine some of the unique structures and associations through slides and live and dried specimens.

Define: conidia, hyphae Slides: Penicillium spp. (conidia) Live specimens: as available

Lichens Cells and structures to recognize: algal cells, fungal hyphae, thallus Slides: lichen ascocarp

3 Lab: Exploring the Fungi Mycorrhizae Define: fungal hyphae, plant roots Slides: endomycorrhizae, ectomyccorhizae

POSTLAB QUESTIONS: Please answer these questions on a separate sheet of paper.

1. Each of the three Protistan groups we examined today resembles a true fungus in some way. Briefly explain why each of these three do not belong in the kingdom Fungi.

2. The three most abundant phyla in the kingdom Fungi, the Zygomycota, Ascomycota, and Basiciomycota, are defined not just by differences in their DNA and evolutionary history, but also by significant morphological differences. What would you need to see to definitively identify a member of each of these groups?

3. The diploid stages of the fungal life cycle are usual very brief returning to a haploid condition through meiosis shortly thereafter. Why are the diploid stages important to the sexual life cycle?

4. Define “coenocytic” and “dikaryotic”. Why are these terms significant in the fungal life cycles?

5. What are some of the human commercial uses for fungi? Explain at least two examples!