BIOL227 F2024

Lab 4: The diversity of fungi

Written by: Catherine Calogeropoulos ©

Edited by: Amandeep Glory and Dr. Andrew Wieczorek

General information about fungi

Unlike the protists scattered throughout every eukaryotic supergoup, the fungi all reside within a
single clade, called the Fungi Kingdom. The fungi likely evolved from a flagellated, single- celled
protist related to the nucleuriids of the opisthokont clade. All fungi are exclusively
heterotrophic, incapable of phagotrophy with cell walls composed of chitin. Fungi are specialists at
secreting digestive enzymes into their environments (called exoenzymes) to liquify organic matter
(both living and dead) and absorb the low-molecular weight nutrients (sugars, amino acids, etc.).
Their digestive prowess includes the ability to break down everything from lignin to pollen grains
to house paint.

Fungi are mostly multicellular but single-celled species (e.g. the yeasts) are also represented.
Multicellular fungi are composed of hyphae (thin filaments) tangled together into a dense
mycelium (a network of hyphae). The mycelia of fungi are haploid. The hyphae can be septate or
coenocytic. Septate hyphae have septa (septum, singular) that divide the filaments into cells. The
septa have a large perforation to allow nuclei, ribosomes and other organelles to easily flow through
by way of cytoplasmic streaming. Coenocytic hyphae lack septa; their filaments are
uninterrupted and thus multinucleated. Fungi that are morphologically limited to just hyphal growth
forms are called moulds. Other more elaborate fungi form macroscopic fruiting bodies. The
common names of these fungi are derived from the shape of their fruiting bodies, e.g. cup fungi,
shelf fungi, puff balls, coral fungi, etc.

The mechanisms for sex and reproduction in fungi is quite diversity. The endgame of this
diversity in sex and reproduction is to give ample opportunities for spore dispersal with brief, but
sometimes long-lived, intermissions for genetic recombination (in other words, sex).

Their twin abilities –digestive prowess and spore dispersal– make fungi both a friend and foe to
humans. Their capacity to digest almost any organic matter, makes them indispensable in
biogeochemical cycling in natural ecosystems and equally indispensable in works of
bioremediation (e.g. cleaning up oil spills) and the food industry (e.g. wine/beer/cheese/kimchi
making). But their spore production makes them problematic: spores are tiny (in fact, microscopic)
remaining aloft in the air for prolonged periods of time allowing them to travel
great distances. This makes pathogenic plant fungi difficult to contain when they infect our
crops. Furthermore, fungal spores in ventilation systems or homes have serious impacts on
human health.

This lab will introduce the five major phyla within the Fungi kingdom: the Chytridiomycota,
Zygomycota, Glomeromycota, Ascomycota and Basidiomycota. The focus will be to identify the
unique features of each group and describe their respective life cycles with an emphasis on
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where sex occurs (if it occurs) and the various stages in which spores are dispersed.

Figure 1: The tree shows fungal diversity. Molecular analyses helped clarify evolutionary
relationships among fungal groups, although areas of uncertainty remain. Recent metagenomics
studies estimate fungal diversity at around 1.5 million species.

Source: Campbell Biology. 3rd edition. Chapter 31.

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1. Chytrids

The defining feature of the opisthokonts clade (in which the fungi reside) is a posteriorly located
flagellum. The chytrids are the only members of the phylum to retain this feature; all other
fungal phyla lack a flagellum. The chytrids are mostly unicellular, but there are some
filamentous forms. Unlike the fungi in all other phyla, the chytrids do not form elaborate
mycelia. Instead, they have a few short filaments with root-like structures called rhizoids for
anchoring into substrates. The substrates are living or decaying organisms on which they feed. The
body of such chytrids is described as a thallus rather than a mycelium.

Members of the Allomyces genus are thallus-forming chytrids. A unique feature of the Allomyces is
the alternation between multicellular haploid and diploid thalli. This alternation, called alternation
of generations, is present throughout the entire plant kingdom, but unique to the Allomyces genus
within the chytrid phylum. In the diagram below, flagellated spores (called zoospores, because of
their flagellum) are produced on several occasions. The gametophyte
generation (a haploid thallus) ❶ erects short upright hyphae that terminate into spore producing
structures called gametangia. Here, the larger female gametangium is located below the smaller male
gametangium. Within the gametangia, zoospores are mitotically produced and discharged.
Zoospores produced by gametangia are referred to as gametes. The gametes fuse ❷ to form a
zygote that divides mitotically to produce the sporophyte generation –a diploid thallus ❸. The
sporophyte generation erects upright hyphae that differentiate into spore producing structures
called sporangia. Mitosporangia ❹ produce zoospores mitotically. These diploid spores grow into
diploid thalli. Meiosporangia ❺ are darker in colour and produce haploid spores -thus meiosis
occurs within meiosporangia. The haploid spores develop into haploid thalli which
brings us back to the gametophyte generation ❶.

Figure 2: Life cycle of Allomyces macrogynus. See text for details.

(Source: https://www.researchgate.net/figure/The-Allomyces-macrogynus-life-cycle-The-sporophyte-generation-is-
maintained-asexually_fig13_44635842 )

Representative chytrid included in this lab: Allomyces

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2. Zygomycetes

The zygomycetes are coenocytic moulds. As with all moulds, the zygomycetes do not produce any
distinct fruiting bodies. The most elaborate part of the mycelium are the sporangiophores ❶– erected
filaments (hyphae) on which sporangia are borne. Contained inside the sporangia are thousands of
haploid, mitotically-derived, spores. Once discharged, the spores disperse (often
great distances) and germinate if they land on a suitable substrate. This process, a form of
asexual reproduction, is the most dominant in the life cycle of zygomycetes. Sex occurs when
mycelia from two opposite mating strands unite ❷. The hyphae of opposite mating strands
produce buds that differentiate into gametangia ❸. The cytoplasm of these buds is separated from the
rest of the mycelium by a septum. Within the gametangia, are several haploid nuclei. When the
gametangia of the two mating strands fuse, in a process called plasmogamy ❹, the entire
structure (now containing two types of haploid nuclei) differentiates into a resistant and
conspicuous structure called a zygosporangium. Within the zygosporangium, the nuclei fuse to
temporarily restore the diploid condition. This process of nuclei fusion is called karyogamy ❺.
Meiosis immediately follows karyogamy. When conditions for dispersal are suitable, a
sporangium emerges ❻ out of the zygosporangium. It is within this sporangium where haploid spores
are discharged. In some species the spores are dispersed in specific directions and with significant
force. Once settled on a suitable substrate, the spores germinate and grow to form a coenocytic haploid
mycelium.

Figure 3: The life cycle of the zygomycete Rhizopus stolonifer (black bread mold). Source: Campbell Biology.
3rd edition. Chapter 31.

Representative zygomycetes included in this lab: Phycomyces, Zygorhynchus, and Mucor.

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3. Glomeromycetes

The members of this group are important plant mutualists; glomeromycetes form arbuscular
mycorrhiza with the roots of nearly 90% of all plant. The hyphae of glomeromycetes are
coenocytic and produce haustoria– specialized terminal ends of hyphae that are tree-like in shape
(hence its name: arbuscular) that push into the plasma membrane of cortical cells in plant roots.
This tree-like form increases the rate of nutrient exchanges with the plant; the plant received
mineral nutrients while the fungus laps up fixed carbon. Glomeromycetes do not produce fruiting
bodies and are limited to only asexual modes of reproduction.

Figure 4: Arbuscular mycorrhizae.

Source: Campbell Biology. 3rd edition. Chapter 31.

Representatives included: none

4. Ascomycetes
The ascomycetes phylum includes fungi that produce elaborate fruiting bodies (e.g. truffles),
moulds that are restricted to hyphal growth forms (e.g. Penicillium), and single cells (e.g. yeasts).
Despite the diversity in morphology, the defining features of this phylum include septate hyphae and
distinct spore producing structures associated with sexual and asexual reproduction. Sexual
reproduction in ascomycetes is marked by the production of resistant structures called asci (ascus,
singular) within which meiosis occurs to produce ascospores. Spores produced asexually (i.e.
mitotically) are called conidia and are borne on specialized hyphal tips called conidiophores. The
ascus and conidiophores are therefore unique structures to this phylum. In what follows are the life
cycles of each type of ascomycete.

An ascocarp is a structure produced by a unique weaving of hyphae that may take a number of
different shapes: an open cup-shaped apothecium; a mostly-enclosed flask shaped perithecium with
a single pore at the top, or a closed cleistothecium with no opening to the outside.
Regardless asci form a fertile layer called the hymenium on the ascocarp.

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http://pakiagriculture.blogspot.com/2011/12/kingdom-fungi-and-phytopathology-unique.html

Diploid yeast cells (also called vegetative cells) reproduce by binary fission or budding. Sometimes
yeast cells differentiate into resistant structures called asci within which meiosis occurs. Meiosis
produces 4 haploid nuclei within each ascus. Following meiosis, one round of mitosis doubles the
number of nuclei to 8. Once discharged, the haploid spores become haploid yeast cells
undistinguishable from the diploid variety. Haploid cells of opposite mating types can fuse to restore
the diploid condition.

All other members of this phylum are multicellular. This includes the moulds (fungi limited to a
hyphal morphology, e.g. Penicillium) and fungi that produce large fruiting bodies. Multicellular
ascomycetes reproduce asexually by erecting specialized hyphae, called conidiophores ❶, from a
network of septate haploid hyphae. The terminal cells of the conidiophores differentiate into
spores called conidia ❷. If or when the conidia land on a suitable substrate, they will germinate and
grow to produce a new mycelium ❸. While most moulds in this phylum are limited to asexual
reproduction, the more complex ascomycetes include as sexual stage that leads to the formation of
large fruiting bodies. The sexual stage begins when a conidium lands on the hypha of an opposite
mating type ❹. In an act of plasmogamy, the cytoplasm of the conidium fuses with that of the cell at
the tip of a hypha. Containing two haploid nuclei, the terminal hyphal cell is now dikaryotic ❺. From
this single cell grows an elaborate dikaryotic mycelium. The terminal end cells of some of these
filaments will differentiate into asci. Inside each ascus is where karyogamy occurs ❻. The
short-lived diploid stage is followed by meiosis producing 4 haploid nuclei. A single round of
mitosis follows to yield 8 ascospores within each ascus ❼. The asci are always densely crowded in an
elaborate cup-like structure called an ascocarp ❽. The ascocarp also contains dikaryotic hyphae that
did not differentiate into asci; these are called sterile paraphyses (not shown in the diagram). In the
species featured in the diagram below, the ascospores (once ejected from the asci) shoot out from an
aperture on the ascocarp.

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Figure 5: The life cycle of Neurospora crassa, an ascomycete. Source: Campbell Biology. 3rd edition.
Chapter 31.
Representative ascomycetes included in this lab: Yeasts: Saccharomyces, Schizosaccharomyces;
Moulds: Penicillium; Cup-fungi: Sordaria, Morchella.

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5. Basidiomycetes

Like the ascomycetes, the basidiomycetes also produce large conspicuous fruiting bodies (think
shitake mushrooms). When the haploid septate hyphae of opposite mating strands fuse together
❶, a long-lived dikaryotic mycelium begins to grow and spread ❷. Under certain environmental
conditions, the mycelium will produce large conspicuous fruiting bodies (e.g. mushrooms) called
basidiocarps ❸. The underside of the mushroom basidiocarp is lined with basidia ❹. It is within
the basidia where karyogamy will occur ❺. The diploid phase is very short live and immediately
followed by meiosis to yield four basidiospores ❻. These spores rest on top of tiny buds called
sterigmata that sit on top of each the basidium. It is from this location that the basidiospores will be
discharged.

Figure 6: The life cycle of a mushroom-forming basidiomycete. Source: Campbell Biology. 3rd edition. Chapter 31.

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Lacking from the above life cycle, is an asexual stage; many basidiomycetes reproduce only
sexually. Those that reproduce asexually include the rusts and smuts –a group of plant parasites that
affect agricultural yields globally. Puccinia graminis –a basidiomycete and an infamous member of
the rusts– has both sexual and sexual life stages that together product 3 kinds of
dispersing spores. The asexual stage occurs when dikaryotic spores of P. graminis infect the stems
of grasses (most notably, wheat grass). The growth of the dikaryotic mycelium is visible as orange
streaks (hence the name of this pathogen -a rust) on the stems of wheat plants. The orange streaks
form distinct structures called uredinia on which dikaryotic spores, called urediospores, are
produced and discharged. These deep red-coloured spores continue to re-infect wheat stems. The
sexual stage begins in late summer, when uredinia differentiate into resistant structures called telia,
within which and over winter, karyogamy occurs. The diploid spores are called teliospores. In the
spring, the teliospores, undergo meiosis to produce four haploid basidiospores.
The basidiospores will be discharged and go on to infect an alternate host plant (the barberry plant).
The haploid mycelium growing in the leaves of these dicot hosts, forms pustules called pycnia
(pycnium, singular). The haploid hyphae of pycnia produce pycniospores. When these
haploid spores land on a hyphae of an opposite mating type, plasmogamy will occur and turn the
hypha, dikaryotic. This dikaryotic mycelium will grow on the underside of the same leaf forming
another pustule called an aecium. The hyphae of the aecium will produce dikaryotic spores that will
infect wheat stems.

Figure 7: Life cycle of Puccinia graminis (Source: https://www.ars.usda.gov/midwest-area/stpaul/cereal-

disease-lab/docs/barberry/life-cycle-of-puccinia- graminis/)
Representative basidiomycetes included in this lab: Coprinus, Puccinia graminis

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Fungi as Mutualistic: copyright Pearson Canada publication (2021).
Fungi form mutualistic relationships with plants, algae, cyanobacteria, and animals. All of these
relationships have profound ecological effects. Some plants harbour harmless symbiotic endophytes,
fungi that live inside leaves or other plant parts. Endophytes make toxins that deter
herbivores and defend against pathogens. Most endophytes are ascomycetes. Some fungi share their
digestive services with animals. These fungi help break down plant material in the guts of cows and
other grazing mammals. Another important symbiotic association of fungi is in the form of lichens.
Lichens are important pioneers on new rock and soil surfaces. Lichens may have helped
colonization of land by plants 550–600 million years ago. A lichen is a symbiotic association
between a photosynthetic microorganism and a fungus. The photosynthetic component is green algae
or cyanobacteria. The fungal component is most often an ascomycete. The algae provide carbon
compounds, cyanobacteria also provide organic nitrogen, and fungi provide the
environment for growth. The fungi of lichens can reproduce sexually and asexually. Asexual
reproduction is by fragmentation or formation of soredia, small clusters of hyphae with embedded
algae.

Figure 8. Anatomy of an ascomycete lichen (colourized SEM). Source: Campbell Biology, 3rd edition. Chapter
31.
Mycorrhizae is another example symbiotic relationship of fungi with the roots of plants. Fungal
association with the roots increases the surface area for absorption of water and nutrients from soil
while plant provides fungi with the ready to use carbohydrates from their photosynthetic abilities. One
type of mycorrhizae is arbuscular endomycorrhiza, where fungi penetrate the cortical layers of the roots
of a vascular plant forming arbuscules which increase the surface area for efficient
resource exchange.

Figure 8: Plant fungal association showing arbuscular myccorhizae.

Figure source: https://microbewiki.kenyon.edu/images/1/11/Amf.jpg?20221209001228

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Lab 4: The diversity of fungi Tu W Th

Name: Bench #:

Note: Always write the total magnification under the slide drawing. Always draw to scale and
labels should be clear and outside the circle.

1. Sketch your observations of the stained slides of the Allomyces sporophyte and gametophyte
generation. Make sure to identify and label the following features:
mitosporangia, meiosporangia, male gametangium, female gametangium and hyphae.

Sporophyte Gametophyte

Allomyces belongs to phylum:

Name all the structures that are diploid:

Name all the structure that are haploid:

Are there any structure that are flagellated. If so, name it/them:

Give 2 differences between sporophyte vs gametophyte generation (in terms of ploidy, cell division,
structures produced)

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2. Two opposite mating strands of Phycomyces were grown on an agar plate. Near the center of
the plate, were the two strands meet, we find the structures typically associated with sexual
reproduction. Further away from this center, you can observe the sporangiophores that
emerge from the mycelium during asexual reproduction. Sketch your observations of both
sexual and asexual reproduction. Make sure to identify and label the following
features: zygosporangium (sexual), suspensors, thorns and sporangium (asexual).

Phycomyces belongs to phylum:

Is Phycomyces homothallic or heterothallic:

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3. Sketch your observations of the stained and wet-mount slides of Zygorhynchus

moelleri. Make sure to identify, draw and label the following features:
zygosporangium, microsuspensor and macrosuspensor. Write the total magnification.

Zygorhynchus belongs to phylum:

Is Zygorhynchus homothallic or heterothallic:

How are suspensors different in Zygorhynchus in comparison to Phycomyces?

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4. Sketch your observations of pre-stained slide of Mucor. Make sure to identify, draw and
clearly label the following structures: sporangium, sporangiophore, columella (junction
between the sporangia and sporangiophore) Write the total magnification.

Mucor belongs to phylum:

Does the cultures show Mucor in asexual or sexual phase of their life cycle:

Are the cultures homothallic or heterothallic:

5. Sketch your observations of the wet-mount slide of Saccharomyces. Make sure to identify,
draw and label the vegetative and budding cells. Write the total magnification.

Saccharomyces belongs to phylum:

Give one use of Saccharomyces cerevisiae in your kitchen:

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6. Sketch your observations of the stained slide of Schizosaccharomyces octosporus. Make sure to
identify, draw and label the vegetative cells. A video will be presented to demonstrate the cells
undergoing fission. Label the ascus and ascospores (which are used for sexual reproduction).

Schizosaccharomyces belongs to phylum:

How many ascospores are present in one ascus:

What is the difference between cell division) asexual) in Saccharomyces vs

Schizosaccharomyces. You can draw and point on the cells to show the difference.

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7. Sketch your observations of the stained and wet-mount slide of Sordaria. Make sure to
identify, draw and clearly label the following structures: ascocarp, ascus, ascospores in an ascus.
Write the total magnification.

Sordaria belongs to phylum:

How many ascospores are present in one ascus:

What is the specific shape of ascocarp in Sordaria:

8. Sketch your observations of the stained slide of Morchella esculenta. Make sure to identify
the fruiting body known as apothecium. Draw and label the following features: ascus,
ascospores, sterile paraphyses. Write the total magnification.

Morchella belongs to phylum:

How many ascopores are produced in each asus and are the spores haploid or diploid:

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9. Sketch your observations of the stained slide of Penicillium. Make sure to identify one
conidiophore, draw and clearly label the following features: conidiophore, stipe (branch),
metulae, sterigmata and conidia. Write the total magnification.

Penicillin belongs to phylum:

What does Penicillin notatum produces that kills the bacteria:

10. Sketch your observations of the stained slide of Coprinus fruiting body basiodiocarp.
Make sure to identify, draw and clearly label the following features: basidium,
sterigmata, basidiospores, and paraphyses (sterile tissue surrounding the basidium).
Write the total magnification.

Coprinus belong to phylum:

How many spores in each basidium. Why this number is different from ascospores.

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11. Sketch your observations of the stained slide of Puccinia graminis. Make sure to identify all
distinct features of pycnium, pycniospores, aecium, aeciospores, uredium, urediospores,
telium and teliospores. Write the total magnification.
Note: the shape of the cells is important in these drawings. Draw the leave and the respective
structures and label as the figure given in the introduction.

Pycnium Aecium

Uredium Telium

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Which spores of Puccinia are diploid:

Which spores of Puccinia are haploid:

Which spores of Puccinia are dikaryotic:

Which plant is the primary host of Puccinia graminis?

12. What are Mycorrhizae? What is the main difference between ectomycorrhiza and
endomycorrhiza (based on the slides).

13. What are lichens.

14. Name any lab (professor from Biology or Genomics) that studies fungi (S. cerevisiae excluded).

15. What is the objective of today’s lab?

16. Circle all the correct options. At the end of today’s lab, you have learned about:
a. bacteria that can associate with fungi
b. different types of fruiting bodies in fungi
c. that fungi are autotrophic
d. fungal association with plants
e. fungi are not important for the ecological balance
f. fungi can be unicellular or multicellular

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