Lab 12 Building Phylogenies Objectives In this laboratory exercise, you will examine six species of agaricomycetes and predict the evolutionary relationships among them. After completing this exercise you will be able to • define ancestral characteristics, derived characteristics, branch point, and phylogeny. • predict ancestral and derived characteristics for agaricomycetes. • construct a phylogeny (phylogenetic tree). • support the phylogeny with data. • explain how evolutionary biologists discover evolutionary relationships. Introduction One of the most compelling pieces of evidence for evolution is that organisms have amazing similarities. An example that almost everyone has heard before is that the limbs of birds, bats, horses, moles, cats, frogs, humans, turtles, and other vertebrates have virtually the same skeletal plan. Furthermore, even snakes and whales show structural remnants of the limbs of their ancestors. The evolutionary interpretation of these similarities is that the vertebrate limb has been modified by natural selection to perform different functions (for example, running, digging, flying). Another commonly used example is that the embryos of turtles, mice, humans, chickens, and many other vertebrates are amazingly similar. Furthermore, the proteins and DNA of organisms are remarkably similar. Why, do you suppose, can human diabetics use insulin extracted from pigs to control their blood sugar levels? Well, the reason is that the chemical structure of human and pig insulin is very similar. In addition to these similarities, we discover that organisms that appear similar in one respect are often similar in other respects (we can say the patterns are “concordant”). For example, organisms that are similar morphologically (in shape) have similar protein structures. Organisms that are less similar morphologically have less similar protein structures. This pattern holds for traits that are not easily modified by evolution, but not so often by traits that are easily modified by selection. For example, flower color might not be a good trait to use when looking for concordance because it is easily changed genetically. The concordance of traits is an important support of evolution. Imagine that we saw that organisms similar in one set of characteristics were very different in a second set of characteristics and different again in a third set of characteristics. This situation would be chaotic and we would be forced to question the reality 1 of evolution. The development of methods of DNA and protein analysis has shown dramatically that organisms that are similar morphologically are also similar at the genetic level. So, similarity among organisms provides evidence for evolution. We can then turn around and use the similarities to try to reconstruct evolutionary relationships. That is the purpose of today’s lab: to construct a hypothes ...

1. Lab 12
Building Phylogenies
Objectives
In this laboratory exercise, you will examine six species of
agaricomycetes and predict the evolutionary
relationships among them. After completing this exercise you
will be able to
• define ancestral characteristics, derived characteristics, branch
point, and phylogeny.
• predict ancestral and derived characteristics for
agaricomycetes.
• construct a phylogeny (phylogenetic tree).
• support the phylogeny with data.
• explain how evolutionary biologists discover evolutionary
relationships.
Introduction
One of the most compelling pieces of evidence for evolution is
that organisms have amazing similarities. An

2. example that almost everyone has heard before is that the limbs
of birds, bats, horses, moles, cats, frogs,
humans, turtles, and other vertebrates have virtually the same
skeletal plan. Furthermore, even snakes and
whales show structural remnants of the limbs of their ancestors.
The evolutionary interpretation of these
similarities is that the vertebrate limb has been modified by
natural selection to perform different functions
(for example, running, digging, flying). Another commonly used
example is that the embryos of turtles,
mice, humans, chickens, and many other vertebrates are
amazingly similar. Furthermore, the proteins and
DNA of organisms are remarkably similar. Why, do you
suppose, can human diabetics use insulin extracted
from pigs to control their blood sugar levels? Well, the reason
is that the chemical structure of human and
pig insulin is very similar.
In addition to these similarities, we discover that organisms that
appear similar in one respect are often
similar in other respects (we can say the patterns are
“concordant”). For example, organisms that are
similar morphologically (in shape) have similar protein
structures. Organisms that are less similar
morphologically have less similar protein structures. This
pattern holds for traits that are not easily

3. modified by evolution, but not so often by traits that are easily
modified by selection. For example, flower
color might not be a good trait to use when looking for
concordance because it is easily changed
genetically.
The concordance of traits is an important support of evolution.
Imagine that we saw that organisms similar
in one set of characteristics were very different in a second set
of characteristics and different again in a
third set of characteristics. This situation would be chaotic and
we would be forced to question the reality
1
of evolution. The development of methods of DNA and protein
analysis has shown dramatically that
organisms that are similar morphologically are also similar at
the genetic level.
So, similarity among organisms provides evidence for
evolution. We can then turn around and use the
similarities to try to reconstruct evolutionary relationships. That
is the purpose of today’s lab: to construct a

4. hypothesized evolutionary relationship (a phylogeny or
phylogenetic tree) of a group of organisms you
probably know very little about: fungi.
Fungi are eukaryotic, heterotrophic, achlorophyllous organisms
closely related to animals within Domain
Eukarya. A unifying character among fungi is that they are
osmotrophic and possess cell walls containing
chitin. There are at least eleven major phylogenetic fungal
lineages (Hibbett et al, 2007).
Fig. 1. Image modified from Hibbett et al. 2007. A higher level
phylogenetic classification of the Fungi. Mycol.
Res. 111:509-547.
Fungi are highly diverse in form and function and truly are
cosmopolitan. Some fungi are aquatic and have
flagellate spores (Chytridiomycota) and are responsible for the
demise of amphibians worldwide. Others
are beneficial living inside digestive systems of herbivore
mammals (Neocallimastigomycota) or hindguts of
arthropods (Kickxellomycotina) while others kill nematodes
(Zoopagomycotina). Most fungi are important
as decomposers in all ecosystems such as the sugar fungi
(Mucormycotina). Almost all vascular plants
benefit from associations with fungi to their roots for increased
water and nutrient uptake

5. (Glomeromycota) and humans have domesticated important
strains of yeast and molds for industrial
purposes (Ascomycota). And, people have always wondered
about the magic emergence of mushrooms
and their effects on the human body (Basidiomycota).
Evolutionary relationships in fungi have been reconstructed
using morphology and DNA. Milestones in the
evolution of fungal characters are shown In Fig. 2. Spores
(reproductive and dispersal units of fungi) and
hyphae (unit of filamentous fungi) are shown as earliest signs of
fungal presence. Other traits emerged later
giving rise to specialized structures and defining groups who
exhibit them. This is the case of Basidiomycota
2
and Ascomycota, the most recently evolved fungal taxa, both of
which share presence of dikaryotic hyphae
together with complex sexual and asexual reproductive
structures.
Fig. 2. Evolution of fungal characters, based on molecular clock
dating. a)Flagellate spore; b)Coenocytic
filament; c)Septa; d)Clamp connections; e)Phragmobasidia;
f)Asexual spores; g)
Asci; h)Ascoma; i)Holobasidium; j)Basidioma. From Berbee &

6. Taylor. 1993. Dating evolutionary relations of
the true fungi. Can.J.Bot. 71:1114-1127
For this laboratory exercise you will study a handful of
Basidiomycota, in the class Agaricomycetes. Fungi in
this group include the mushrooms, puffballs, shelf fungi, jelly
fungi, chanterelles, stinkhorns and others.
These fungi represent 98% of all described species in the
Basidiomycota. Observe a working classification of
this group in the image below. The specimens you have in front
of you may be placed in several of the
orders within class Agaricomycetes (Fig. 3).
3
Fig. 3. Classification of Basidiomycota in its sub-lineages.
Images taken from the website Tree of Life. URL:
http://tolweb.org/Fungi/2377
How to reconstruct a phylogeny
Think about the kind of diagram (phylogeny) you need to draw.
Your goal is to develop and draw a
phylogeny. A phylogeny is a reconstruction of relationships
between taxa. The more characters organisms
share between them, the closer related they are. The basic
assumption involved in making an evolutionary

7. tree (phylogeny) is that two organisms that have a more recent
common ancestor will share more
characters than two organisms with a less recent common
ancestor. A character can be any descriptor:
from anatomical structures to DNA. One of the greatest
strengths of constructing an evolutionary tree
using this method is that it is testable. The discovery or addition
of characters should strengthen your
phylogenetic hypothesis. If, for example, new evidence is found
you could add it to your evolutionary tree
to either support or reject your hypothesized evolutionary
sequence.
How would you approach the reconstruction of a phylogeny for
a lamprey, a shark, a frog, a dog, a cow, a
monkey and a human? Probably you already know that the dog,
cow, monkey and human are more related
between themselves than to the rest, but how so? How can you
justify your assumption? First, you need to
construct a table in which you write as many characters you can
find and then score them (ideally with
0=absent, 1=present).
Lamprey Shark Frog Monkey Cow Human
Vertebral column 1 1 1 1 1 1
Jaws 0 1 1 1 1 1
Hair 0 0 0 1 1 1

8. Loss of tail 0 0 0 0 0 1
Habitat marine marine amphibian arboreal terrestrial terrestrial
Based on your results you then decide which characters are
ancestral (shared by more than one taxon) or
derived (if they are unique to the taxon) to design your
phylogeny (Fig 4a). The lamprey shares the one
character with the rest and so it is at the base of the
phylogenetic tree, serving as the outgroup, a group
4
that is thought to have traits similar to the ancestor of the group
being studied. In other words, it is the
most ancestral taxon in this analysis. By default, the taxon at
the tip of the phylogenetic tree will be the
most evolved (the one with most derived characters). While
building a phylogeny, it is important you
understand that derived characters are the most useful to
determine the position of taxa and that the more
characters you use the stronger your phylogenetic tree will be.
Case in point: which mammal is more
related to the human: cow or monkey? How can you support a
correct phylogenetic relationship among
them based on the table above? Not possible. You need to
elaborate on additional data. Better phylogenies
have several traits showing the same result. This concordance of

9. traits gives you more confidence in your
conclusions.
Fig. 4. A phylogeny showing evolutionary relationships among
vertebrates. The lines represent evolutionary
pathways that lead to groups. In this example, lamprey is the
“outgroup” for the “jawed” vertebrates. Branching
points show when two groups diverged. The “hatch” marks mark
evolutionary innovations that unite
descendant groups.
Finally, notice that not all characters you choose may end up
being appropriate; this is the case of vertebral
column because all taxa (in our example) have it, or in the case
of habitat a character harder to justify; in
the end these may not be needed in your phylogeny. The
phylogeny in Fig 4b is the same as in Fig 4a but it
appears to be more concordant in that it relies on more derived
characters (resulting from a more detailed
character table).
Phylogenetic relationships also vary depending on the number
of taxa used. Observe Fig 5 and notice how
4a
4b

10. 5
the use of characters changes its placement within the
phylogeny.
Fig. 5. Two possible phylogenies showing evolutionary
relationships after the addition of a horse to the earlier
phylogeny. In 4a, we hypothesize that the lengthened foot
evolved once, in a lineage leading to both cow and
horse. In 4b, we hypothesize that the lengthened foot arose
twice, once in the lineage leading to the cow and
once in the lineage leading to the horse. (Note that we wouldn’t
put this character on the slanted line near the
appearance of hair because it would then have to “disappear”
before the divergence of monkeys.) So, because
we have a complicated trait that is likely to evolve only once,
we would use the first phylogeny—a.
It is likely you will end up building more than one phylogeny,
all of which correctly address the problem.
But, which one is the best one? A better phylogeny preferably
does not have reversals of character states,
that is, you hypothesize fewer evolutionary changes to explain
your phylogeny. This idea is called the

11. principle of parsimony. In Fig 5b, “walking on toes” requires an
extra evolutionary step to separate cow
from horse, whereas in Fig 5a it is assumed that the foot and
toes only changed once.
Assignment: Build a Phylogeny of Fungi
Your job is to construct a phylogeny for six agaricomycetes
species. Your goal is to hypothesize evolutionary
relationships among these species and show the characters that
led you to your hypothesis. You will show
your hypothesis and the table of characters that support your
hypothesis in an evolutionary (or
phylogenetic) tree, a diagram that shows where evolutionary
innovations arose.
1. Examine the boxed set of agaricomycetes. Notice one of the
specimens is labeled as “outgroup”. Write
down the names for all species and look for their file names in
the “Phylogeny of Fungi” SAKAI webpage
(see “instructions” below). Each file will include an image and
a description. See the accompanying
diagrams (Figs 5-7) to learn what the traits are.
2. Examine the characters of the agaricomycetes and of the
outgroup (Cladina). Characters that are similar
5a
5b
6

12. between the outgroup and agaricomycetes (e.g., living on soil,
dijkaryon) will not help build a
phylogeny. You will use only evolutionary innovations in
building your phylogeny; ancestral traits will
not help you.
3. Next, choose characters that you think might be useful in
building the phylogeny. Choosing which
characters to use is one of the most important parts of building
a phylogeny. The following tips will help
you choose characters, but different people will probably use
different characters.
a. Avoid traits that you think are easily changed by evolutionary
processes (e.g., color of
basidiomata).
b. Choose traits either from the pictures or the list of traits and
compare the “states” of those
characters to those of the out group. The character state of the
out group is considered to be
the ancestral state. For example, the presence of mycelium (see
Fig. 2) is an ancestral trait
because it also occurs in Cladina lichens. The presence of the
basidium is an evolutionary
innovation in Agaricomycetes because it does not occur in
Cladina. Any change from that state
is an evolutionary innovation. Non-varying traits won’t tell you
anything about evolution within
the group.

13. c. Character states can be “presence” vs. “absence” or variations
of a character that is “present”
(e.g., hymenium enclosed or exposed in different shapes like
gills or tubes).
4. When you find traits that vary among agaricomycetes species,
list the various character states and then
decide which of the character states is likely to be most
advanced, least advanced, and intermediate.
For example, agaricomycetes in terms of basidiocarp shape:
stipitate, sessile, gasteroid, etc. In this case,
you would need to decide which one is the most primitive.
Reference to the character state of the out
group will help you make these decisions. One caution: loss of a
character is relatively easy.
5. Think about the kind of diagram (phylogeny) you need to
draw.
6. To start building a phylogeny, pick one trait (maybe
basidiocarp or spore shape) and draw a phylogeny
using just the one trait. When building a phylogeny based on
just one trait, you will probably not be
able to mark changes at all the points of evolutionary
divergence.
7. Next, pick other traits and draw new phylogenies. You don’t
need to draw phylogenies for all the traits
listed but you should draw a good number. It is important to
realize that some phylogenies are the
same even though the order of species is not the same.

14. 8. Now you need to choose the better phylogeny using the
principle of parsimony (the least amount of
evolutionary changes to explain your hypothesis).
9. Strengthen your phylogeny by adding several traits to each
taxon. Note that concordance of traits gives
you more confidence in your conclusions.
10. Turn in your phylogeny (follow directions from your
instructor). Also attach a list of the characters you
used and show the ancestral and derived states of those
characters.
Instructions on how to access Resources
Login to USAOnline (Sakai). In “My Workspace” click on
“Memberships” found on the left-hand column.
Click on “Joinable Sites” and look for “Phylogeny of Fungi”
(listed alphabetically). Upon joining the site you
will see the link in your upper tab of Sakai. Under “Resources”
you will find descriptions for all fungi;
download and print (optional) files in your box. If you need
extra help, there are useful weblinks and videos
related to this assignment. Once you are finished with this class,
you can unjoin this webpage.
7
Fig. 5. General line drawings for typical agaricomycetes. Taken
from Hawksworth et al. 1983. Ainsworth &

15. Bisby’s Dictionary of the Fungi.
8
Fig. 6. Terminology associated to mushrooms. Image taken from
www.urbanmushrooms.com.
9
Fig. 7. Lichen forming fungi. Images taken and modified from:
digilibraries.com
A, Thallus of foliose lichen (Parmelia) with apotthecia.
B, Cross section of one apothecium, × 5.

16. C, Section through the body of a gelatinous lichen (Collema),
showing the photobiont (Nostoc) individuals surrounded by
the fungus filaments, × 300.
D, a spermagonium of Collema, × 25.
E, a single Nostoc thread.
F, spore sacs and paraphyses of Usnea, × 300.
G, Protococcus cells and fungus filaments of Usnea.
A, Fruticose lichens on wood
B, Usnea, with apothecia
C, Sticta,
D, Peltigera, with apothecia
E, cross section of a single apothecium
F, Cladina, with apothecia
G, Crustose lichen (Graphis) on bark
H, Soredium of a lichen.
10
Your Name___________________________________________

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