Laboratory 9
Seed Plant Phylogeny
In this laboratory exercise, which extends over the next two or three weeks, we will become familiar
with the problems facing systematic biologists who want to develop ideas about the evolutionary
history of groups of living things. Though you might think that figuring out the evolutionary history of
groups like the dinosaurs or the seed plants would be easy, it turns out that it’s not. Both theoretical
and practical problems make inferring evolutionary history one of the most challenging of the life
science disciplines.
Like other disciplines in biology, the study of evolution proceeds through the experimental cycle,
depending on the construction of hypotheses from observations and the rejection or retention of these
ideas based on experimental work. What’s unique is that these hypotheses address events in the past,
often millions of years ago, but they are based on observations made today - a process known as remote
inference. We shall in all likelihood never see major groups of organisms evolving over long periods
of time, so we are stuck with guessing at the origin of the major groups through reconstructing history
from the fragments of evidence still unobscured by later events.
Perhaps the most important thing to understand about constructing evolutionary hypotheses is that the
work is quite simply a comparison of groups of living things. Consequently, all of this work depends
on defining what groups of living things you are studying and what characters of those living things
you are going to use in your analysis. Thus, at the heart of any evolutionary study is a table of
characters for different groups of organisms - you should fix this table in your mind as a central feature
of this work - it symbolizes the set of observations from which the hypothesis can be constructed.
Our plan will be to begin by becoming familiar with an evolutionary group of living things, then learn
the steps in collecting evidence from these organisms by observation. These observations are the
characters that can be used to construct a hypothesis about evolutionary history. Constructing the
hypothesis we will work on in two ways, first manually and second using computer programs. Finally,
we will consider alternatives to our hypothesis to learn more about the quality of our ideas.
PLAN OF WORK - SEED PLANT LAB
Week One (week three of lab)
I. Introduction to taxa - tour of trees on campus and greenhouse
II.Introduction to characters, scoring a few character states in the lab.
III.Building a tree manually for three taxa.
A. Principles of Phylogenetic inference
B. Data Table
C. Constructing the tree
Week Two (week four of lab)
IV.Building the full tree via computer and manipulating it to test ideas.
NOTES ON LAB WORK. Help your fellow students in the lab as much as possible, working in
groups where possible. Take time to share results of the different steps in this lab exercise on the
board, with help from your teaching fellow.
I. Introduction to the Study Group - the Seed Plants
The Seed Plants are a group of some 70 orders of plants, including familiar groups like the pine trees
and bizarre and unusual plants from exotic places like Welwitschia, a plant with an underground trunk
and two leaves that pile up like giant piles of ribbons as they grow, from the Namibian desert in
southwestern Africa. Most of the diversity is in the flowering plants, which have seeds enclosed in
fruits - there are 83 orders and 370,000 species of flowering plants. Of the other eighteen orders, most
(eleven) are extinct - leaving seven orders and about 600 species of living seed plants excluding
flowers.
Eleven major living groups of seed plants are included in these seven orders and the angiosperms:
Cycadales
- the cycads, palm-like plants from tropical regions
1. cycads - Cycadaceae
Ginkgoales - the ginkgo, a bizarre tree from China planted at UVM
2. ginkgo- Ginkgo
Taxales
- the yew, a common evergreen shrub with red fleshy seeds grown at UVM
3. yew - Taxaceae
Coniferales - the pines, cedars, redwoods, podocarps, norfolk island pines, and their allies
4. redwoods and cedars - Taxodiaceae plus Cupressaceae “Taxod Cypress”
5. norfolk island pines - Araucariaceae
6. pines - Pinaceae
7. podocarps - Podocarpaceae
Ephedrales 8. Ephedra, a leafless desert shrub
Welwitschiales
9. Welwitschia, the bizarre namibian thing mentioned above
Gnetales
10. Gnetum, woody vines and a tree from tropical rain forests
ANGIOSPERMS
11. angiosperms - 83 orders of flowering plants
The big question has always been, what has the evolutionary history of these plants been and from what
group of seed plants did the flowering plants, which have seeds enclosed in fruits, arise? This question
has been unusually interesting for two reasons - first, these plants are the most prominent on the planet,
and second, it is clear that several of these groups have evolved in concert with major groups of
animals, in particular the cycads, gingkos and conifers with the vegetarian dinosaurs like Apatosaurus
and Triceratops and Stegasaurus, and the angiosperms with the insects. Understanding the evolution of
the major animal groups is directly dependent on understanding the evolution of these groups.
The goal of this lab is for you to work on the problem of seed plant evolution using data from the
recent literature, but in the process familiarizing yourself with the plants, some of their characters, and
the techniques available for constructing hypotheses about evolutionary history.
We will look at the plants and some of their characters first.
A. Tour of Seed Plants on Campus
1. Begin by taking a walking tour of the area of campus around Marsh Life Sciences Building with the
goal of capturing an image of some of the plants we are going to work on. Start with a brief visit to the
greenhouse.
Look at the cycad (Cycas revoluta, order Cycadales). This cycad is common in the Pacific Islands and
tropical Asia.
The key features to notice in building your image are ---a. a single, unbranched trunk and one cluster of leaves at the top
b. fern-like leaves - that is leaves divided into leaflets
c. these leaves arranged in a helix, not in pairs along the stem
d. seeds (in this case abortive) borne right on the edges of modified leaves - look carefully up
among the green leaves to find these tan, fuzzy, fertile leaves)
This plant is female - it produces seeds, but it does not produce pollen.
2. The other plant to see in the greenhouse is the norfolk island pine, which is in the genus Araucaria in
the family Araucariacae. Notice the following:
a. strong central axis with well-developed branches to the side (notice that the branches are in
whorls - giving the plant a very organized appearance)
b. small needle-like leaves arranged helically on the stems
3. The rest of the trees are outside. First, look for the pruned evergreen shrubs around the library,
especially on the side towards Marsh Life Sciences Building. These are yew trees, in the genus
Taxus. Look for the following features:
a. Yew is a tree with branches arising from a single central main axis.
b. evergreen leaves with a single vein running along the middle of the leaf
c. leaves arranged in a helix, not in pairs opposite one another across the stem
d. if you are lucky, “berries”, which are actually small seeds surrounded by juicy red stem
outgrowths to attract birds
Yew is famous for the quality of its wood, specifically for making bows. The famous longbow of
European history was made of yew. The species planted at UVM is common through the forests of
Europe and has been used for thousands of years. These plants are perhaps the most poisonous plants
planted at UVM - all except for the red flesh on the seed. This plant is now famous as a source of
taxol, a leukemia cure.
4. Now walk across to the bookstore area. Beyond the bookstore, on the lawn to the north of Morrill
Hall, are two ginkgo trees (Ginkgo biloba, order Ginkgoales). The key features to notice in building
your image of the ginkgo are ---a. these trees are branched from a single central main axis
b. the major shoots bear short little shoots at long intervals - and the short shoots, in the active
season, bear all the leaves and reproductive structures.
c. once again, the leaves are attached in a helix around the short shoot - you can tell because
the leafbases leave scars on last year’s long shoots when they fall off
d. on the ground under one of the trees (the female one) you will find the seeds - either with
their fleshy seed-coat layer or without, in which case they are hard and tan.
e. you will also find the leaves, which look like fans about four inches across - look for
forking veins in the leaf tissue - keep one of these as a memento if it’s useful.
The ginkgo survives in a few totally obscure places in interior China - one well-known remaining wild
population is entirely confined to a cliff face. The tree survives best in gardens - the oldest cultivated
individuals are in the parks of temples and such. The tree turns out to be very useful as an inner-city
planting, because it is very pollution resistant.
5. Now walk out onto the UVM green. Find the dawn redwood (genus Metasequoia, order Coniferales,
family Taxodiaceae), a conical tree with reddish brown bark and bare branches. For our study, take in
an image that includes --a. again, a single central axis bears many lateral branches
b. small branches shed on the ground along with the leaves
c. some small shoots on the branches persist to increase branch size
d. look for shoots on ground and on the tree with leaves still attached: the leaves are in two
rows on opposite sides of the small branches; they are paired
Dawn redwood is a relative of the California redwood, big tree, and bald cypress. This tree was
discovered in China when a botanist realized that a specimen collected by a field botanist in China was
actually this plant. The dawn redwood had been known for years as a fossil - when the Chinese
returned to the interior valley, they found the living plants prospering! Now, 50 years later, with
greatly increased population pressure, the wild populations of dawn redwood are endangered - no new
young plants are germinating in the wild stands.
6. On the way back to the lab, look at the conifers. There are spruce trees between the entrance to the
bookstore and the theatre, and there are pine trees just beyond the bookstore on the right, near the
board for posting notices. Fix an image of the Coniferales using these trees. They have --a. The same strong central axis with ample branches.
b. Evergreen leaves arranged helically along the stems.
c. These leaves are very narrow; they have only one or two veins per leaf.
d. Each tree makes both seeds (in pine cones) and pollen (not evident this time of year).
7. On the green in front of Marsh Life Science Building, in from of the biology offices, is a tree with
brown-purple woody bean-like fruits (some years they are missing). This is the kentucky coffee tree, in
the genus Gymnocladus, order Fabales, which we’ll use to represent the flowering plants
(angiosperms). This tree has ---a. a spreading form, without a single prominent central axis
b. fruits, in this case woody beans - a closed container inside of which the seeds are protected
--- the seed inside this bean is equivalent to the whole seed borne by the ginkgo tree
The Kentucky coffee tree makes a good symbol for the flowering plants because its order, the Fabales,
is one of the largest and most important groups in the angiosperms, including over 20,000 species (over
5% of the whole diversity) and numerous economically important plants, both for foods, timbers, dyes,
and medicines. Beans are foremost among these economically important plants. This tree was used as
a coffee substitute by the early immigrants to Kentucky and Tennessee - but it made a poor substitute.
The roasted seeds were eaten by various native American groups.
II. Characters
Once you have chosen a group of living things, the first thing to do is to identify a set of varying
characters. Now that you have some images of the plants in the study group for this exercise, spend
some time looking at some of their characters in the lab to become familiar with the kinds of structures
that we will be talking about in an evolutionary context.
The simplest way to do this is to compare the pines and the flowering plants - from the analysis that we
will perform there are five characters that are fairly easy to see and differ between these two groups.
We’ll look at them in order of difficulty.
1. Woody Cones Absent versus Present. (character 64) This first character is a good place to start, since
you can decide which groups have woody cones from a distance of at least 10 feet away from the plant.
The conifers get their name from bearing these woody cones - the cones in the labs will remind you
that you know this character well from your own experience. Angiosperms (flowering plants) never
have cones like these - obviously the flowering plants from the greenhouse in the lab don’t have cones.
Starting with the obvious is wise, because characters can be extremely hard to see and understand.
2. Unisexual versus Bisexual Strobili. (character 35) Put simply, flowers can be simultaneously male
and female, but pinecones are either male or female, never both.
The large cones in the lab are entirely female - that is they produce seeds but not pollen. In the seed
plants, the female household is responsible for producing ovules, which when fertilized become the
seeds that characterize the whole evolutionary group. In the pines, these ovules, and later seeds, are
borne in the big, woody cones, whereas the pollen, which is the functionally male part of the plant, is
produced in a much smaller cone-like structure that is not at all woody.
Now look at the flowers in the lab. These have both male and female structures. Try to equate the
structures of the flower and cone. Both are shoot tips with reproductive structures; in other words each
consists of leaves and stems with attached seeds and pollen-producing structures.
3. Vessels versus tracheids. (character 13) This is a microscopic character. Vessels and tracheids are
two kinds of cells called tracheary elements found in the tissue called xylem. These tracheary elements
are designed to carry out two jobs at once, provide structural support to the plants and provide transport
pathways for water and dissolved inorganic nutrients absorbed from the soil. The best place to look for
these cells is in the wood, which is xylem produced by the cambium of trees. The two cell types are
dramatically different and easy to tell apart under the microscope - here is a table of differences:
tracheids
smaller diameter
pointed, closed ends; not in long
tubes
vessel elements
larger diameter
open ends, fit into long tubes
called vessels
a. Look at wood of pine and a flowering plant, the maple, under the microscope. Both have
tracheids, which are small elongate cells with pointed, closed ends. The vessel elements jump
out at you in the maple - they are these big hollow places in the wood. Look closely to see
that they are in fact pipe-shaped cells fitting into long tubes - the vessels.
b. Check the pine to prove to yourself that the vessel is absent from the pine wood. Be
careful, there is another structure, a resin duct, that looks sort of like a vessel in pine wood,
visible in the transverse sections, but you can distinguish these from vessels.
4. Free-nuclear versus Cellular Early Embryogeny: (character 89) First of all, what is free-nuclear.
Free-nuclear refers to tissue resulting from nuclear division without cell division, so that you end up
with what amounts to a soup of nuclei in a large cell. Some animals have free-nuclear tissues, but
zoologist use the word syncoetium to describe them.
Look at the slide of a conifer embryo. In the conifer slide, the young embryo inside the seed is made
up of nuclei in a large cell. Now compare the slide of the young embryo of the flowering plants. It is
made entirely of cells, each with a single nucleus.
III. Building a Tree
You should have the beginning of the idea of the two dimensions to the problem presented by this lab the groups of organisms and the characters. So the question is --- how do you use the varying
characters to construct hypotheses about evolutionary history for groups of organisms? Obviously the
answer is, compare the groups using the characters, but how to make the comparisons carefully?
A. Using Characters to Distinguish Groups.
In this lab we are seeking a hypothesis for the evolutionary relationships of the living seed plants,
including the groups you’ve looked at outside. Each group has a set of characters - some of these are
variable (different species have different character states) and some are constant (all species of the
group are the same.) Comparing the groups leads to discovering whether the character varies between
the groups. So, for instance,
1. Leaf size varies between species of pines, angiosperms, and cycads,
2. Woody cones are either absent or present in a group - all pines have them, no angiosperms
have them, no cycads have them,
3. Seeds are present in all of the groups we are studying.
It is the characters that are constant within a group, but vary among the groups, that are important in
developing evolutionary histories. Each of the characters you just looked at with the materials in the
lab are constant within a group but vary between groups, and we will use them - and others - in
constructing our hypothesis of evolutionary history.
We said at the beginning that the heart of any evolutionary study is a table of characters for different
groups of organisms. To start with, here’s a list and a table of just the characters you looked at for the
groups of seed plants in our study.
List of Characters
64. Woody cones absent (0) versus present (1)
35. Strobili unisexual (0) versus bisexual (1)
13. Vessel elements absent (0) versus present (1)
89. Free-nuclear (0) versus cellular (1) early embryogeny
Character (by number above)
cycads - Cycadaceae
ginkgo- Ginkgo
yew - Taxaceae
redwoods - Taxod Cypress
norfolk island pines - Araucariaceae
pines - Pinaceae
podocarps - Podocarpaceae
Ephedra
Welwitschia
Gnetum
angiosperms
64
0
0
0
1
1
1
1
0
0
0
0
35
0
0
0
0
0
0
0
0
0
0
1
13
0
0
0
0
0
0
0
1
1
1
1
89
0
0
0
0
0
0
0
0
1
1
1
B. Using Characters to Decide Relative Similarity of Groups
Once you have a table like this one, you can start to think comparatively about the groups of organisms
in the study set. For instance, you can answer questions like, which groups are most similar, which are
most different? To focus your attention on the problem, answer the following questions based solely
on the table above ----
1. How many groups of groups are there - that is groups of groups that are the same for all five
characters?
2. Which is more similar to Ephedra -- Gnetum or the angiosperms?
3. Are conifers like the redwoods and pines more similar to Ephedra or to cycads and
ginkgos?
However, the best way to assess similarity is all at once, considering all possible relationships.
Strangely, we proceed backwards, counting up the number of differences between groups. For this part
of the lab, build a matrix of distances. The distance equals the number of character states that are
different. To begin with, combine the groups that are identical for the five characters so that you have
a smaller number of groups each of which has all the same character states. Next, for each possible
pair of groups, make a list of the characters for which they are different. Now build a matrix of
distances between all the combined groups by counting the number of characters that differ. Use the
following example :
Ephedra
0
Gnetum
1
0
Angiosperms
2
1
0
Ephedra Gnetum angiosperms
Once you have constructed your matrix, you are ready for the critical step that introduces the
fundamental modern images associated with charting evolutionary history. This step is to construct a
network graph that represents all the similarity relationships. For the three groups in the matrix above,
the network looks like this:
FIG. 1
distance 1
distance 1
Ephedra
Gnetum
character 89
change: 0-1
angiosperms
character 35
change: 0-1
Notice that the distance in this graph between Ephedra and the angiosperms is two, just as in the data
matrix, because you add the one character change between Ephedra and Welwitschia to the one
character change between Welwitschia and the angiosperms. .
Now construct a matrix and then a network using all five groups of groups (yes, five was the right
answer to question 1 above). - Hint: the network must include two branches from the combined group
cycads plus ginkgo plus yews (Taxaceae).
C. Constructing an Evolutionary Hypothesis
Now, how does this network become a hypothesis for an evolutionary history? The answer is, by going
through a process known as rooting the tree. The key to this process is understanding primitive and
derived characters and groups. You need some definitions:
A primitive character state is transformed by the evolutionary process into a derived character
state, for instance - a forelimb of a dinosaur is transformed into the wing of a bird.
A primitive evolutionary group is one that has few derived character states;
A derived evolutionary group is one that has many derived character states.
So far, we have said nothing about which groups are most primitive or most derived evolutionarily. All
we have done is to talk about the changes in characters necessary. But these changes, if we could
decide primitive versus derived, would yield a hypothesis for the evolutionary changes in the history of
the group.
The process of rooting a tree is almost always done by specifying an outgroup, a group that we are
fairly certain is not part of the group we are interested in (what is called the study group) but is closely
related to it, and comparing its character states to those of the organisms in the study group. The
convention that is used is that character states shared by some of the members of the study group and
the outgroup are primitive for the study group. The alternative character state, not shared with the
outgroup, is the derived character state.
In the example groups above, the cycads are usually considered to be the most primitive members, because they
are different from the rest of the groups in lacking some key derived character states. Among these are some
you can understand - for instance, the cycads do not branch, whereas all of the other groups have extensive
branching of stems - you saw this yourself on the walk. Another feature is the wood - cycad wood is weak and
full of parenchyma cells, whereas the wood of the remaining groups is strong - it has a small, carefully placed
set of parenchyma cells. From this arises the idea that the cycads are more primitive than the rest and also that
they are the outgroup, so that they can be used to interpret the character states in the remainder of the groups.
As you can see, the cycads’ character states have all been scored as zeros. Let’s look at our condensed
data table:
Character (by number above)
cycads, ginkgos, yews
redwoods, araucarias, pines, podocarps
Ephedra
Welwitschia & Gnetum
angiosperms
64
0
1
0
0
0
35
0
0
0
0
1
13
0
0
1
1
1
From this chart, you can see how to interpret the characters. For instance, Ephedra is derived for
character 13, and the redwoods and their allies are all derived for character 64.
89
0
0
0
1
1
Based on this assessment of primitive and derived character states, you can now root the
network to produce a tree, that is an evolutionary hypothesis. Here are two ways of
portraying this hypothesis, each showing the five groups and the characters that are changing.
angiosperms
35
Welw. and Gnetum
FIG. 2
89
Ephedra
pines etc.
64
13
cycads
cycads
pines etc.
Ephedra
angiosperms
Welw. and Gnetum
35
64
89
FIG. 3
13
The first one (Fig. 2) probably makes more sense to you, since it is much like the network you made.
It shows the cycads as the ancestor of the rest of the plants and the two lineages, pines and their allies
on the one hand and all the rest on the other hand, evolving from the cycads.
However, it is conventional these days to show all the groups as descendants and to leave the ancestors
unidentified, for a philosophical reason. It is simply very unlikely that we would ever have an actual
ancestor to study. Virtually everything we look at, even fossils, has probably evolved by divergence
from the common ancestors indicated by the nodes (meeting places of lines) on the trees. So, we
redraw the tree like the second one (Fig. 3), still showing the changes, but showing all the groups, even
the cycads, as descendants.
D. Complicating the Problem: Incongruent Characters.
If data tables always looked like the one we have been working with, hypothesizing the history of
groups would be easy. However, there is a major problem - many data sets include incongruent
characters - by which we mean characters that support different evolutionary histories. Here is our data
table again, but with two incongruent characters added.
Character 51: pollen outer coat made up of interwoven threads(0) versus small granular particles(1)
Character 79: seed coat simple(0) versus differentiated into an outer fleshy and inner stony layer(1)
You can see the two character states of character 79 displayed in the lab.
Character (by number above)
cycads - Cycadaceae
ginkgo- Ginkgo
yew - Taxaceae
redwoods - Taxod Cypress
norfolk island pines - Araucariaceae
pines - Pinaceae
podocarps - Podocarpaceae
Ephedra
Welwitschia
Gnetum
angiosperms
64
0
0
0
1
1
1
0
0
0
0
0
35
0
0
0
0
0
0
0
0
0
0
1
13
0
0
0
0
0
0
0
1
1
1
1
89
0
0
0
0
0
0
0
0
1
1
1
51
0
0
1
1
1
0
0
1
1
1
1
79
1
1
0
0
0
0
1
0
0
0
0
A. Draw a tree like the one in Figure 2 above, but include more groups (there are now eight different
combinations of characters). Start with a matrix, go on to a network, then root the network using the
cycads. Label the tree with the character changes for each character.
B. You will discover a serious difficulty when you try to show the changes in the network and the tree there is no network in which each character changes only once. In labeling the evolutionary tree, you
will either have to
1) change the character back to primitive once it’s derived (reversal) OR
2) make the character originate more than once (parallelism)
(Note, these two choices are the two possible solutions to a single problem, the incongruence
of characters: each solution yields the same total number of transformations. Incongruence
means that the characters support different phylogenetic histories: only one can be correct.)
These two kinds of evolutionary change, which certainly happen in the history of lineages, are called
homoplasies. Homoplasies are especially common in plant evolution, perhaps less so in vertebrates.
Because homoplasies exist, more than one network and tree are possible. The question is, which is the
best hypothesis for an evolutionary history when there are homoplasies? In other words, which is the
right tree?
E. The Solution to Homoplasy: Select the Simplest Solution.
The traditional and modern answers to this question are the same - choose the tree with the fewest
changes, which is the same as saying choose the simplest explanation. The rationale for this is not
based on biology --- evolution rarely takes the simplest route. The rationale is tactical - the simplest
tree is the easiest one to disprove.
Beginning in the lab, construct three trees for the evolutionary history of this group that differ in their
total number of steps. How are the evolutionary changes represented by the character changes different
in each tree? How many steps long is the shortest tree? (This may be assigned as homework between
the weeks.) Make sure that you complete your first tree and show it to your teaching fellow
before you leave lab.