Lab 1: Classification and Systematics

History of Classification:
Humans have had a practical need for naming organisms around them for as long as oral language
has existed. For example, we have long needed to categorize organisms into groups, such as “edible”,
“toxic”, “dangerous” or “medicinal” that describe how those organisms might be used by humans, or
whether they represent a hazard. Having shared classification schemes has allowed humans to both
describe and order the natural world, as well as communicate with each other about that world. As
expected, early written accounts indicate a continuous interest in the diversity of living forms.

The fantastic array of described species known today exceeds one million species, and possibly
millions more are not yet described. This does not include those that are now extinct and are part of
the fossil history of the earth. During the past three centuries, numerous schemes have been proposed
to organize and classify life forms. Before the 1700’s, there was no universally accepted system of
nomenclature, so scientists often had many names for one species. These names were often lengthy
descriptions of the organism. For example, one name for peppermint was Mentha floribus capitatus
foliis lanceolatis serratis subpetiolatis, which can be translated as “the mint plant with flowers in a
head, leaves lance shaped, saw toothed with very short petioles.” As you can imagine, using these
names was very cumbersome.

In 1735 Swedish scientist Carolus Linnaeus published Systema Naturae, which outlined a
classification system in which different organisms were grouped using particular shared
characteristics, including morphological (physical characteristics), developmental, behavioral, or
ecological traits. This work focused primarily on animals. In 1753, Linnaeus published Species
Plantarum, which focused on plants. In his work, Linnaeus developed the first formal methods of
taxonomy, the branch of biology that is concerned with the discovering, describing, naming, and
classifying organisms. He created a hierarchical classification system in which very similar
organisms were placed into small groups, which were then grouped into larger, increasingly less-
similar groups. We use essentially the same system today. Linnaeus also popularized the use of
binomial nomenclature, or the use of species names comprised of two parts.

Modern classification schemes are based on the early work of Linnaeus, which described two
kingdoms of life (Table 1). Over the past 2 ½ centuries, many scientists have contributed to the
field, and the current scheme that we use was proposed by Woese and involves three domains
(Eukarya, Archaea, Bacteria) and six kingdoms (Animalia, Plantae, Fungi, Protista,
Archaebacteria, Eubacteria). However, recent molecular work (e.g., Williams et al. 2013) has
indicated that Eukarya may be nested within Archaea, and that a two-domain system (Archaea,
Bacteria) would more accurately reflect evolutionary relationships. In this course, we will continue
to use the three Domain system. As you can see, it is a difficult task to find the best system, and it
changes as we learn more about organisms.

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Table 1. History of Classification of Life
Linnaeus (1735) Haeckel (1866) Whittaker (1969) Woese (1990)
2 Kingdoms 3 Kingdoms 5 Kingdoms 3 Domains
Animalia Animalia Animalia Eukarya
Plantae
Vegetabilia Plantae Fungi Archaea
Protista
Protista Monera Bacteria

The Hierarchical Classification System:

The systems developed by Linnaeus and his immediate successors are known as “artificial”
classification systems because they were based purely on morphological similarities, rather than
on evolutionary/genetic relatedness. Today, the goal of classification is to reflect the evolutionary
relationships among species. A system that reflects these relationships is called a “Natural”
classification system. Our current hierarchical classification system is shown below, using
humans as an example.

Classification of Humans (Homo sapiens):

Domain-Eukarya
Kingdom-Animalia
Phylum-Chordata (subphylum Vertebrata)
Class-Mammalia
Order-Primates
Family-Hominidae
Genus- Homo
Species- Homo sapiens

There are many mnemonic devices for remembering the order of these nested groups. One
example is “Do Keep Pots Clean Or Families Get Sick. Note that the general term taxon (pl.
taxa) can be used to refer to organisms any level of organization. Also, note the correct plural
forms for three of the groups below:
phylum - (pl., phyla)
genus - (pl., genera)
species - (pl., species)

In the current system, species is considered the basic unit of biological classification. Groups share
increasingly more characteristics as they progress down the hierarchy. For example, organisms in
the same genus share more characteristics than those in different genera in the same family, while
those in the same family are more similar than those in different families in the same order. This is
because the system reflects evolutionary relationships, and organisms in the same genus share a
more recent common ancestor, and are thus more closely related, than those just in the same
family. Similarly, those in the same family are more closely related than those in the same order.

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Nomenclature:
A system of classification requires a set of rules for naming organisms that, ideally, reflects the
classification system and provides information regarding that system. The study of names and the
rules governing names is called Nomenclature.

All species' names are composed of two elements, hence the term binomial nomenclature. The
first term represents the genus to which the organism belongs. The genus name is a Latin noun
which always begins with a capital letter. The second term is the specific epithet, and is a Latin or
Latinized adjective. The specific epithet term is not capitalized. When both terms are used
together, they specify a single, unique species (e.g., Homo sapiens). Furthermore, when the
complete name (genus and specific epithet) is written, both terms must be underlined or italicized.
Complete scientific names also include an authority. The authority is the name of the person or
persons who first used that name. Some authority names are so common, that they are given as
abbreviations. For example, scientific names that were first used by Linnaeus are designated by
the letter “L.” as the authority. The authority is not italicized. Some examples of scientific names,
with the Genus, specific epithet, authority format are given below:

Puma concolor L. Mountain Lion

Canis lupus L. Gray Wolf
Canis latrans Say Coyote
Tamiasciurus hudsonicus Erxleben American Red Squirrel
Nymphaea odorata Aiton American White Water Lily
Populus deltoides L. Cottonwood

In practice, the authority is often either not included or is only included the first time the name is
written out in a scientific article. The genus name is often abbreviated after the first mention of the
name as well. For example, Puma concolor L. will often be written as P. concolor after the first
use of the name.

There are numerous rules regarding the structure and use of scientific names, developed and
monitored by international organizations of working scientists. The International Code of
Nomenclature (ICN), by the International Botanical Congress, governs the rules for naming algae,
fungi, and plants. The International Code of Zoological Nomenclature (ICZN), by the International
Commission on Zoological Nomenclature, governs the rules for naming animals. The International
Code of Nomenclature of Bacteria (ICNB) governs the rules for naming Bacteria and Archaea.

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 In-Lab Part 1: Systematics & Phylogenies
Background
Systematics is the branch of biology that tries to uncover the evolutionary history, or phylogeny, of
groups of organisms. A diagram that summarizes such history is called a phylogenetic tree.

Modern Systematics Uses both Morphological and Molecular Data

We have long used morphological characteristics to determine relationships between organisms, but
they can be misleading. Complex eyes are an example. It seems logical that a simple eyespot arose
once, then slowly changed into different, more complex eyes in response to various selective
pressures. So, it should be possible to use eye structure as a morphological feature to assign
organisms to groups, right? Wrong! Evidence suggests that primitive light gathering structures
evolved as many as 60 different times! Complex eyes (like ours) have evolved independently at least
three times, possibly more. So, if we used eye structure alone to place organisms in various taxa, the
groupings probably would not represent evolutionary history. This does not mean morphological
features cannot be used to determine evolutionary relationships, but rather that a modern systematist
must examine many different features in order to create a phylogenetic tree that reflects true
evolutionary history. It is important to remember that every phylogenetic tree is a hypothesis about
the evolutionary relationships of the organisms depicted on that tree.

Over the last few decades, systematists have started using the tools of molecular biology.
Inexpensive sequencing methods and powerful bioinformatics software allow them to compare long
stretches of DNA or proteins from several organisms, calculate relative similarities between them,
and create a phylogenetic tree that maps their genetic relatedness to one another. Figure 1 shows the
possible evolutionary relationships between several bony and cartilaginous fishes, based on the
sequence of amino acids in the insulin precursor protein. Molecular sequence data have settled many
arguments about who is related to whom, and overturned taxonomic relationships that were based on
physical similarities alone. However, molecular data are not infallible! Like morphological features,
DNA and protein analyses only indicate likely genetic relations between two or more organisms.

So, which type of data is most reliable in determining evolutionary histories? Most modern
phylogenetic relationships are determined using mathematical algorithms that compile multiple
individual trees (each built using morphological
features or molecular sequence data) to create a single
consensus tree. Figure 2 is an example of a consensus
tree. It shows the relationships between classes of
insects and is based on a combination of several sets
of molecular sequence and morphological data.

Phylogenetic Trees
Phylogenetic trees can be drawn in many different ways.
Compare Figures 1 and 2, for example. Several different
formats are shown here. However, all phylogenetic trees
share fundamental characteristics. All trees have
branches and nodes (see Figure 3). Living, or extant,
Figure 1. Phylogeny of fish based on sequence of
taxa are placed at the ends of branches. Depending on
insulin precursor protein. the analysis, these taxa might represent any of the
hierarchical groups from our classification scheme
(e.g., individuals, species, genera, families), or even individual genes. Note that the taxa in Figure 2 are

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 orders, the taxa in Figure 3 are classes, and the taxa in Figure 4 are individuals. Branches represent
lineages, or species that form a line of descent. Sometimes, phylogenetic trees are drawn so that the
length of the branch indicates how much time has passed since the divergence of that lineage from a node
(Figure 4). In other trees, branches are drawn so that they are proportional to each other (Figures 2, 3).

Nodes are branching points, or points at which two lineages

diverge. Each node represents an actual historical common
ancestor. Ideally, each node branches into only two
distinct lineages. However, sometimes we don’t have
enough information to be able to say for certain how three
or more taxa are related to each other. When a phylogeny is
constructed with three or more taxa originating from the
same node, it is called a polytomy. Systematists continually
add more data to try to resolve polytomies.

Taxa that arise from the same common ancestor form a

clade (indicated by brackets in Figure 3). A clade is a
group of organisms that includes a common ancestor and all
the descendants of that ancestor. Taxa within a clade are
more closely related to each other than to any other taxa
depicted on the tree. For example, turtles are more closely
related to birds than to mammals, because birds, turtles, and
lizards form a clade that excludes mammals. In other words,
turtles share a more recent common ancestor (node) with
birds than with mammals. However, turtles are more closely
Figure 2. Consensus phylogenetic tree for the
related to mammals than to amphibians.
major classes on insects.
Mammals

Polytomy

Node

Branch

Figure 4. Phylogenetic tree for the genus Sium (water parsnips) modified
Figure 3. Phylogenetic tree for select taxa of from Spalik and Downie (2006) with branch lengths estimating time since
chordata divergence.

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A group of organisms that is closely related to a group that is under study is called an outgroup. For
example, in Figure 3, mammals would be the outgroup relative to the clade consisting of birds,
lizards, and turtles. In Figure 1, tuna would be the appropriate outgroup for the clade consisting of
flounder and zebrafish. The outgroup is compared to the group under study to help the investigator
determine whether a particular trait is likely ancestral in the clade.

In-Lab Activity #1

The goal of this activity is to introduce you to the basics of generating a phylogenetic tree.

You are given a data matrix of character states for four species (all birds) and four characters/traits.
In this example, a character state of “1” indicates the presence of a particular trait, while a character
state of “0” indicates the absence of a particular trait. A character state of “1” also indicates a derived
trait, which means that it evolved in the current group (birds) and would not be present in a closely
related outgroup. An appropriate outgroup for birds would be dinosaurs, which have character states
of “0” for all four traits.

You will first need to determine the number of synapomorphies (shared, derived traits) between
groups of species in your table, and then use those values to construct a phylogenetic tree. We know
that species that share more derived traits are more closely related because it means that they have
evolved along a common lineage/branch/clade together for a longer period of time; in other words,
they have a more recent common ancestor.

Rather than having you draw a tree from scratch, we have provided you with a template of a
phylogenetic tree. This tree can accommodate six species (one at each terminal tip); as you only have
four species, not all branches will be used as you fill in the tree. There are multiple correct ways to
complete the tree, so it’s not necessarily cause for concern if your phylogenetic tree looks different
than the others around you. In addition, there are five horizontal lines on branches of the tree that
indicate the general location on the tree where a specific trait evolves. As you have only four
characters in the matrix, one of these horizontal lines will be left empty. Remember that all taxa on a
branch (=clade) above the horizontal lines should possess that specific trait. *** There are special
circumstances when the preceding rule is not the case, but we will not introduce that into your
practice trees.

Table 1. Data Matrix of Character States

Species
Character Red-tailed
Canada Goose Mallard Ostrich
Hawk
Has beak (no teeth) 1 1 1 1
Capable of flight 1 1 0 1
Has talons 0 0 0 1
Has webbed feet 1 1 0 0

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In-Lab Activity #2

In this activity, as in the first, you are given a data matrix of character states. Here we undertake a
broader examination of the evolutionary history of vertebrates, and thus the characters separating the
eight species are more distinctive. As before, character states indicate the presence (“1”) or absence
(“0”) of a particular trait. Character states of “1” also indicate derived traits, so the more 1s that two
species share (i.e., synapomorphies) the more closely related the species are.

In this activity you are again provided with a preconstructed phylogenetic tree. However, this tree has
the exact number of branch tips for the list of species, and all horizontal lines will be used. When
properly constructed, there is only one correct organization of the tree.

*** One character state will be used twice in this tree, highlighting a special situation known as
convergent evolution.

As stated in Activity #1, a morphological feature defining a branch must apply to every organism on
the particular branch of the tree and this must NOT be true for organisms that are not on that
particular branch, with an exception…If two organisms share a derived trait due to common descent,
that trait is a synapomorphy (a shared, derived trait). The more synapomorphies two organisms
share, the more closely related they are. Note, however, that two organisms may share a derived trait
due to convergent evolution, rather than due to common descent. For example, in plants, both some
Cacti and some Euphorbs have evolved succulent stems and spines. However, these traits, which are
adaptations to the desert environment, were not present in the common ancestor of Cacti and
Euphorbs and have evolved independently in each group. The possibility of convergent evolution is
one reason why it is important to use many characters when determining relationships, as it is
unlikely that convergent evolution will affect all characters examined.

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 Table 2. Data Matrix of Character States for Vertebrates
Species
Character
Bat Bird Chimpanzee Fish Frog Gorilla Human Lizard
Gills 0 0 0 1 0 0 0 0
Vertebrae 1 1 1 1 1 1 1 1
Limbs 1 1 1 0 1 1 1 1
Keratinized (waterproof) skin 1 1 1 0 0 1 1 1
Scales on body/feet 0 1 0 0 0 0 0 1
Lays shelled eggs 0 1 0 0 0 0 0 1
Wings 1 1 0 0 0 0 0 0
Mammary Glands 1 0 1 0 0 1 1 0
Opposable Thumbs 0 0 1 0 0 1 1 0

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Interpretation:

1. Of the nine characters used to organize species onto the tree, some are more useful in
understanding evolutionary relationships than others. In particular, “wings” and two other
characters should have been least helpful in your placement of the species onto your tree.
Identify the “two other” characters and explain why they were less helpful.

2. “Wings” represented a unique incongruity in your tree. This is because they represent an
example of convergent evolution.

(A) Based on the position of this character on your tree, explain in your words what
convergent evolution means.

(B) How can you determine from your data matrix/tree that wings are in fact an example of
convergent evolution and are not a synapomorphy of the two species that possess them?

3. Three taxa possessed opposable thumbs: Chimpanzee, Gorilla, Human. However, based on
how the tree was drawn, two of these species are more closely related to each other, than
either of them is to the third.

(A) Based on your tree, which two species are most closely related? How did you come to
this conclusion?

(B) What addition type of characters might help you to better determine the relationship
between such groups of closely related species?

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In-Lab Activity #3

Biologists have many ways to study evolution. They can use fossils to learn about ancient
species. They can compare the anatomy of modern species. They can observe the order in which
cells develop in embryos. All of these clues reflect what took place over time at the molecular
level. DNA and proteins, the genes and the products of genes, provide powerful evidence for
descent with modification. As DNA changes over time, the proteins that are produced by the
DNA change too. The result is that many organisms have similar, but not identical, versions of a
given protein. Differences among these homologous proteins provide clues to evolution.

This activity uses a handout that guides you through the process associated with producing
proteins from DNA, so that you can better understand the steps involved and see how
changes/mistakes can lead to patterns that can been used to understand evolutionary
relationships.

Specifically, in this activity you will (using a key and by hand) take a starting DNA template and
work it through to the Proteins (amino acid sequences) that are produced from it. This process
was described in the online video that you watched as part of your pre-lab for this week.
*** RNA can be produced directly from the original DNA…we have added in the additional step
of producing complementary DNA to illustrate the full potential complexity of the process.

Template DNA (starting point) ® Complementary DNA ® RNA ® Proteins

Note that when such changes/mistakes occur in germline cells, they can be passed to subsequent
generations. These changes can accumulate over time and lead to patterns that we can use to
determine levels of relatedness.

In-Lab Activity #4a

In Part 4 of this lab, you will compare amino acid sequences of hemoglobin from the eight
animals used in Activity #2. Hemoglobin is the molecule in blood that carries oxygen. This
complex molecule contains four protein chains. Table 3a shows the complete amino acid
sequence for one of those chains, called beta. Each letter stands for a different amino acid. Each
column is a location on the protein chain. NOTE: Locations where the amino acids are identical
in all eight animals are shaded gray.

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Table 3a. Complete beta hemoglobin amino acid sequence alignment. Shaded columns indicate
no differences.

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Analysis:
1. In Table 3b, use the row labeled Human as your control. Compare Animal # Differences
the amino acids (AA) at position 3 (first column) for each animal to Iguana
the AA at position 3 (first column) for humans. Circle differences. Chimp
Bat
2. Repeat Step 1 for all the other positions (columns). Be sure to Frog
compare each animal’s AA to human. Gorilla
3. Calculate (sum) the total number of differences for each animal Salmon
compared to humans and enter your data in table to right. Duck

Table 3b. Condensed beta hemoglobin amino acid sequence alignment. Only positions that
differ are included.

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In-Lab Activity #4b

UniProt database

You are now going to use molecular evidence to answer questions about how the eight different
organisms are related. Follow along with your TAs as they demonstrate how to use a protein
sequence database called UniProt to build phylogenetic trees. Start by building a tree for the
eight animals you considered above.

Protocol
1. Begin by navigating to the database at legacy.uniprot.org
2. Find the “Search” box and verify the default “UniProtKB” is selected.
3. Enter the phrase hemoglobin beta then click search.
4. Set the items per page from 25 to 250.
5. Use the right-hand scroll bar to scroll through the names of the entries. Find and select the 7
animals listed below: (once the boxes are checked, the yellow information bar will indicate “7
selected”)
Animal Entry Entry name
Atlantic salmon Q91473 HBB_SALSA
African clawed frog P02137 HBBL_XENLA
Muscovy duck P14260 HBB_CAIMO
Cave bat P11758 HBB_MYOVE
Western lowland gorilla P02024 HBB_GORGO
Human P68871 HBB_HUMAN
Chimpanzee P68873 HBB_PANTR
6. Once all seven animals have been selected, click “Add to basket.”
7. Click the right arrow to advance from entries 1-250 to 251-500.
8. Now find and select the animal below.
Animal Entry Entry name
Common iguana P18987 HBB_IGUIG
9. Click “Add to basket.”
10. Check your basket to make sure you’ve selected the correct organisms.
11. Check the box next to each animal, then click on the “Align” button to run a comparison.
12. A multiple sequence alignment will appear at the top of the results page. This is the same
analysis you did in Part 4a when comparing animal sequences to the human sequence.
13. A phylogenetic tree will appear near the bottom of the screen.

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Analysis:
Sketch and label the phylogenetic tree in the space below.

Interpretation:
1. Based on the tree produced from the hemoglobin data, which mammal listed is most closely related
to humans? What is the evidence for your conclusion?

2. Does the data support the organization that you made in Question 3 for Activity 2? If not, how
would you explain any differences?

3. In terms of descent, what does it mean to say that humans are more closely related to gorillas than
to bats?

4. Below is a table of all pairwise comparisons, you can fill in the blanks from your table above.
A student used the hemoglobin data to conclude that iguanas and ducks are more closely
related than are iguanas and humans or ducks and humans. Was the student correct?

Human Chimp Gorilla Bat Iguana Duck Frog Salmon

Human 0
Chimp 0
Gorilla 1 0
Bat 27 28 0
Iguana 52 53 47 0
Duck 45 46 45 35 0
Frog 65 64 65 76 70 0
Salmon 76 77 78 74 67 76 0

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5. Nearly 50 locations were not listed in Table 2b because the amino acid was the same in all
eight species. One possible explanation is that no mutations occurred in the codons for those
locations. Suggest another possible explanation. Hint: Think about why some mutations might
be harmful.

In-Lab Activity #5

Shark Dolphin Human

Predict:
Consider if you think dolphins are more closely related to sharks or to humans, then sketch a line
to complete the phylogenetic tree above based on your prediction.

Provide evidence:
List three traits common to both sharks and dolphins.

List three traits common to both humans and dolphins.

Test:
You’ll again use Hemoglobin beta amino acid sequences in UniProt to build a phylogenetic tree.

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Protocol:
1. Begin by returning to the UniProt database.
2. Find the “Search” box and verify the default “UniProtKB” is selected.
3. Enter the phrase hemoglobin beta then click search.
4. Set the items per page from 25 to 250.
5. Use the right-hand scroll bar to scroll through the names of the entries. Find and select the
animals listed below:
Animal Entry Entry name
Human P68871 HBB_HUMAN
Atlantic salmon Q91473 HBB_SALSA
Bottle-nosed dolphin P18990 HBB_TURTR
6. Click “Add to basket” in the top toolbar.
7. Click the right arrow to advance from entries 1-250 to 251-500.
8. Now find and select the animal below.
Animal Entry Entry name
Port Jackson shark P02143 HBB_HETPO
9. Click “Add to basket” in the top toolbar.
10. Click “Basket” in the top right corner, select all four animals, then click “Align”.

Analysis:
Sketch and label the phylogenetic tree in the space below.

Interpretation:
1. Use molecular evidence to make a claim that dolphins are more closely related to either sharks
or humans.

2. What morphological evidence supports this claim?

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3. How could you explain the morphological evidence that refutes this claim?

4. What does this suggest about the use of morphological versus molecular data in producing
trees?

In-Lab Activity #6

Phylogenetic trees do more than just illustrate how species are related. They also provide biologists
with an important tool to test predictions about how animals have evolved. For example, at a very
simple level, it could be difficult to identify cases of convergent evolution without first knowing how
species are related. In this activity we will use molecular data to answer a question focused on
morphology.

Dolphin toes:
A student wants to determine what the feet of the ancestors of dolphins were like. She knows that
dolphins are mammals and that based on earlier studies, they likely evolved from terrestrial four-
legged hoofed mammals (ungulates), which over many generations returned to the seas. The
problem is that ungulates are divided into two major clades: perissodactyls (= odd-toe ungulates)
and artiodactyls (= even-toed ungulates). This means that the ancestor of dolphins and other
cetaceans either had an odd or even number of toes on their feet; of course, as modern cetaceans
lack feet there is no way to determine the answer to this question morphologically. Help her use
hemoglobin beta chain sequence data to evaluate these two hypotheses.

Protocol:
1. Choose 2 even-toed ungulates, 2 odd-toed ungulates, and 1 cetacean (a dolphin or whale)
from the list below.

*** Note: When you find an animal, check to make sure that it is the hemoglobin beta chain
(preferably without a number after it) and not another hemoglobin type such as alpha or
gamma.

2. Once you’ve selected your animals, click “Align”

Even-toed ungulates Odd-toed ungulates Cetaceans

Pig (Sus scrofa) Rhinoceros (Ceratotherium simum) Bottle-nosed dolphin (Delphinus truncates)
Hippo (Hippopotamus amphibius) Horse (Equus caballus) Sperm whale (Physeter macrocephalus)
Bison (Bison bonasus) Tapir (Tapirus terrestris) Common minke whale (Balaena rostrata)
Deer (Odocoileus virginianus) Zebra (Equus zebra)

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Analysis:
Sketch and label the phylogenetic tree in the space below.

Interpretation:
1. Use evidence to make a claim that dolphins’ (and whales’) feet would have either an even or
odd number of toes.

2. What other types of evidence could be gathered to support or refute this claim?

Wrap-Up Discussion:

Before leaving the lab each week, it is good to have a discussion to solidify what was learned. Your
TAs will lead this discussion to go over what was learned today. Be sure to ask any questions you
have, and be sure that all questions in this manual have answers written before you go! This will
provide study material for the in-class exams.

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