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Biology 1108L – Laboratory Exercises

Phylogenetics

Kennesaw State University Departments of Ecology, Evolution, and Organismal Biology

& Molecular & Cell Biology

Lab modified from Gendron, R. P. 2000. The classification and evolution of Caminalcules. The American Biology Teacher 62: 570-576. Edits made by Joe Dirnberger, Sigurdur Griepsson, Paula Jackson, Thomas McElroy, Joel McNeal, and Heather Sutton.

CLASSIFICATION AND EVOLUTION Humans classify almost everything, including each other. This habit can be quite useful. For example, when talking about a car someone might describe it as a 4-door sedan with a fuel injected V-8 engine. A knowledgeable listener who has not seen the car will still have a good idea of what it is like because of certain characteristics it shares with other familiar cars. Humans have been classifying plants and animals for a lot longer than they have been classifying cars, but the principle is much the same. In fact, one of the central problems in biology is the classification of organisms on the basis of shared characteristics. As an example, biologists classify all organisms with a backbone as "vertebrates." In this case the backbone is a characteristic that defines the group. If, in addition to a backbone, an organism has mammary glands and hair it is a mammal, a subcategory of the vertebrates. This mammal can be further assigned to smaller and smaller categories down to the level of the species. The classification of organisms in this way aids the biologist by bringing order to what would otherwise be a bewildering diversity of species. The field devoted to the classification of organisms is called taxonomy [Greek. taxis, to arrange, put in order + nomos, law]. The modern taxonomic system was devised by Carolus Linnaeus (1707-1778). It is a hierarchical system because organisms are grouped into ever more inclusive categories from species up to kingdom. Figure 1 illustrates how four species are classified using this taxonomic system. (Note that it is standard practice to italicize the genus and species names.) Figure 1

Keep in mind that Linnaeus’ system of classification does not imply inherited relationships between different taxa. Indeed Linneaus, like most other scientists of his time, considered species to be fixed entities that had been created in their present form. According to this view, Linnaeus' system of classification was simply a useful means of cataloging the diversity of life. This static view of taxonomy began to change at the 1859 publication of Charles Darwin’s On The Origin of Species. As a consequence of Darwin's work it is now recognized that taxonomic classifications are actually reflections of evolutionary history. For example, Linnaeus put humans and wolves in the class Mammalia within the phylum Chordata

KINGDOM Animalia Plantae PHYLUM Chordata Arthropoda Angiospermophyta

CLASS Mammalia Insecta Monocotyledoneae

ORDER Primate Carnivora Hymenoptera Asparagales

FAMILY Hominidae Canidae Apidae Alliaceae GENUS Homo Canis Apis Alium SPECIES sapiens

(human) lupus (wolf)

mellifera (honeybee)

sativum (garlic)

because they share certain characteristics (e.g. backbone, hair, mammary glands, etc.). We now know that this similarity is not a coincidence; both species inherited these traits from the same common ancestor. In general, the greater the resemblance between two species, the more recently they diverged from a common ancestor. Thus, when we say that the human and wolf are more closely related to each other than either is to the honeybee, we mean that they share a common ancestor that is not shared with the honeybee. Another way of showing the evolutionary relationship between organisms is in the form of a phylogenetic tree (Greek. phylon, stock, tribe + genus, birth, origin): Figure 2

The vertical axis in this figure represents time. The point at which two lines on the phylogeny separate indicates when a particular lineage split (an ancestral speciation event!). For example, we see that the lineage leading to mammals diverged from the lineage leading to reptiles about 350 million years ago. In other words, the most recent common ancestor shared by mammals and reptiles lived around 350 million years ago. The horizontal axis represents, in a general way, the amount of divergence that has occurred between different groups; the greater the distance, the more different their appearance. Note that because they share a fairly recent ancestor, species within the same taxonomic group (e.g. the group Archosauria, which originated around 250 million years ago) tend to be closer to each other at the top of the tree than they are to members of other groups. Several types of evidence can elucidate the evolutionary relationship between organisms, whether in the form of a taxonomic classification (Fig. 1) or a phylogenetic tree (Fig. 2). One approach, as already discussed, is to compare living species. The greater the differences between them, the longer ago they presumably diverged. There are, however, pitfalls with this approach. For example, some species resemble each other because they independently evolved similar structures in response to similar environments or ways of life, not because they share a recent common ancestor. This is called convergent

evolution because distantly related species seem to converge in appearance (become more similar). Examples of convergent evolution include the wings of bats, birds, and insects, or the streamlined shape of whales and fish. At first glance it might appear that whales are a type of fish. Upon further examination it becomes apparent that this resemblance is superficial, resulting from the fact that whales and fish have adapted to the same environment. The presence of hair, the ability to lactate, and endothermy clearly demonstrate that whales are mammals. Thus, the taxonomist must take into account a whole suite of characteristics, not just a single one. The fossil record can also be helpful for constructing phylogenetic trees. For example, birds are so unique in their characteristics when compared to other living taxa that taxonismists placed them in a distinct group, Class Aves. However, the fossil record reveals that birds are dinosaurs, having diverged from other saurischian dinosaurs around 150 million years ago. The use of fossils is not without its problems, however. The most notable of these is that the fossil record is incomplete. This is more of a problem for some organisms than others. For example, organisms with shells or bony skeletons are more likely to be preserved than those without hard body parts. CLASSIFICATION AND EVOLUTION OF ARTIFICIAL ORGANISMS In this lab you will develop a taxonomic classification and phylogenetic tree for a group of imaginary organisms named Caminalcules after the taxonomist Joseph Camin who devised them. Towards the end of this protocol are pictures of the 14 "living" species that you will use. Take a look at the pictures and note the variety of appendages, shell shape, color pattern, etc. Each species is identified by a number rather than a name. For fossil Caminalcules, there will also be a number in parentheses indicating the geological age of each specimen in millions of years. The purpose of this lab is to illustrate the principles of classification and some of the processes of evolution (e.g. convergent evolution). We do these exercises with artificial organisms so that you will approach the task with no preconceived notion as to how they should be classified. This means that you will have to deal with problems such as convergent evolution just as a taxonomist would. With real organisms you would probably already have a pretty good idea of how they should be classified and thus miss some of the benefit of the exercise. Exercise 1: The Taxonomic Classification of Living Caminalcules Carefully examine the fourteen living species and note the many similarities and differences between them. On a sheet of paper, create a hierarchical classification of these species using the format in Figure 3. Instead of using letters (A, B, ...), as in this example, use the number of each Caminalcules species. Keep in mind that Figure 3 is just a hypothetical example. Your classification may look quite different than this one; there is no single correct answer to this exercise.

Figure 3

The first step in this exercise is to decide which species belong in the same genus. Species within the same genus share characteristics not found in any other genera (plural of genus). The Caminalcules numbered 24 and 28 are a good example; they are clearly more similar to each other than either is to any of the other living species so we would put them together in their own genus. Use the same procedure to combine the genera into families. Again, the different genera within a family should be more similar to each other than they are to genera in other families. Families can then be combined into orders, orders into classes and so on. Depending on how you organize the species, you may only reach the level of order or class before all species are grouped into a single taxon. Exercise 2. The Comparative Approach to Phylogenetic Analysis Now, you can use your classification of the 14 living Caminalcules to construct a phylogenetic tree. This tree should reflect your taxonomic classification. For example, let us say you have put species A and G into the same genus because you think they evolved from a common ancestor (x). Their part of the tree would look like the diagram below. Figure 4

When there are three or more species in a genus, you must decide which two of the species share a common ancestor not shared by the other(s) (see “b” under “Hints, Suggestions, and Warnings” below). This diagram indicates that species E and K are more closely related to each other than either is to C. We hypothesize that E and K have a common ancestor (Y) that is not shared by C. Similarly, two genera that more closely resemble each other than they do other genera presumably share a common ancestor. Thus, even in the absence of a fossil record it is possible to develop a phylogenetic tree. We can even infer what a common ancestor like Y might have looked like.

PHYLUM CAMINALCULA CLASS 1 CLASS 2 ORDER 1 ORDER 2 ORDER 3

FAMILY 1 FAMILY 2 FAMILY 3 FAMILY 4 GENUS 1 GENUS 2 GENUS 3 GENUS 4 GENUS 5 GENUS 6

A G H D B J L E K C F I

Exercise 3. Molecular Phylogeny of the Caminalcules Species with fingers Modern systematists compare DNA sequences to reconstruct phylogenetic trees. A DNA dataset of informative characters is provided below for the 5 living Caminalcules species that have digits or “fingers” on their forelimbs and large black spots on their back. Data from Species 2 is provided as an outgroup taxon, which roots the tree to a “tentacled” taxon outside the group in order to better ascertain which DNA character states are ancestral vs. derived within the ingroup of taxa possessing forelimb digits. 1 2 3 4 Species 2 (Outgroup) A G C G Species 20 C G T T Species 1 A G T G Species 16 A G C G Species 3 C A C T Species 14 C A T T Trees representing two hypothetical relationships of these taxa are provided at the end of the protocol. You will tally the simplest, most parsimonious number of steps to explain each of the four DNA characters above on both of the trees provided. For Character 1, there is no way a single mutation, or even two mutations, can explain the Cytosine shared by Species 20, 3, & 14 in Tree II below.

However, there are a number of ways in which the Cytosine could have evolved in these taxa via three mutational steps; a mutation from A to C could occur independently in each species (left) or a mutation from A to C in the common ancestor of the ingroup could have been followed by mutations reverting back to an A in species 1 and 16 (right). No matter what, a minimum of three evolutionary steps are necessary to explain Character 1 on Tree II.

In Tree I, by contrast, only a single mutation is necessary to explain Character 1.

In this tree, Character 1 serves as a synapomorphy supporting Species 20, 3, & 14 as a monophyletic group. That means it is a character shared only by the modern species descended from a single point on the tree, and the mutation from an A to the C which species 20, 3, & 14 share is presumed to have occurred on the branch that represents their most recent common ancestor.

Calculate the minimum number of steps required to explain Trees I & II for the remaining three characters. It will help to write the character state for each species above the species number on the tree (see the example done for you, Character 1, on the tree sheet at the back of the protocol). Tally which tree or trees require the fewest total mutations to explain (like a golf scorecard but with 4 holes/rounds, the lowest cumulative score wins). Hint: You can assume the outgroup has the ancestral character state for this exercise. Char. 1

steps Char. 2 steps

Char. 3 steps

Char. 4 steps

Total steps

Tree I 1 Tree II 3 Exercise 4. Fossil Reconstruction of Caminalcules Phylogeny Using a large sheet of posterboard, newsprint, or any large, flat surface, construct a phylogenetic tree for the fossil + living Caminalcules. Use a ruler to draw 20 equally spaced horizontal line on the paper or, if working on a table that you can’t write on, use some sort of placeholders (like coins, skittles, paperclips, etc.) to mark the ends of invisible gridlines. Each line will be used to indicate an interval of one million years. The line at the bottom of the tree represents an age of 19 million years and the top line represents the present (0 years ago). Cut out all the Caminalcules (including the living species). Each fossil Caminalcules species is identified by its species number and its age (in millions of years) in parentheses. Make certain to not cut off these numbers. Put the species on the gridlines you’ve made according to their age (the number in parentheses). Some years may completely lack fossils. Beginning with the oldest fossils, connect species into a phylogenetic tree according to their evolutionary relationship. Figure 5 shows how to get started. Continue the tree from there. Make sure you at least take a good resolution image of your tree before you disassemble it. You’ll need the organisms’ characteristics to complete the worksheet. Figure 5

52

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Hints, Suggestions, and Warnings a. Draw lines faintly in pencil to indicate the path of evolution. If you’re working on a

surface on which you don’t want to write, you can use string, strips of paper, or even real twigs as branches to connect species.

b. Branching must involve only two lines at a time:

c. There are gaps in the fossil record, so there won’t be a fossil connection at every line

(i.e. you will have connect species across time gaps of multiple millions of years). Also, some species may have gone extinct without leaving any descendants; their lines will end before the present.

d. The Caminalcules were numbered at random; the numbers provide no clues to

evolutionary relationships. e. There is only one correct phylogenetic tree in this exercise. This is because of the

way that Joseph Camin derived his imaginary animals. He started with the most primitive form (#52) and gradually modified it using a process that mimics evolution in real organisms.

ASSIGNMENT: You will turn in the following:

• The table for your taxonomic classification of living Caminalcules (similar to the one in Figure 3)

• Your phylogenetic tree based on only on your classification of the 14 living Caminalcules

• The molecular trees with mutations mapped as hash marks on the branches and the total tally of mutations necessary to explain Tree I vs. Tree II

• Your phylogenetic tree based on the 40 fossil Caminalcules + the 14 living Caminalcules (don’t turn in the large sheet with the pasted pictures but instead draw the tree on notebook paper using the species numbers as shown in Figure 5

• Answers to the worksheet questions (fossil Caminalcules and the worksheet will be provided separately in week 2 of the lab)

References cited: Sokal, R.R. 1983. A phylogenetic analysis of the Caminalcules. I. The data base. Systematic Zoology 32: 159–184. Lab modified from: Gendron, R. P. 2000. The classification and evolution of Caminicules. The American Biology Teacher 62: 570-576. Figure 2 modified from: Freeman, S. and J. C. Herron. 2005. Evolutionary Analysis, 4th ed. Pearson, New Jersey.

Tree I Character 1 Tree II

Character 2

Character 3

Character 4