Lab 5 pre-lab.

In our last lab, you created a simplified hominid phylogeny based on a table of important characteristics. Your task in Lab 5 will be to learn more about the sub-discipline of systematics and to practice constructing more formalized phylogenetic tress (evolutionary maps) for particular sets of taxa and traits. We will be relying on morphological (physical) traits to construct our trees in this lab, but it is important to understand that modern systematics now relies heavily on genetics (e.g., shared genes and alleles to determine) to proposed evolutionary relationships among various taxa. 

• Introduction
• Do you know enough?
• What we will do in lab?
• LABridge

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How do we illustrate evolutionary relationships among related taxa?

Taxonomy names, describes, and sorts taxa into groups but the sub-discipline of phylogenetic systematics identifies and illustrates the relatedness of those taxa through evolutionary time. Systematists attempt to determine the level of relatedness amongst taxa based on their degree of similarity or differences, either morphologically, genetically, or with some combination of both type of traits. Systematists then build a hypothesis about the evolution of these taxa, known as a phylogenetic tree, or phylogeny. Such diagrams display the lines of evolutionary descent and speciation of various taxa, organisms, or genes form a common ancestor. You might be familiar with drawings of the "Tree of Life" (in the sidebar), which are actually stylized interpretations of a giant phylogeny tracing the decent of all life from our single common ancestor; the first self-replicating organism on the planet.
To date, there are millions of identified species, and there are likely millions more to be discovered. Phylogenies organize these species into groups that share similar genetic, biochemical, physical, or behavioral traits based on a process known as circumscription (i.e., a process that groups species with other similar species based on their degree of similarity), and there position and proximity on the tree represent the closeness of their shared evolutionary history. Mordern phylogenies can take several forms as pictured below.

Phylogeny images from Kahn Academy (link to article), originally sourced from "Taxonomy and phylogeny," by Robert Bear, et. al. 

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Please review these other important terms and concepts. 
​Character - An observable trait of an organism; can be anatomical, behavioral, etc.
Character State - Different manifestations of a character. Different eye colors are different character states.
Apomorphy - A new, derived character that appears in a taxon but was not present in its ancestral taxon. Loss of a character can also be an apomorphy.
Synapomorphy - A derived character state shared among two or more taxa. These are used to establish evolutionary relationships among those taxa.
Plesiomorphy - An ancestral character state. This may change to become an apomorphy.
Monophyletic - A monophyletic taxon contains all the descendant taxa of a particular ancestral taxon.
Paraphyletic - A paraphyletic taxon contains only part of the descendant of an ancestral taxon, and is thus not a natural grouping.
Outgroup - A taxon thought to have the character states of an ancestral taxon to the taxa you are considering. The outgroup will be used to determine which of the character states in your taxa are apomorphic.
Parsimony - The simplest method of doing things, or the simplest route to get to something or somewhere.

Tree of life representation by Studio W© World Art Group.

Charles Darwin's first diagram of an evolutionary tree sketch (1837), from his First Notebook on Transmutation of Species, adapted from wikipedia.org.

Be sure you know all the bold-faced concepts on this page before moving on.

DO you know enough about interpreting phylogenies?

Review Example 1 in the sidebar, in which evolutionary time moves from right to left. Colored lines show the evolutionary trajectory of five taxa descended from their most recent common ancestor (abbreviated MRCA). Pink for A and B (with lighter pink for A and darker pink for B), purple for C and D (with lighter purple for C and darker purple for D), and blue for taxa E. The taxa on a tree are often specific species, but can also be larger taxonomic groups like genera, families, kingdoms, etc. Each big red dot signifies a divergence, or split, among these taxa. We can see that first, the larger group diverged from E, then A and B split from C and D. Lastly, A diverged from B and C diverged from D. Phylogenies can be made more informative if we place the character-states possessed by our taxa of interest, as they appeared through time, causing each split. Example 2, in the sidebar, shows what this might look like using a vertical tree design, similar to what we will use in lab.
In Example 2, we see some type of rodent as the common ancestor at the bottom, and five, extant (living), derived (new) species at the top of the tree. Labeled generically as taxa A through E. In these types of vertical trees, time moves in an upward direction, and we can follow the development of new (apomorphic), shared character states (synapomorphies) up the tree in various paths. First notice taxa A has no apomorphies. It is therefore the outgroup and is shown on the first branch. The original divergence, or break away, from the common ancestor resulted in taxa A and a new group of organisms that all have a fuzzy tail. This means that taxa B, C, D and E all have fuzzy tails, a synapomorhic trait among them. We can also see that only taxa D and E have the new trait of whiskers. Next, another group diverged with the development of big ears (which includes taxa C, D, and E), and lastly D and E diverged with evolution of whickers. Taxa A - E represent a monophyletic group for this common ancestor. If we were to only include taxa B - E, it would be an incomplete paraphyletic group.  Also please note, we never put plesiomorphic (ancestral) traits on the tree. If the trait does not appear, one assumes it is the same as it was for the MRCA. 

​You should be able to trace the evolutionary lineage of a taxa from the MRCA to the extent species or group (genus, etc.).

You should understand how the placement of character states on a tree helps define evolutionary relationships and taxa divergence.

If you are still struggling with interpreting tress, please review the Khan Academy tutorial in the sidebar. It is excellent.

Example 1.

Example 2.

Khan Academy tutorial. Click for link.

Phylogeny images from Kahn Academy (link to article), originally sourced from "Taxonomy and phylogeny," by Robert Bear, et. al. 

What will we do in lab & how will we do iT?

Lab 5 contains three exercises.

1. ​Tree Practice: You will practice constructing a tree together as a class.
2. You will be provided character matrix to construct a new phylogeny with your lab group. 
3. You will practice constructing another tree on your own.

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Homologous vs analogous traits

​Systematics is ruled by parsimony and consequently based on the assumption that fewer evolutionary events are more likely to occur than lots of them. Therefore, systematists assume that most similar traits are homologous, meaning they exists in different taxa due to a close evolutionary relationship; that they were inherited from the same most recent common ancestor. However, this is not always the case. The character trait of wings does not exist in both bats and birds because it was inherited by both in a shared common ancestor where it originated. Instead, flight evolved twice, once in the lineage of bats and again in the lineage of birds. This goes against the rule of parsimony and is an example of an analogous traits. Review the comparison below from lab last week. We will explore these traits again in our next lab. Understand that homologous traits are what dictate the relationships in a phylogeny and are only on a phylogeny one time; analogous traits can appear multiple times as they have evolved multiple times in different taxa. Mistaking an analogous trait for a homologous trait would lead to a very incorrect tree, and because parsimony is the rule, this mistake is common and must be guarded against. 

Homologous vs. analogous traits. Click to enlarge.

How systematics is linked with other pursuits and disciplines (from Wen, et al.). Click for article link.

Four proposed phylogentic tress of the mbuna cichlids from Lake Malâwi. Click to enlarge. Source below.

Article on mbuna cichlid evolution. Click for article link (Scherz, et al.).

​You should be able to trace the evolutionary lineage of a taxa from the MRCA to the extent species or group (genus, etc.).

*SPECIAL NOTE: You will be asked to upload your Vestigial Structures Extension assignment as a Word document along with this week's LABridge.
If you feel confident with this material, click the bridge icon below and navigate to Blackboard to take the LABridge for this week. Be ready to be tested on this material before you go to the quiz, and make sure you have your Lab Notebook Guide ready to submit as well.

Click here to get to WKU's blackboard to take your LABridge for this week.

Lab 5 Protocol

Following this lab you should be able to...

• Define and understand the concept of systematics
• Learn the terminology associated with phylogenetic analyses
• Understand how to use phylogenetic data
• ​Learn to build a phylogenetic tree

Overview. In today's lab you will practicing making and using phylogenetic trees.

1. Exercise I. Create a phylogeny of amniotes as a class
2. Exercise II. Construct a Phylogenetic Tree of Cichlids
3. Exercise III. Practice making phylogenies from coded matrices. 

• Exercise I
• Exercise II
• Exercise III

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Exercise I. creatE a phYlogeny of amniotes as a class

Most systematists now use genetics to determine the level of relatedness amongst taxa. This is the most robust method and, with the right amount of genetic material sequenced, can establish 99.99% confidence in a determination of relatedness. However, genetic analyses are very costly and require sophisticated equipment. When conducting studies in the field or on species from which a scientist can not collect tissue samples, like threatened or endangered species, then observable, measurable, physical features must be used to build phylogenies. This approach is known as cladistics and is accomplished by coding characters with numbers to determine how species might be related to one another. You will use this technique to build phylogenies in lab today.
​In this activity you will build a phylogeny of the amniotic vertebrates. The amniotes constitute a monophyletic group that is defined, in part, by the presence of an amniotic egg (a newly derived trait that keeps eggs from drying out).  All vertebrates that existed before the evolution of the amnion (e.g. fish and amphibians) were intimately tied to the water; otherwise their eggs would dry-out. Reptiles, birds, and mammals are all amniotes; they possess an amnion which makes it possible for their eggs to be laid in dry places (for those that lay eggs). For this analysis, you will use amphibians as the outgroup because they are assumed to be closely related to terrestrial taxa, yet they still rely on moist places to lay their eggs because they lack an amnion. In this way, they are considered a transitional group between fully aquatic and fully terrestrial vertebrates.

We will use these six taxonomic groups for our first phylogeny. Please note that they are not all on the same taxonomic level; we are using classes, an order, and two sub-orders.

Procedure.​
1. Open your Lab Notebook Guide. ​You will find three tables.

• Table 1 describes the traits we use for this tree. They are import characters that signify the evolutionary relationships among our taxa. These are observable morphological characters that can be confirmed as present or not-present, and can be measured or counted.
• Table 2 provides the states of each character for each of our taxa.
• Table 3 will be completed by you.

2. Review all three tables and follow along with your TA. 
3. Based on the answers provided in character matrix, fill-in the coded character matrix in your Lab Notebook Guide (Table 3) with the proper codes, which are listed in your guide. A few have been done for you as examples.
4. Get TA approval on your coded character matrix before you continue.
5. Using a blank sheet of paper, follow along with the class and construct the phylogeny.

• Hint: You never put ancestral (or pleisiomorphic) states on the tree. These are ones coded to zeros (e.g., 1-0 or 8-0).
• Hint: Character stat 6-1 in an analogous trait. It will appear on your tree twice. All other traits will only be placed one time.
• Hint: You can always check your tree by starting at one taxa and tracing back to the common ancestor for your group. Cross off character-sates as you go. Once your are at the beginning of your tree, you should have accounted for every apomorphic/synapomorphic trait. 
• Refer to the steps in the sidebar of you get stuck.

​6. Take a picture of your phylogeny to add to your Lab Notebook Guide.

Steps to refer to as you build your tree. Click to enlarge.

Click to download.

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Portions of this lab were adapted from: ​Brooks, D., D.A. McClennan, J.P Carney, M.D. Dennison, & C.A. Goldman. 1995. Phylogenetic Systematics. Pages 239-258 in Tested Studies for Laboratory Teaching, Vol. 15 (C.A. Goldman, ed.). Proc. of the 15th Workshop/ Conference of the Association for Biology Laboratory Education (ABLE), 390pp.

Exercise Ii. Construct A phylogeny of cichlids

Now it's time to try making a phylogeny on your own. You have been provided with character matrix and the coded character matrix (below) of nine species of fish from the family Cichlidae. Cichlids are an amazing group of freshwater fish, found in the South America, Africa, and southern Asia. Notice that we have four of these taxa in our lab tank: Petrotilapia tridentiger, Labeotropheus fuelleborni, Labidochromis joanjohnsonae, and Metriaclima pulpican. They are all mbuna-type cichlids. We will explore cichlids more in the next lab. Review the information below as some of these traits might not be intuitive. 

Visual Cones

Visual cones: They are well known for their bright colors and are sometimes referred to as "freshwater coral fish," making them a favorite in the pet trade. Most are yellow and blue (directly across from each on the color wheel), but cichlids that live deeper in the water column will have more red and silver, as the available light spectrum differs. Visual cones are a type of photoreceptor cell in the retina and provide for color vision. The amazing variation in cichlid color is due, in part, to their ability to recognize so many colors due to high numbers of these cones.

Bowers

Bowers: Character 1, bower type, describes the mating habits of many male cichlids who build sand structures (or bowers) to attract females. The structure of these bowers, classified as "castles" or "pits", and the number of structures can help differentiate species from each other. The practice is a type of pre-zygotic reproductive barrier, in which mating behavior can isolate species from each other. Characters 2, 6, and 7 also refer to bower-use and other characteristics (e.g., number of outer rings and pit shape). Click for article (Kocher, 2004).

Jaw Types

Jaw Morphology: Character 8, jaw type, is a homoplasy. It should go one your tree last. It is highly analogous and 8-1 will appear twice. Within each small microcosm in the lake, different lineages of cichlid have developed various yet similar jaw morphologies to fill available niches. For example, many different species of cichlid can all graze on algae on the same rock, as their diverse mouth parts allow each one to specialize on the top, sides, or bottom. Character 4, teeth type, is also associated with jaw morphology (Jaw types, from the Florida Museum of Natural History).

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Materials.

Basic tree shape. Click to view.

Character and coded character matrices. Click to view.

Species ID cards

Steps for tree creation.

Procedure.

1. ​Please note: You are constructing a partial phylogeny for this lab exercise. A more complete cichlid phylogeny is depicted in the image in the sidebar. It is massive!
2. Download all necessary materials.
3. Review the three main trait-types: visual cones, bowers, and jaw-type.
4. Use the coded character matrix to construct your phylogeny (the basic shape is provided here). 
5. Remember: You never put ancestral (or pleisiomorphic) states on the tree. These are ones coded to zeros (e.g., 1-0 or 7-0).
6. Remember: Character 8-1. It will not help you sort these taxa; place it last.
7. Remember: You can always check your tree by starting at one taxa and tracing back to the common ancestor for you group. Cross off character-sates as you go. Once your are at the beginning of your tree, you should have accounted for every apomorphic trait. 
8. Refer to the steps we discussed if you get stuck.
9. ​Take a picture of your phylogeny to add to your Lab Notebook Guide
10. Complete the Lab Notebook Guide for Exercise II. 

Cichlid speciation rates on macroevolutionary timescale (Monash University). We are focusing on the Lake Malawi group.

This article does an excellent job explaining divergent evolution of cichlid jaw and teeth morphology, along with other traits.

Exercise iii. More Practice

Procedure. Use these various coded-character matrices for practice. They are similar to what you can expect to see on our exam and will give you some good practice. Alternatively, you can create a tree, then the matching coded-character matrix. You can trade the matrix with your group and get some more practice. If you like to mark on the matrix as you work out the tree, you can print these off, or recreate them in Word, Excel, or on a scratch paper. 

Character 6-1 in analogous.

Faculty Spotlight: Keith Philips

​Keith Philips is our resident systematist and specializes in insect diversity in highly threatened habitats. He works to understand the effect of habitat alteration on particular groups of interest, such as the scarabaeine dung beetles, and to discover new species before they have gone extinct. He conducts phylogenetic and biogeographic analyses, revisions of poorly known taxa, and behavioral and ecological studies. Current emphasis in his lab is the acquisition of molecular sequence data, but morphological data is also gathered to produce the most robust hypotheses of evolution. If you are interested in systemtaics or insect ecology and diversity, send him an email @ [email protected].
Notaferrum n. gen. (Coleoptera: Ptinidae): the first known spider beetle associated with weaver ants...
A phylogenetic analysis of dung beetles (Scarabaeinae: Scarabaeidae): unrolling an evolutionary history...