Biology 1012 K Lab Manual

The diversity of today’s biological world is the result of nearly 4 billion years of evolution. In an attempt to reconstruct the evolutionary history of life, biologists analyze patterns of change in the heritable features of organisms. Heritable features are those that can be passed down from one generation to the next. The proposed evolutionary relationships that arise from analyzing heritable traits among organisms are referred to as phylogeny. Biologists who work to reconstruct phylogeny are called systematists. Systematic analyses help create classification systems that convey information about the evolutionary relationships of organisms.  

Hypothesized lineages of organisms can be represented as branching trees, called phylogenetic trees. A phylogenetic tree is a diagram of the hypothesized evolutionary history of a group of organisms (Figure 1). Many types of data can be used to generate phylogenetic trees, including morphological characteristics such as shape of body parts and molecular data such as nucleotide sequences of DNA. These different types of data don’t always tell the same phylogenetic story, so any phylogenetic tree is a hypothesis based on the type of data that was used and so is always subject to further scientific analysis and revision in light of new data.  

Taxonomy is the branch of biology that describes, names, and classifies species and groups of species. A species is typically defined as a group of organisms in which genetic exchange through reproduction is possible. Humans, for example, are a separate species from chimpanzees, because although humans can reproduce with each other (and thereby exchange genetic information), they cannot reproduce with chimps. Because of this genetic isolation, individual species typically don’t pass on heritable traits to each other, and are, therefore, thought to be independent evolutionary units.  

Taxonomists use the Linnaean system of classification, which gives each species a scientific name composed of two parts (binomial nomenclature). The first word in each scientific name identifies the genus (plural = genera) to which the organism belongs, while the second word is the specific epithet. The genus name is always capitalized, while the specific epithet is not capitalized. Both parts of the binomial are either underlined or italicized (e.g. Homo sapiens or Escherichia coli). The Linnaean taxonomic system is hierarchical in that it groups species and genera into more inclusive categories: family, order, class, phylum, kingdom, domain (Fig. 2). A named taxonomic group at any level of the hierarchical system is called a taxon (plural = taxa). Although the Linnaean system of classification was developed before Darwin published his theory of evolution by natural selection, modern systematists strive to incorporate evolutionary history into taxonomic classification schemes and vice versa. Ultimately, taxonomy should reflect the true evolutionary history of an organism: its phylogeny (and vice versa).  

Systematics 

Systematics is the study of biological diversity in an evolutionary context. Systematists are biologists who work to integrate classification systems, evolutionary processes and geologic history. Therefore, the modern field of systematics strives to generate phylogenetic trees by analyzing evolutionary changes in characters of organisms and incorporating this information into taxonomic classification schemes. The resulting phylogenetic trees are diagrams of data, which describe hypothesized patterns of evolutionary change in inherited characters among nested groups of organisms.  

Cladistics analysis 

Today, most systematists use a method called cladistic analysis to generate phylogenetic trees, which can also be referred to as cladograms. Each branch point (or node) on a phylogenetic tree represents a divergence of two taxa from a common ancestor and each branch stemming from a branch point is called a lineage (or clade), and each lineage gives rise to a taxon at the tip of each branch (Fig. 3).  A phylogenetic tree helps to depict the evolutionary relationships of a group of organisms. Phylogenetic trees typically proceed chronologically from the past to the present (Fig. 4). Those taxa that share a more recent common ancestor are more closely related. Those that share a more ancient common ancestor are less related (Fig. 4).  Take a good long look at Figures 3 and 4. Read all the captions and be sure you understand them before you go on. 

To build a phylogenetic tree, systematists must first identify characters (also called traits) that are shared by taxonomic groups. A character is any feature that a particular taxon possesses. Characters may be anatomical structures, behavioral patterns, DNA sequences, or any other HERITABLE trait. For example, all birds share the trait of feathers, while all mammals share the trait of hair. When these shared traits are placed on a phylogenetic tree, they are placed below the branch(es) of the taxa that have these traits. For example, feathers are a trait unique to birds, so this trait only goes on the branch right before birds (Fig. 5). Hair, on the other hand, is a trait shared by marsupials, primates and and rodents (they all have hair), so this character is placed on the tree right before the common ancestor of these three groups (Fig. 5).  

Unfortunately, identifying homologous characters is not an easy task. First of all, finding similar traits in the first place can be quite difficult. Secondly, not all similarities among taxa qualify as homologies! In fact, taxa can share similar traits that they DO NOT inherit from a shared common ancestor. Sometimes taxa from very different branches of the tree of life can independently evolve similar traits due to similar environmental pressures. For example, the external structure and function of the enlarged dorsal fin of a killer whale (mammal) is similar to that of the enlarged dorsal fin of a shark (cartilaginous fish). This fin helps to stabilize them while they are moving fast in water hunting prey. However, the common ancestor of a shark and a killer whale did NOT have a similarly enlarged dorsal fin.  In other words, these two animals have a common trait (an enlarged dorsal fin) that was NOT inherited from their common ancestor. Thus, their dorsal fin is not a homologous trait, but rather is called an analogous trait (Fig. 6). Analogous traits are traits that are shared by two or more taxa but are NOT inherited from the common ancestor of these two taxa. Analogous traits typically arise via a process called convergent evolution in which similar characteristics arise in often distantly related taxa due to similar selective pressures in similar environments. For example, the enlarged dorsal fins in whales and sharks function to stabilize movement in an aquatic environment. One can infer that similar advantages would be gained by sharks and whales that had evolved dorsal fins that aid in stabilization. Thus these two different groups of organisms “converged” on the same trait . It’s convergent evolution!  

Convergent evolution has been documented in a variety of taxa. For example, distantly related plant families - the Euphorbiaceae (spurge family) and the Cactaceae (cactus family) - have members that grow in desert environments of Africa and the Americas, respectively. Despite not being closely related and living in very different parts of the world, some members of both cacti and spurges have fleshy, columnar stems for water storage, protective spines and reduced leaves. These structural similarities are analogous adaptations that evolved independently in response to the similar environmental pressures (low water availability) of a desert habitat. 

Determining whether a trait is analogous or homologous is important because homologous traits are shared traits that can define a taxonomic group (ex. hair in mammals), whereas analogous traits are found in distantly related taxa so do not indicate close evolutionary relationships (ex. dorsal fin of shark and whale). 

Since taxa are often defined on the basis of shared heritable characters, we can only recognize new or different lineages when characters change. Consequently, the next step in a cladistic analysis after identifying an important character that defines a taxonomic group, is to identify the “original state” of the character (the ancestral form) versus the more recent, “changed state” of the character (the derived form). The appropriate application of the term ancestral or derived to a given character state depends on the group of taxa being examined. For example, systematists conclude that scales (the ancestral form) evolved in one lineage into feathers (a derived form) and in another lineage into hair (a derived form).  They made this conclusion by observing that feathers in birds and hair in mammals are both epidermal structures made of the protein keratin (Fig. 5).  When we look at reptiles and the shared common ancestor of all 3 of these taxonomic groups we see that they have a more primitive keratinized epidermal structure called scales. Because scales are present in the more ancient common ancestor of these groups, but hair and feathers are not, scales are considered to be the ancestral state of this epidermal covering character while hair and feathers are considered to be the derived state of this character. 

It is often difficult to determine whether a trait is ancestral versus derived. One way to do this is to compare the taxa being studied to an outgroup. An outgroup is a taxon that is closely related to the study group (ingroup), but branched off from the lineage of the ingroup prior to the origin of the common ancestor of the ingroup. For example, in Figure 5 marsupials would serve as an outgroup in an analysis of primates and rodents. The outgroup often, but not always, possesses ancestral character state(s) and lacks the derived character state(s). Thus, we can use the outgroup to determine if a character state is derived or ancestral. If the outgroup has the character state, then that character state is likely to be ancestral. If the outgroup lacks the character state, then that character state is likely to be derived. For example, in Figure 5 marsupials serve as an outgroup in an analysis of primates and rodents, and we can use marsupials to determine whether a placenta is a derived or ancestral trait. Because the outgroup (marsupials) lack a placenta, we can reasonably conclude that a placenta is a “derived character state” that is shared by primates and rodents. 

The key to generating a well-supported cladogram is being able to identify shared derived characters. Shared derived characters are important because they define the true evolutionary relationships (aka natural groups or lineages) of organisms. Defining and naming natural groups through the use of shared derived characters enables systematists to create a classification system that conveys information about the true evolutionary relationships of taxa. 

Since the number of possible hypotheses about phylogenetic relationships increases with the size of the ingroup, it is very likely that a data set could generate more than one phylogenetic tree. For example, a cladistic analysis of three taxa could generate a maximum of three different trees. If only two additional taxa are added to the analysis (for a total of five taxa in the ingroup), then suddenly 105 trees are possible. An analysis of 10 taxa could yield 34,459,425 trees! Systematists resolve conflicting hypotheses by utilizing the principle of parsimony, or preferring the simplest among a set of plausible explanations of a phenomenon. When applied to reconstructing phylogeny, applying the principle of parsimony means that systematists select the tree (hypothesis) that minimizes the number of times that a character state changes. For example, it makes more sense for hair to have evolved once in a common ancestor (Fig. 7, tree B) than to have independently evolved two separate times (Fig. 7, tree A). Thus, tree B below is considered to be the most parsimonious tree and therefore the best supported hypothesis for the true evolutionary relationships of these taxa.  

Figure 7. Tree A, which says primates are more closely related to birds than rodents is less parsimonious than tree B, which says that primates are more closely related to rodents than birds. This is because in tree B the trait, “hair”, only had to evolve once.  

ACTIVITY #1A: Generating Phylogenetic Hypotheses 

At your lab bench, you will find a set of envelopes: one envelope is labeled INGROUP and the other envelope is labeled OUTGROUP. Inside the INGROUP envelope, you will find 5 “taxa.” These 5 taxa represent your focal study group. Inside the OUTGROUP envelope, you will find a single taxon labeled O, which represents your outgroup. Work in pairs! 

1. Your goal is to generate a hypothesis about how these six taxa are related using the cladistic method.  Using these taxa fill in the character table and build a hypothesized cladogram. 

Fill in the following table with: 

• Characters – general description of a heritable trait  (e.g. shape of organism) 

• Character states – the alternative forms of a character (e.g. square or rectangle). Determine which character state is ancestral (this should also describe the outgroup) and which is derived. 

Place each taxon name (e.g. king of spades) and the derived character states on the cladogram below.  Remember your cladogram is one hypothesis that explains the evolutionary relationships of the taxa assigned to your group. 

ACTIVITY #1B: Generating Phylogenetic Hypotheses 

2. Using the same set of cards, generate an alternative hypothesis about how these six taxa are related using the cladistic method. 

Fill in the following table with: 

• Characters – general description of a heritable trait (e.g. shape of organism) 

• Character states – the alternative forms of a character (e.g. square or rectangle). Determine which character state is ancestral (this should also describe the outgroup) and which is derived. 

Build an alternative phylogenetic tree with each taxon name (e.g. king of spades) and the derived character states in the space provided below.  

3. Does convergent evolution need to occur for this hypothesized phylogeny to be correct? (In other words, is the same character evolving twice on your tree?) 

4. Does the principle of parsimony allow you to select only one of your hypotheses? Why or why not? 

5. From years of research, you have discovered that Redness, Diamond Shape, and Having a Face are the three most important characteristics for defining the evolutionary relationships of card organisms. Place these three traits on the trees below. 

6. Now that you have placed these important traits on the tree, which hypothesized tree, A or B, is the most parsimonious? This is the tree that more likely depicts the true evolutionary relationships of these “organisms”?             

ACTIVITY #2: Taxonomy and Phylogeny 

Work in groups of 4 for this Activity. 

As discussed in the introduction, we know that systematists work to integrate taxonomy (classification systems) with the phylogeny (genealogical relationships among organisms). Since humans are organisms they, too, are classified under the hierarchical taxonomic system. The scientific name for humans is Homo sapiens (sapiens is the specific epithet; Homo is the genus name).  The complete taxonomic information on humans is: 

Family: Hominidae Phylum: Chordata 

Order: Primates Kingdom: Animalia 

Class: Mammalia Domain: Eukarya 

Below is a list of CHARACTERS that define humans.  

• dorsal spinal cord and notochord                                      

• multicellular heterotroph 

• hair, mammary glands, and 3 bones in middle ear 

• loss of tail 

• membrane-bound organelles 

• opposable big toe, forwardly directed eyes, all toes and fingers have nails rather than claws 

1.  Match the list of characters above to the appropriate taxonomic group:

2. Now, place the CHARACTERS on to the tree below at the appropriate location. 

3. Finally, circle the group of organisms that belongs to EACH LEVEL of the hierarchical classification scheme and label the group with its corresponding taxonomic name (we have already circled the members of the Domain Eukarya).

4.  Now that you have experienced constructing and interpreting phylogenetic trees, it is finally time to look at some “real” organisms!  Observe the butterfly, pillbug and fly provided to identify characters that unite and differentiate them.  Use your dissecting microscope to look carefully at external morphology.  Do NOT unpin or touch the butterfly or the fly! 

Generate a phylogenetic tree for these three organisms using the characters you observed.  Be sure to place characters on the tree that are true of all three, just two and one unique to each organism. 

5. When you have completed your phylogeny, show it to your instructor.  They will then give you the proper taxonomy for each organisms; use it to fill in the table below.   

Is the phylogeny you drew consistent with the taxonomy provided?  Give a thorough explanation for WHY you answered yes or no. 

If no, did you draw an incorrect phylogeny?  Explain. 

If yes, describe how your characters and their placement allowed you to make this determination. 

ACTIVITY #3: Using phylogenies to identify unknowns 

Work individually for this activity to prepare you for identification of your unknown DNA barcoded organism later in the semester. 

You are now familiar with building and interpreting hypothesized phylogenies, including the fundamental concepts that: 

• each branch point (node) on a phylogenetic tree represents a common ancestor from which two taxa evolved  

• one can identify the most recent common ancestor for specific taxa (see Figure 4 in the introduction).   

You also learned that the evolutionary history of taxa, as depicted in a phylogeny, should be reflected in the hierarchical taxonomy (classification) of those taxa. 

Later in the semester, you will each collect a living specimen and sequence the DNA of a particular region. You will then use this to generate a unique DNA barcode to identify your specimen. Part of this identification will require that you interpret a computer-generated phylogeny containing your unknown specimen and other related taxa identified by a database search (see phylogenies below). You will be asked to name the most specific taxonomic level (Species? Genus? Family? etc.) to which you are confident your specimen belongs. The following activity allows you to practice answering that question.  

Activity 3A: Consider the computer-generated phylogeny in the figure below. 

Our unknown shares its most recent common ancestor with Canis lupus (domestic dog) at node 2 but can we conclude from just the circled part of the phylogeny that our unknown is Canis lupus (domestic dog)?  No, because a speciation event could have occurred at node 2.   

If instead we move back to the next most recent common ancestor (node 1), we can more confidently conclude that all of the descendants of the common ancestor at node 1 are Canis lupus, including our unknown. We can therefore more confidently identify our unknown as the species Canis lupus. This is because our unknown is more similar to Canis lupus (the dog) than another individual of the SAME species (the wolf). Without the wolf as an outgroup, it is impossible to make this determination.  

Activity 3B: Consider the next computer-generated phylogeny in the figure below. 

What is the most specific taxonomic level to which you are confident the unknown belongs?   

(Hint: What taxonomic level do all descendants of the common ancestor at node 1 share?) 

Answer: ______________ Therefore, we can identify our unknown to this level. 

Activity 3C: Consider the computer-generated phylogeny in the figure below. 

When some taxa on the phylogeny do not share the same genus (or species), we need to know the higher levels of taxonomy for each species to identify our unknown (see Table below).   What is the most specific taxonomic level shared by all descendants of the common ancestor at node 1? 

Answer: _____________   Therefore, that is the most specific taxonomic level to which we can identify our unknown. 

Activity 3D: Consider the computer-generated phylogeny in the figure below and the table provided. 

Name the most specific taxonomic level (Species? Genus? Family? etc.) to which you are confident your specimen belongs.  ___________________ 

Hint: To answer this question use the examples to help you determine what common ancestor you should evaluate in this phylogeny.