Title: Phylogenetic Methods and Vertebrate Phylogeny Central Question: How can evolutionary relationships be determined objectively? Sub-questions: 1. What affect does the selection of the outgroup have on the determination of primitive and derived characters states? 2. What are the evolutionary relationships among representatives of terrestrial tetrapods traditionally placed in the classes Reptilia, Aves (birds), and Mammalia? 3. From examination of your evolutionary tree, what characters does your hypothesis identify as evolving by convergent evolution (arising independently along different evolutionary branches)? 4. From examination of your evolutionary tree, what characters does your hypothesis identify as being lost during the evolution of the taxa in the ingroup? 5. What characters would allow you to differentiate among reptiles, birds, and mammals? Can you identify some of the characteristics of each of these three classes among the characters used to construct your evolutionary tree? 6. How could you test your phylogenetic hypotheses? Introduction and Background: In this laboratory exercise we will become familiar with some of the techniques used by systematic biologists who want to objectively develop hypothesis about the evolutionary history of living things. Both theoretical and practical problems make inferring evolutionary history (a discipline known as systematics) one of the most challenging of the life science disciplines. Like other disciplines in biology, systematics proceeds through the experimental cycle, depending on the construction of hypotheses from observations and the rejection or retention of these hypotheses based on experimental work. What s unique is that while these hypotheses address events in the past, often million of years ago, they are based on observations made today -- a process known as remote inference; a process will be employing in this lab. We shall in all likelihood never see major groups of organisms evolving over long periods of time, so we are stuck with making educated guesses at the origin of the major groups through reconstructing histories from the fragments of evidence still unobscured by later events. Terms: Taxon or taxa. A term for any group (taxa = groups) of living things of any taxonomic rank (species, genus, family, order, class, etc.). Cladogram. A branching diagram where the branching is based on the inferred historical connections between the entities as evidenced by shared derived characters. Phylogenetic tree. A branching diagram in which the branching portrays the hypothesized evolutionary relationships and the sequence of hypothetical ancestors linking taxa. Character state matrix. A table of characters where the state of the character in each taxon is coded as being primitive (usually with a zero) or derived (usually with a 1).
Part 1: Introduction to procedures In this section you will be led through the steps of constructing an evolutionary tree. Along the way you will need to answer questions and complete tables. Please refer to your notes from class whenever you need to! Information provided in boxes is for your reference. Questions that you will need to answer in writing will be presented in italics. Overview of how to construct an evolutionary tree: In this lab you will infer the evolutionary relationships among a selection of vertebrates (animals with vertebral columns or backbones). To infer phylogenies, systematists use information provided by both fossil and living organisms. This work depends on defining what groups of living things you are studying (ingroup) and what characters of those organisms you are going to include in your analysis. In order to construct an evolutionary hypothesis (tree) you will need to do the following: 1. Make some observations about characters found in the organisms you are studying. 2. Use outgroup comparisons to determine which characters are primitive and which are derived. 3. Construct a character state matrix of primitive and derived characters. 4. Use the Wagner algorithm to construct an evolutionary tree from the data in your character state matrix. 1. Observations. The data for the basis of any evolutionary study is a table of characters for the different taxa (both ingroup and outgroup) being studied. This table organizes the set of observations from which the phylogeny can be constructed (see Table 1.1) Table 1.1 Morphological characters of four taxa of carnivores using Creodont as an outgroup. Taxa Character Outgroup Ingroup creodont wolf cat bear hyaena Auditory bullae incomplete complete complete complete complete Stance plantigrade digitigrade digitigrade plantigrade digitigrade # digits front 5 5 5 5 4 foot # digits hind foot 5 4 4 5 5 Claws non-retractile non-retractile retractile non-retractile non-retractile Alisphenoid absent present absent present absent canal Rostrum long long short long long Carnasial well developed well developed well developed poorly well developed developed Last molar large large small large small # lower molars 3 3 1 3 1
2. Outgroup Comparison. An outgroup is included to provide an objective mechanism to differentiate primitive characters from derived character state. Any character shared between the outgroup and any taxon in the ingroup is considered primitive. Once the primitive character state has been identified, the alternative states present in the ingroup are the derived character states. Review Table 1.2 for further illustration of the use of the outgroup comparison to identify primitive and derived character states. Please fill in all blanks with the appropriate term, primitive or derived. Table 1.2. Designation of the primitive and derived character states of the ten morphological characters of four taxa of carnivores (Table 1.1) by outgroup comparison. Taxa Character Outgroup wolf cat bear hyaena Auditory bullae incomplete complete complete complete complete primitive derived derived derived derived Stance plantigrade digitigrade digitigrade plantigrade digitigrade primitive derived derived primitive derived # digits front foot 5 5 5 5 4 primitive # digits hind foot 5 4 4 5 5 primitive Claws non-retractile non-retractile retractile non-retractile non-retractile primitive primitive derived primitive primitive Alisphenoid canal absent present absent present absent Rostrum long long short long long primitive primitive derived primitive primitive Carnasial well developed well developed well developed poorly well developed developed Last molar large large small large small # lower molars 3 3 1 3 1 Question: If your outgroup has character that is NOT shared with any of the organisms in the ingroup, can you assume that it is a primitive character? Please thoroughly explain your answer. Please check this page with your TA before proceeding:
3. Construction of a character state matrix. Once the primitive character states are differentiated from the derived character states, the data are coded with 0 s for the primitive state and 1 s for the derived states and the coded data placed in a character state matrix (see Table 1.3). Please fill in the blanks with the appropriate codes. Table 1.3a Character state matrix for 10 morphological characters of the four taxa of carnivore and the outgroup Creodontia. Character polarity determined by outgroup comparison (Table 1.2). Taxa Character Outgroup Ingroup wolf cat bear hyaena Auditory bullae 0 1 1 1 1 Stance # digits front foot 0 0 0 0 1 # digits hind foot Claws Alisphenoid canal 0 1 0 1 0 Rostrum 0 0 1 0 0 Carnasial 0 0 0 1 0 Last molar 0 0 1 0 1 # lower molars 4. Construction of an evolutionary tree by use of the Wagner algorithm. The Wagner algorithm is used to construct a phylogenetic (Wagner) tree under the assumption that the tree that requires the smallest number of character changes is desirable. This assumption is based on the principle of simplicity or parsimony. Outlined below are the steps or procedure of the Wagner algorithm. Steps of the Wagner algorithm- GENERAL EQUATION: D (A,B) = X(A,i) X(B,i) Step 1. Calculate the distance of each taxon from the outgroup (note that if the outgroup has the primitive character state for each of the characters used, the distance between a taxon and the outgroup is identical to the number of derived character states of that taxon). Step 2. Select the taxon with the least distance (fewest number of derived characters) from the outgroup. Step 3. Connect the outgroup to the taxon selected with a branch or line. Step 4. Select the taxon with the next lowest distance from the outgroup. Step 5. Connect this taxon to the branch constructed in step 3. Step 6. Select the taxon with the next lowest distance from the outgroup. Step 7. Calculate the distance from this taxon to each branch now on the tree. Step 8. Connect the taxon with the next lowest distance from the outgroup to the branch with the lowest calculated distance. Step 9. If taxa remain that have not been connected to the tree, repeat steps 6 through 9 until all taxa are places of the Wagner tree.
Please look over the table below. Then calculate the distance of each taxon from the outgroup. Table 1.3b-- Construction of a Wagner tree from the data in the character state matrix. Taxa Character Outgroup wolf cat bear hyaena Auditory bullae 0 1 1 1 1 Stance 0 1 1 0 1 # digits front foot 0 0 0 0 1 # digits hind foot 0 1 1 0 0 Claws 0 0 1 0 0 Alisphenoid canal 0 1 0 1 0 Rostrum 0 0 1 0 0 Carnasial 0 0 0 1 0 Last molar 0 0 1 0 1 # lower molars 0 0 1 0 1 Distance from OG Step 2. Bear has the lowest distance from the outgroup. Bear has a distance of. Step 3. Bear is connected to the outgroup with a branch (as shown below).
Step 4. Wolf has the next lowest distance from the outgroup. Wolf s distance is. Step 5. Wolf is connected to the branch connecting bear and the outgroup and the character states of the hypothetical ancestor (HA-1 1 ) are inferred. Step 6. Hyaena is the taxon with the next lowest distance from the outgroup, with a distance of (see step 1). Step 7. Calculate the distances from hyaena to branches A, B, and C of the tree above. Distance from hyaena to branch A: dist (hyaena, bear) + dist (hyaena, HA-1) dist (bear, HA-1) /2 = (6 + 5 1)/2 = 5 Distance from hyaena to branch B: dist (hyaena, wolf) + dist (hyaena, HA-1) dist (wolf, HA-1) /2 = (5 + 5 2)/2 = 4 Distance from hyaena to branch C: dist (hyaena, HA-1) + dist (hyaena, OG) dist (HA-1, OG) /2 = (5 + 5 2)/2 = 4 Step 8. Attach hyaena to the branch with the lowest distance, branch B in this example. What would happen if you attached to branch C? Please thoroughly explain your answer. Please check this answer with your TA: 1 Please note: HA-1 means hypothetical ancestor #1, not hypothetical ancestor minus 1.
Step 9. Cat is the final taxon to be added to the tree with a distance of from the outgroup. Step 10. Calculate the distances from cat to branches A, B, C, D, and E on the tree above. Distance from cat to branch A: dist (cat, bear) + dist (cat, HA-1) dist (bear, HA-1) /2 = (8 + 7 1)/2 = 7 Distance from cat to branch B: dist (cat, wolf) + dist (cat, HA-2) dist (wolf, HA-2) /2 = (5 + 6 1)/2 = 5 Distance from cat to branch C: dist (cat, HA-1) + dist (cat, OG) dist (HA-1, OG) /2 = (7 + 7 2)/2 = 6 Distance from cat to branch D: dist (cat, hyaena) + dist (cat, HA-2) dist (hyaena, HA-2) /2 = (4 + 6 4)/2 = 3 Distance from cat to branch E: dist (cat, HA-2) + dist (cat, HA-1) dist (HA-1, HA-2) /2 = (6 + 7 1)/2 = 6
Step 11. Attach cat to the branch with the lowest distance, branch D in this example. Finally, you should redraw the tree, keeping it rooted in the outgroup. Please note that the trees shown on this page (above and below) are identical in their meaning. Figure 1-- Wagner tree showing the phylogenetic relationship (hypothesis) of four taxa of Carnivora.
Part 2: How are turtles, crocodiles, birds and mammals related? In this part of the lab you will infer the evolutionary relationships among a selected group of vertebrates (ingroup). A primitive amphibian will be used as an outgroup and the characters of the outgroup will be determined from fossils and living forms. Please choose one or two partners. Dispersed around the lab room are specimens, skulls, skeletons, and diagrams of amphibians (outgroup), turtles, crocodiles, birds, and mammals. 1. Your first job is to collect data on the characteristics listed in Table 1.4 for the outgroup and ingroup taxa. Some of the data has already placed in the table. Use observations to fill in the rest of the table. Most of the questions concerning characters have yes or no answers based on the presence or absence of the character. The character concerning the projection of the ischium refers to whether it projects toward the head (anterior) or toward the tail (posterior). Your lab instructor will identify anatomical characters such as temporal fossa, ilium, and ischium. If you are having difficulty discerning any of these characters please ask your TA for help. Table 1.4 Characters of selected vertebrates. Taxa Characteristic Outgroup Ingroup Amphibian Turtle Crocodile Bird Mammal type of egg anamniotic amniotic amniotic amniotic amniotic 1 scales present feathers present hair present red blood cells nucleated nucleated nucleated nucleated anucleate teeth set in sockets 2 lower jaw more than single bone single temporal fossa two temporal fossas illium projected only anteriorly rod-like ischium 3 chambered heart no yes yes no no 4 chambered heart no no no yes yes Please note: 1. Although most mammals do not lay eggs, the duckbilled platypus and the spiny echidna of the Australian region lay amniotic eggs. 2. Although modern birds do not have teeth, many fossil birds had teeth which were placed in sockets. Please check in with your TA before proceeding to step 2:
2. Using outgroup comparison, determine which character states (for each character listed in Table 1.4) are primitive and which are derived. Remember that any character shared between the outgroup and any taxon of the ingroup is the primitive character. A character present in the outgroup and not present in any taxa of the ingroup is often considered primitive also. Those characters not identified as primitive are derived. Coding the primitive character state with a 0 and derived states with a 1 allows you to construct a character state matrix in the table below from your observational data in Table 1.4. Table 1.5-- Character State Matrix Taxa Characteristic Outgroup Ingroup Amphibian Turtle Crocodile Bird Mammal type of egg scales present feathers present hair present red blood cells teeth set in sockets 2 lower jaw more than single bone single temporal fossa two temporal fossas illium projected only anteriorly rod-like ischium 3 chambered heart 4 chambered heart Differences from outgroup 3. Calculate the distance (number of derived characters) between each taxon of the ingroup and the outgroup. Place this distance in the final column of the character state matrix (above). Please check this with your TA before proceeding to step 4
4. Use the Wagner algorithm to construct an evolutionary tree and infer the derived characters present in each hypothetical ancestor from the data in your character state matrix. If you are having problems see your TA, however, your TA will not be able to be of much assistance unless you can show them your work. Have your TA review your phylogenetic tree.
Closing questions: Please thoroughly explain all your answers. 1. What inferences or evolutionary hypotheses are shown in your evolutionary tree? Do the reptiles (turtles and crocodiles) share a common ancestor that is not shared with members of any other class (birds or mammals) of vertebrates examined? 2. What characters are inferred to be convergent (i.e. evolved independently along different evolutionary lineages)? Does your evolutionary tree make any inferences of character reversal (a derived character state that reverts back to the primitive state)? 3. What characters are diagnostic of reptiles? 4. What characters are diagnostic of birds? 5. What characters are diagnostic of mammals? 6. How could you test the phylogenetic hypotheses you have made in your evolutionary tree? 7. So what do you think, can evolutionary relationships be determined objectively? (Be sure to explain your position.) Please turn in your answers for your TA to review. Your responses will be evaluated and will count as your first lab assignment.