Reconstructing Evolutionary Relationships S-1 Practice Exercise: Phylogeny of Terrestrial Vertebrates In this example we will construct a phylogenetic hypothesis of the relationships between seven taxa of terrestrial vertebrates: frogs, turtles, mammals, birds, crocodiles, lizards, and snakes. We will use another taxon (ray-finned fish) which has some similarities with your terrestrial vertebrates but which is not part of that group as our out-group to polarize the characters chosen for the analysis. Identifying Characters The first step in every analysis is identification of the characters. This is often the most important and most difficult part of the work (not to mention time and labour consuming). Below is the list of the characters, each of which is accompanied by the corresponding number used in Table 1. Table 1 contains a list of the character states found in each taxon. (You are not expected to learn these characters and their states for the quiz or test.) Amnion (1): Does the egg have the amnion, an extra-embryonic membrane that evolved to overcome desiccation? Possible states: amnion present, absent. Appendages (2): Early vertebrates evolved fins to swim more efficiently in the water. Limbs allowed movement on land. Although snakes lack external limbs, several groups of snakes have rudimentary appendages, proof of the fact that the ancestors of snakes did have limbs. Possible states: fins, limbs. Body covering (3): Possible states: scales, scutes, hair, feathers. Metabolism (4): Maintenance of a steady internal state (homeostasis) is critical. In vertebrates, body temperature is controlled either internally by metabolic processes (endothermy) or is dependent on the external environment (ectothermy). Possible states: endothermy, ectothermy. Internal nostrils (5): The nostrils of most fishes do not connect with the mouth. The nostril of lungfishes and of all terrestrial vertebrates do. Possible states: internal nostrils present, absent. Temporal fenestrae (6): Number of temporal holes in the skull, which allow for the expansion of jaw muscles (enhancing biting strength). Possible states: none, one, or two. Male genitalia (7): Presence or absence of a hemipenes, or split penis. Snakes and lizards are unusual in that they have a double penis, whereas other vertebrate groups have a single penis. Possible states: hemipenes present, absent. Digestive system (8): Presence or absence of a specialized gizzard, a very muscular portion of the stomach. The muscle action, together with a tough lining of cuticle, and especially grit that is ingested, aid in the grinding of fibrous foods such as seeds. Possible states: gizzard present, absent. Nitrogenous waste (9): Type of nitrogenous waste. Ammonia is converted to either urea or before excretion. Possible states: urea,. Digits on hind limb (10): Possible states: zero, four, or five digits. Urogenital system (11): Presence or absence of a urinary bladder, a distensible sac in which urine is stored before being excreted. Possible states: bladder present, absent.
S-2 Reconstructing Evolutionary Relationships Table 1. Character states for seven terrestrial vertebrates and ray-finned fish. Taxon Amnion 1 2 Body covering 3 4 Internal nostrils 5 Temp. fenestrae 6 Appendages Metabolism Hemipenis 7 Gizzard 8 Waste type 9 Hind digits 10 Bladder 11 Fish absent fins scales ecto absent 0 no absent urea 0 present Frogs absent limbs smooth ecto present 0 no absent urea 5 present Turtles present limbs scutes ecto present 0 no absent Crocodiles present limbs scutes ecto present 2 no present Lizards present limbs scutes ecto present 2 yes absent Birds present limbs feather endo present 2 no present 5 present 4 absent 5 present 4 absent Mammals present limbs hair endo present 1 no absent urea 5 present Snakes present limbs scutes ecto present 2 yes absent 0 present Out-Group Comparison Now that you have gathered the original data, use the out-group comparison method to polarize the characters. By definition, the out-group taxon, fish, is coded 0 for each character. Any character state of an in-group taxa which is the same as the out-group is thus coded 0, and if it is different from the out-group it is a derived character and is coded 1 (or if it is multistate, 2, 3, etc. ). Activity: Based on the characteristics exhibited by each taxon, complete Table 2. The multistate characters have been coded for you. Table 2. Character states recoded with respect to the out-group. Taxon 1 2 3 4 5 6 7 8 9 10 11 Fish 0 0 0 0 0 0 0 0 0 0 0 Frogs 1 0 1 Turtles 2 0 1 Crocodiles 2 1 2 Lizards 2 1 1 Birds 3 1 2 Mammals 4 2 1 Snakes 2 1 0
Reconstructing Evolutionary Relationships S-3 Getting Started Initially, there are no relationships known among the in-group and out-group taxa. Thus, if you were to draw a tree representing what you know of their relationships it would look like tree A. A There are several ways to go about building a tree. One of the easiest ways is to do so on a character by character basis, that is, drawing a tree for each character. Each one of these trees is a character tree, where the relationships between our taxa are depicted in terms of a single character. This means that you have to group together all the taxa that share the same state for the character considered. Once all character trees are generated, we can start combining them together to generate the tree that includes all characters, that is, the final phylogenetic tree. In our analysis we will treat characters 3 and 6 as unordered, and character 10 as ordered. Character Trees Character tree 1 (amnion) depicts the topology or tree shape resulting from grouping taxa together on the basis of character 1. In this tree, ray-finned fish and frogs are basal (they share the ancestral condition) and all other taxa share the shared derived character of presence of an amnion. Character tree 2 (appendages) shows that the character state presence of limbs groups all taxa of our in-group together, whereas our out-group (ray-finned fish) has fins. This character is important because it separates our in-group from the out-group and represents a shared derived character for the in-group. Character tree 3 (body covering) is a little different from the previous two trees because it contains more than two character states. In this case, we proceed in very much the same way, grouping taxa on the basis of shared character states. Remember, since this is an unordered character it is not necessary to progress through states 3(1) and 3(2) before getting to 3(3) (i.e., 0 ÿ 3 is only one step).
S-4 Reconstructing Evolutionary Relationships Character tree 4 (metabolism) groups birds and mammals as belonging to the same clade, whereas all other taxa are basal. Character tree 5 (internal nostrils) defines the out-group from the in-group. If you look carefully, it is the same tree as character tree 2. When you see that you have more than one character tree which depicts the same set of relationships, you can combine them in a single character tree. This makes building the final tree easier in that you can see which relationships are best supported by a greater number of shared derived characters. Character tree 6 (temporal fenestra) also presents more than two character states. In this case, character state 6(2) constitutes a unique derived character for mammals, character 6(1) is a derived character shared by snakes, lizards, crocodiles, and birds, whereas ray-finned fish, frogs, and turtles have the ancestral state. Activity: For the remaining character trees at left, add the characters and taxon labels, and fill in the blanks in the text below (add name of taxon, you can use abbreviations, such as FI, FR, T, etc.) by referring to Tables 1 and 2. Character tree 7 (hemipenes) group and together through sharing hemipenes while all other taxa have an absence of this character. Character tree 8 (gizzard) shows that and possess a gizzard, which is absent in all other taxa.
Reconstructing Evolutionary Relationships S-5 Character tree 9 (waste type):,,, and have as waste type, whereas ray-finned fish, and share the ancestral character of urea as a waste type. Character tree 10 (hind digits) is another multistate character. In this case, based on the information provided from the fossil record, we have confidence in the fact that four hind digits evolved from five hind digits, and not from zero, hence we treat this character as ordered (i.e.,10:0 ÿ 1 and 10:1ÿ 2). and have four hind digits, whereas,,, and all have five digits and and have none. Character tree 11 (bladder) groups and together (they do not have a bladder) and all other taxa are basal, as with character 8. We now have 11 character trees. Building the Phylogenetic Tree How do we combine all the character trees into one single tree? We suggest that you start by finding a character that defines the in-group and separates it from the out-group. Do this by identifying those derived characetrs that only members of the in-group possess (i.e., shared derived characters for the in-group). From our character trees, we can see that two characters separate the in-group from out-group. B Activity: The first step is to start building your final tree by plotting, on tree B, the two characters that define the in-group. Character 2 is done for you. What is the other character that defines the in-group? Add this character to trees B to G. C The next stage is to see if any character trees support a grouping that has all the in-group taxa except one (i.e., one taxon is grouped with the out-group). From our character trees, we can see that there are two character trees that show this pattern. Character 1(1) defines all taxa except frogs, and character 10(1) defines all except snakes. How do you decide now which one to use? Look to the other character trees, and see if there are any that support a grouping of frogs with other taxa, to the exclusion of snakes; or a grouping of snakes with other taxa to the exclusion of frogs. You will see that there are several characters that support a grouping of snakes with other taxa [6(1), 7(1), 9(1)], while
S-6 Reconstructing Evolutionary Relationships no other character supports a grouping of frogs with anything else to the exclusion of snakes. Frogs are hence the most basal of the terrestrial vertebrates, hence tree C. ~ Add character 1 to tree C (as well as character 5). D Next look for a character that defines all taxa, except fish, frogs, and at least one other taxon. Character trees 3 and do this: character states 3(2) and are present in all taxa except fishes, frogs, and mammals. (Feathers are a unique derived character in birds.) When you add these characters to tree C you obtain tree D. Note that you must also include a step each for characters 3(1), 3(3), and 3(4). These are all unique derived characters, and although unique derived characters are not useful characters for reconstructing phylogenetic relationships, they can tell us something important about the life and evolution of our taxa. ~ The multi states of character 3 have been added to tree D, but you must add character. E The next characters to add would be character 6, which pulls off frogs, mammals, and turtles with the out-group. When you add character 6 to tree D you obtain tree E. We must also add character state 6(2), an a unique derived character in mammals. Next, look and see if there are character trees that show the relationships of the unresolved taxa. In our case, character trees and do this, supporting birds and crocodiles as being sister groups. This illustrates the point that if you find multiple character trees that support the same relationship then this relationship will probably be upheld in the final tree. When we add character trees and to tree E we obtain tree F. F You can see that we still have not been able to resolve the relationships between snakes, lizards, and the monophyletic group formed by birds+crocodiles. We can do this by adding character, which gives us tree G. G 6: 0 ÿ 2 3: 0 ÿ 1 3: 0 ÿ 4 2: 0 ÿ 1 3: 0 ÿ 3 3: 0 ÿ 2 6: 0 ÿ 1 Now we have a tree that includes all taxa. However, we have not added all characters. We then have to add the remaining two characters to tree G. When you build a cladogram for the first time, it may be easier to leave the multistate characters last (although you can not always do this). Character 10 evolves state 10(1) in the ancestor of the land vertebrates, and state 10(2) in the ancestor of crocodiles and birds, and has a reversal to 0 (from 1) in snakes (as shown in tree H).
Reconstructing Evolutionary Relationships S-7 At this point you will also find that, because of the relationships depicted by our tree (referred to as the topology of the tree), it will be impossible to add character state 4(1) only once. Character tree 4 shows that birds and mammals are the only two taxa to have an endothermic metabolism, hence they are represented (in character tree 4 ) as being sister groups. There are however many more characters that support the sister group relationships birds+crocodiles, and mammals+(turtles, lizards, snakes, birds, crocodiles). In order to change the topology of tree G to show the sister group relationship birds+mammals, we would have to re-evaluate the evolution of all characters and we would find that some other states would have to evolve independently more than once. Which topology should we choose? We solve this problem using the principle of parsimony. Principle of Parsimony The principle of parsimony is used to determine which tree best fits the data during a phylogenetic analysis when more than one tree is obtained. Recall that a character state is one of two or more alternate forms of the same character. For example, character 3 (body covering) has four different forms, thus four character states. A specific character state can evolve more than once, as in the case of homoplasious characters. The optimal tree is the tree that minimizes the number of homoplasious characters, and hence requires the least number of character state transformations. This is also referred to as the shortest tree. The length of the tree is measured in number of character states that have evolved. Each character state transformation is called a step and when you measure the length of the tree you count the number of steps. In our vertebrate example we have to hypothesize that endothermy, character state 4(1), has evolved twice independently in mammals and birds. This character is thus a homoplasy. Tree H is our final tree. Activity: ~ To tree H, add character 4, a homoplasious character that evolved independently in both mammal and birds. ~ Count all of the character transformations mapped onto tree H. How many steps are in our final tree? We did not choose the alternate tree that grouped mammals and birds because it would have resulted in many more steps than it took to obtain tree H.
S-8 Reconstructing Evolutionary Relationships H Add character 4 to this tree. It is important to realize that the resulting phylogenetic tree depicts relationships among taxa. There are many ways in which these relationships are represented. It is important that you be able to look at a tree and recognize the relationships that are being indicated. Keep in mind that the branches coming out of one node can be rotated 180E and the relationships do not change. ~ Note that the tree at left depicts the same relationships as in tree H. In the tree below the node for crocodiles+birds has been rotated, as has the node for lizards+snakes and the node for crocodiles+birds+lizards+snakes. All nodes can be rotated and the relationships remain the same. In this analysis we have built our phylogenetic tree by hand, but most analyses are so complicated that they can only be performed using computers and specific software programs, such as PAUP and Hennig86. These programs, in addition to constructing the shortest trees, also give information needed to evaluate the trees obtained, such as the length and other statistics that will allow estimates of the level of homoplasy in the data (i.e., how many of the characters used are homologies and how many are homoplasies).