BIOA01 Lab 2 Introduction to Phylogenetic Analysis F2021 PDF

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BIOA01H3 F – Fall 2021

LAB 2 An Introduction to Phylogenetic Analysis Overview In this lab you will analyze evolutionary relationships of species from molecular data, as well as by using cladistic techniques. The molecular analysis will involve a comparison of the amino acid sequence of the hemoglobin beta chain of eight vertebrates. The cladistics exercise will introduce you to important principles of cladistics analysis and then allow you to apply these principles to build a phylogenetic tree.

Objectives 1. Gain an understanding of the principles of phylogenetics, as they apply to molecular and cladistics analysis 2. Apply principles of phylogenetic analysis to construct phylogenetic trees based on molecular similarity 3. Apply principles of cladistics analysis to resolve hypothetical evolutionary relationships

Useful Preparations 1. Read sections 14.1 - 14.2 (pgs. 301 - 309), 20.6 (pgs. 442 - 444), and 22.1 – 22.2 (pgs. 467 - 478) in Morris et al. (2019) to familiarize yourself with the terms and concepts outlined in these instructions.

There are two Labster simulations that are required to be completed in Lab 2. The Labster simulations can be found under the “Labs” heading in the Modules section of our BIOA01 Quercus page. The assigned Labster simulations are titled “Evolution: Are you related to a Sea Monster?” and “Evolution: Journey of the Canids”. These simulations must be completed by Friday October 1st at 12:00pm (noon) EST. There will be no extensions given for missing the deadline. The score from your first completed attempt will be counted as your score. Make sure to clear your cache/cookies on your web browser before attempting the simulations. If you experience technical issues, feel free to resume or restart the simulation, as only a complete attempt (completing the simulation) will count as your score. The Lab 5 Assessment is due to be completed on Friday October 1st by 5:00pm EST. You will only have one, three-hour attempt to complete it and no second attempts will be given so only click the “Start Quiz” button when you are prepared to do so. No late submissions will be accepted. Some of the work outlined in this instruction document should be completed prior to beginning your attempt, as you will not have sufficient time with three hours to complete the work outlined here plus the Lab 2 Assessment.

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BIOA01H3 F – Fall 2021

Lab 2 - Background Information One of the most profound implications of Darwin’s theory of evolution is that all life on this planet can be traced back to a common origin. When Charles Darwin observed living species, he recognized that they were modified descendants of earlier ones. As ancestral species’ repeatedly separated and diverged through time, multiple descendant species arose with nested patterns of similarity. This branching history of descent is called phylogeny. Our system of taxonomy is based on phylogeny. That is, we classify organisms together because they have a common evolutionary ancestor. However, in most cases we cannot determine ancestry directly because the fossil record is poor or absent for most species (and time travel is not an option!). For this reason, we must rely on shared, homologous features to build hypotheses around species relatedness. A key assumption is that if two species share a given feature or characteristic, then they inherited the character from a common ancestor somewhere in the past before the two present species separated from each other and evolved differences in other characteristics. The newer characters are that are shared by two species, the more closely related they are. Organisms that share many homologous features are most likely closely related and probably had a relatively recent common ancestor. For example, when analyzing molecular similarity of primates, the chimpanzee (Pan troglodytes) genome sequence and the human (Homo sapiens) genome sequence are about 98% similar. This is because the common ancestor of these two species lived only about 6 million years ago. In the six million years since these two species diverged, there has not been enough time for many differences to appear in their genome sequences. On the other hand, humans and yeast (Saccharomyces cerevisiae), which shared an early eukaryotic ancestor about 1.2 billion years ago, have many more differences in their genome sequences. Our ability to reconstruct and interpret the evolutionary history of organisms has profound implications for all areas of biology. Evolutionary biologists, ecologists, ethologists, physiologists, and molecular biologists who study organisms that are living today only have a single slice of the whole history of life on Earth to examine, as most of the species that have ever lived on Earth are now extinct. The study of evolutionary histories (phylogenies) adds a new important dimension, time, to their studies.

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BIOA01H3 F – Fall 2021

Classifying Based on Molecular Similarity Comparison of sequence differences in either nucleic acids that make up DNA or RNA or the amino acids that make up proteins can indicate evolutionary relatedness. These differences arise among species through mutation. Mutations are defined as any heritable change in the genetic material of an organism. Heritable means that the mutation is stable and is therefore passed on though cell division (mitotic or meiotic). Most mutations are spontaneous and random. They occur by chance without any assignable cause and are unconnected to the needs of an organism (they can be beneficial, detrimental, or have no observable effect). When considering how mutations might help us to infer evolutionary relationships among species, it is important to consider how mutations accumulate in species. Consider a single population of certain species that suddenly becomes two populations that are geographically isolated from one another (perhaps owing to the uprising of a mountain range). This geographic isolation prevents any contact between the two newly formed populations and therefore reproduction only occurs within each population. Over time, different mutations accumulate in each population. How genetically different these populations are from one another (the extent of genetic divergence) is determined by the amount of time that has passed. Two species’ with very few genetic differences are expected to have only recently diverged and therefore considered closely related. In contrast, species that have many genetic differences would be considered more distantly related. In this exercise you are going to draw conclusions about the relationships of humans (Homo sapiens), rhesus monkeys (Macaca mulatta), mice (Mus musculus), rats (Rattus norvegicus), domestic ducks (Anas platyrhynochos), Canada geese (Branta canadensis), Nile crocodiles (Crocodylus niloticus), and alligators (Alligator mississippiensis) by examining the amino acid sequences for the first 60 amino acids of the β (beta) chain of hemoglobin. Hemoglobin is a molecule that has been found in all kingdoms of life. In vertebrates, red blood cells contain this important molecule. Hemoglobin is a globular protein that consists of four subunits, two α (alpha) and two β (beta) subunits. Each subunit contains a heme group that contains iron and will bind oxygen. It is the hemoglobin in red blood cells that will bind oxygen and allow the red blood cells to transport oxygen to the individual cells of vertebrate animals. You may recall that amino acids are the building blocks of proteins and that proteins are encoded in the DNA of an organism. During translation, ribosomes will read a triplet codon from the mRNA transcript and the corresponding amino acid will be added to the polypeptide chain. For simplicity, The International Union of Pure and Applied Chemistry (IUPAC) has assigned a single letter code to represent all 20 amino acids (see Table 2-1). Table 2-1. Single-letter IUPAC codes for the 20 standard amino acids. A - alanine C - cysteine D - aspartic acid E - glutamic acid F - phenylalanine

G - glycine H - histidine I - isoleucine K - lysine L - leucine

M - methionine N - asparagine P - proline Q - glutamine R - arginine 3

S - serine T - threonine V - valine W - tryptophan Y - tyrosine

BIOA01H3 F – Fall 2021

Table 2-2 shows the amino acid sequence for the first 60 amino acids of the beta chain of hemoglobin for the species listed. You will notice that not all amino acids are accounted in this table. Amino acid positions that are identical for all eight species have been omitted from this table (ie. no number 7 amino acid). Table 2-2. The first 60 amino acids in the beta chain of hemoglobin that show sequence variation in eight species of vertebrates.

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BIOA01H3 F – Fall 2021

Procedure Determine the number of differences in the first 60 amino acids of the beta chain of hemoglobin between each pair of species. As you will be asked questions regarding these comparisons in the Lab 2 Assessment, please write them in Table 2-3 below for quick reference:

Table 2-3. Differences in beta chain amino acid sequences using 28 comparisons between pairs of species.

Monkey

Mouse

Rat

Duck

Human Monkey Mouse Rat Duck Goose Crocodile

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Goose

Crocodile

Alligator

BIOA01H3 F – Fall 2021

Classifying Using Cladistic Analysis Cladistic analysis is a method of determining evolutionary relatedness by analyzed shared features. Similar to the method based on molecular similarity, species with many shared homologous features are expected to have had a recent common ancestor and therefore considered closely related while species that have few if any shared homologous features would have an ancient common ancestor and would be considered more distantly related. To expand on this description of cladistics, you must first understand the following terms: Clade: a lineage that arose for one ancestor (a monophyletic lineage). Character: some observable feature of an organism. It can be morphological, molecular, developmental, physiological, behavioural, or ecological (e.g., either presence of absence of legs). Homologous: when a character in two or more taxa can be traced back to the character in their common ancestor, or where one character state in a taxon is derived from that in another (i.e., similarity resulting from common descent). Analogous: when a similar character in two or more taxa is not a result of a common ancestor but instead convergent evolution (eg. wings of an insect and wings of a bird). Ancestral (or plesiomorphic) character state: a character state that was seen in the early ancestors of an organism (e.g., for reptiles, having four legs is an ancestral state because the earliest reptiles had four legs). Derived (or apomorphic) character state: a character state that was not seen in the early ancestors of an organism (e.g., limblessness of snakes is a derived or apomorphic character state for the reptiles). A modified character state (with reference to another, ancestral, character state. Synapomorphy: a shared, homologous, derived character state (e.g., all snakes are limbless, so limblessness is a synapomorphy for snakes). Ingroup: the group (such as snakes) that is now being analyzed. Outgroup: a group that is thought to be closely related to the ancestor of the ingroup, but is not part of the ingroup (e.g., snakes share a recent common ancestor with lizards, so lizards would make a good outgroup for a phylogenetic analysis of snakes). Parsimony: the scientific preference for the simplest possible explanation that will explain some data.

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BIOA01H3 F – Fall 2021

Homoplasy (-ies): similarities in characters of different species resulting from convergent evolution and/or character state reversals, and not resulting from common descent. Convergent evolution occurs when similar characters evolve independently in two or more taxa. Character state reversals occur when a derived character reverts to an ancestral condition. Homoplasies are identified by their lack of congruence with a phylogenetic hypothesis (cladogram), and thus can only be identified after the analysis is completed. A character state is homoplasious if it occurs more than once on a tree. Node: a branching point on a phylogenetic tree or cladogram that represents a speciation event in which one ancestral taxon gave rise to two distinct descendant taxa. Branches above the node can be rotated without changing the relationship of the taxa. Branch: a line on a phylogenetic tree that connects a node to a terminal taxon. Internode: a line on a phylogenetic tree that connects two nodes. Monophyletic group: a phylogenetic lineage composed of two or more taxa including the common ancestral taxon and all descendants. A monophyletic group is also called a clade (eg. amphibians). Paraphyletic group: a group composed of the common ancestral taxon and some, but not all, descendants (eg. reptiles). Polyphyletic group: a group that does not include the common ancestor (eg. flying tetrapods). Sister group: a grouping on a phylogenetic tree where two groups are more closely related to each other than any other group. Cladogram/phylogeny/phylogenetic tree: a tree structure that represents the evolutionary relationships within a group of organisms. Although there are some subtle differences between these terms most biologists will use them interchangeably. For this lab, we will also use these terms this way. Now that you have familiarized yourself with some of the terminology, we can expand on our initial description of cladistics by saying that cladistics is the classification of an ingroup based on synapomorphic characters. If apomorphies start to accumulate once two lineages have separated, then numerous synapomorphies indicate a recent common ancestor and a high degree of relatedness. On the other hand, few synapomorphies indicate an ancient common ancestor and a low degree of relatedness.

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BIOA01H3 F – Fall 2021

Cladistic analysis involves three steps. We’ll go over these steps first, and then work through an example (Cladistic Analysis of Insects; found on page 9), which employs this strategy for resolving evolutionary relationships. 1. Thoughtful identification of homologous characters These characters must be relatively stable, heritable, must show both ancestral and derived states within the ingroup, and must be homologous or not be considered homoplasies. Homoplasy is the collective name for a series of conditions (such as convergences and reversals) that violate the assumptions of cladistic analysis. Cladistic analysis assumes that species share derived characters because they share a common ancestor that had that derived character. However, sometimes convergences occur and there are independent origins of a feature in unrelated lineages. For example, the fins of sharks, the flippers of dolphins, and the wings of penguins look alike but arose independently in the cartilaginous fishes, the mammals, and the birds. These structures are analogous, not homologous and therefore should not be chosen as characters in a cladistics analysis. Additionally, cladistic analysis assumes that if a species does not have a derived character, it is because its ancestor diverged from the lineage that developed the derived character before the derived character originated. However sometimes lineages have reversals – they have a derived character and it reverts to the ancestral type. For example, having hair is a derived character of mammals, as compared with reptiles. Dolphins have no hair because of a reversal – the mammalian ancestors of dolphins had hair and then the dolphin ancestor lost it. Hairlessness in dolphins and alligators (for example) is analogous, not homologous. These characters can be used in cladistic analysis but you must show where the reversal occurred on your cladogram. 2. Choosing an outgroup and coding the character states as either ancestral or derived in a character matrix. The outgroup is used to determine the ancestral state characters. Then any difference from the outgroup is coded as derived. Characters can be present in two states, such as present or absent (these are binary characters) or in more than two different states (multi-state characters). When constructing cladograms binary characters are coded as “0” or “1”, and multistate characters are coded as “0”, “1”, “2”, etc. It is important to understand that a code of “1”, “2”, etc., does not necessarily imply an order in which character states evolved, only that there is more than one derived state. 3. Grouping by shared derived characters to form the most parsimonious cladogram, producing a hypothesis regarding phylogenetic relationships. Parsimony is the scientific preference for explanations that are as simple as possible or involve the fewest number of evolutionary changes.

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BIOA01H3 F – Fall 2021

Solving a Cladistic Problem Example of Cladistic Analysis in Insects In this example the relationships of six groups of insects have been studied. The insects include mantids (Mantodea), true flies (Diptera), beetles (Coleoptera), cockroaches (Blattaria), and butterflies (Lepidoptera). The sixth group, silverfishes (Zygentoma), includes several common species, which you might have also seen scurrying around the floor in damp areas of your house or other buildings. The silverfishes diverged from other insects very early in insect history. We used silverfish as the outgroup. To conduct our analysis, we used five morphological characters, taken from various stages of the life cycle, and seven molecular characters.

1. Thoughtful identification of homologous characters Morphological characters We used five external characters in our analysis (Table 2-4 on next page). For each of the characters the names of the two states are given: Wings (1): The majority of insects have one or two pairs of wings; some are flightless. States: winged , wingless. Caudal Filaments (2): Some insects have several filaments (antennae-like structures) emerging from the last abdominal segment, at the posterior end of the adult; in most, there are no filaments projecting from the abdomen. States: absent , present. Metamorphosis (3): Insects either undergo incomplete metamorphosis, which consists of egg, larval, and adult stages; or complete metamorphosis, which also includes a pupal stage. States: incomplete , complete. Larval type (4): Insect larvae come in various forms: some have long legs like the adults; others are caterpillar-like, with a cylindrical body and short, stumpy legs. States: longlegged , caterpillar-like. Eggs (5): Some insects lay their eggs singly; others lay them together in a single, large egg case. States: single egg , egg case.

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BIOA01H3 F – Fall 2021

Molecular characters Table 2-5 shows a portion of the sequence of 18S rDNA from each of our insect groups. 18S rDNA is transcribed to rRNA which is used to form ribosomes in the cells of insects and of all eukaryotes (organisms whose cells have a nucleus and other membrane-bound organelles, such as animals, plants, fungi and protists.) The complete 18S rDNA sequence in insects is about 2,000 base pairs long; we looked at a portion that consists of 20 base pairs. At each base pair position, each taxon can have one of 4 character states: A (adenine), C (cytosine), G (guanine), or T (thymine) – the four different nitrogenous bases that make up DNA. Most of the base pairs in the region of 18S rDNA we used are identical in all six of the taxa, and hence do not tell us anything about the relationships of these taxa. Seven of the base pair positions do show more than one state among the six taxa. The last row of Table 5-5 shows which characters are phylogenetically informative; these characters have been re-numbered 6 through 12 in Table 2-6.

Table 2-4. Original character states for six insect taxa. Characters Taxa

Caudal filaments Metamorphosis (3) (2)

Wings (1)...