General Biology 2 Lab Manual

 Open Educational Resource

Department of Biological Sciences

 Dmitry Y. Brogun, Azure N. Faucette, Kristin Polizzotto, Farshad Tamari

This Open Educational Resource Laboratory Manual was funded in part by the OER Grant at the Kingsborough Community College - The City University of New York.

Content hosted at:  Manifold CUNY |  | https://cuny.manifoldapp.org/projects/general-biology-oer-laboratory-manual

CUNY Academic Commons | https://generalbiologyoer.commons.gc.cuny.edu/

This publication is licensed under Creative Commons Non-Commercial Share-Alike by Dmitry Y. Brogun, Azure N. Faucette, Kristin Polizzotto, Farshad Tamari

Department of Biological Sciences, Kingsborough Community College, CUNY. Farshad.Tamari@kbcc.cuny.edu , Dmitry.Brogun@kbcc.cuny.edu

Azure.Faucette@kbcc.cuny.edu, Kristin.Polizzotto@kbcc.cuny.edu

Cover Image: by Dmitry Y. Brogun (CC-BY-NC-SA) data analysis after (Letunic and Bork 2019), visualization via iTOL https://itol.embl.de.

About the Authors

Table of Contents

About the Authors        3

Lab Exercise: Evolution: Geological Time, Primate and Human Evolution, and Molecular        11

Objectives:        11

I. Geological and Evolutionary Timeline        11

A. Geological Evolutionary Timeline        11

B. Exercise        12

II. Primate and Human Derived Characteristics        14

A. Primate Derived Characteristics        14

B. Human Derived Characteristics        14

C. Skull fossil exercise        14

III. Molecular Evolution        15

A. The use of molecular data        15

B. Molecular evolution exercise        16

Lab Report: Evolution: Geological Time, Primate and Human Evolution, and Molecular        17

I. Geological and Evolutionary Timeline        17

B. Exercise        17

II. Primate and Human Derived Characteristics        18

C. Skull fossil exercise        18

III. Molecular Evolution        19

B. Molecular evolution exercise        19

Lab Exercise: Evidence of Evolution And Population Genetics        20

Objectives:        20

I. Hardy-Weinberg Theorem        20

A. Introduction        20

B. Some definitions        21

C. Exercise 1: The Effect of Natural Selection        22

D. Exercise 2: The Effect of Genetic Drift        25

II. Types of Selection        26

Lab Report: Evidence of Evolution And Population Genetics        29

I. Hardy-Weinberg Theorem        29

C. Exercise 1: The Effect of Natural Selection        29

D. Exercise 2: The Effect of Genetic Drift        30

II. Types of Selection        30

Lab Exercise: Phylogenetics        31

Objectives:        31

I. Building the Phylogenetic Tree        31

A. Introduction        31

C. Understanding phylogenies        32

B. Definitions        33

Exercise 1. Reconstructing primate evolution        37

Exercise 2. Building the Phylogenetic Tree        38

II. Types of Speciation        40

Lab Report: Phylogenetics        42

Exercise 1.        42

Exercise 2.        43

Building the Phylogenetic Tree        43

II. Types of Speciation        45

Lab Exercise: Bacteria        46

Objectives:        46

I.        Three-Domain System        46

II. Overview of Bacteria        47

A. Introduction        47

B. Some definitions        48

C. Photosynthetic Bacteria        48

III.        Exercises        49

Exercise 1: Prokaryotic survey 1        49

Exercise 2: Prokaryotic survey 2        49

Exercise 3. Bacteria verses conditions.        53

Exercise 4: Adopt a Bacterium        54

Lab Report: Bacteria        56

Exercises        56

Exercise 1: Prokaryotic survey 1        56

Exercise 2: Prokaryotic survey 2        57

Exercise 3. Bacteria verses conditions.        61

Exercise 4: Adopt a Bacterium        61

Lab Exercise: Protista        63

Objectives:        63

I.        Eukaryotic Survey        63

A.        Introduction: Protists - Who are they?        63

B.        Some key definitions        64

C.        Classification and Phylogeny of Protists - Four Eukaryotic Supergroups        65

II.        Four Eukaryotic Supergroups- Excavata,”SAR” clade, Archaeplastida, and Unikonta        65

A.        Supergroup Excavata        65

B.        Supergroup Archaeplastida        69

C.        Supergroup Unikonta        70

III.        Exercises        71

1. Eukaryote phylogeny        71

2. Classifying protists        71

3. Conclusion        72

Lab Report: Protista        73

IV.        Exercises        73

1. Eukaryote phylogeny        73

2. Classifying protists        74

3. Conclusion        75

Lab Exercise: Fungi        76

Objectives:        76

I. Fungal Classification and Phylogeny        76

A. Introduction        76

B. Some definitions        76

C. Classification/Phylogeny of Fungi        77

D. Life cycle of Fungi        78

II. Exercises: Fungal Diversity        79

A. Zygomycota        79

B. Ascomycota        81

C. Basidiomycota        83

D. Imperfect Fungi (Deuteromycota)        85

E. Mutualistic Fungi        85

Lab Exercise: Fungi        89

II. Exercises: Fungal Diversity        89

A. Zygomycota        89

B. Ascomycota        89

C. Basidiomycota        89

D. Imperfect Fungi (Deuteromycota)        90

E. Mutualistic Fungi        90

Lab Exercise: Nonvascular Plants and Seedless Vascular Plants        91

Objectives:        91

I. Kingdom Plantae: Origin and Phylogeny        91

A. Introduction: Evolution, Classification, and Life Cycle        91

B. Some key definitions        93

C. Exercises        93

II. Nonvascular Plants        95

III. Seedless Vascular Plants        97

Lab report: Nonvascular Plants and Seedless Vascular Plants        100

I. Kingdom Plantae: Origin and Phylogeny        100

A. Introduction: Evolution, Classification, and Life Cycle        100

C. Exercises        100

II. Nonvascular Plants        102

III. Seedless Vascular Plants        103

Lab Exercise: Seed Plants        105

Objectives:        105

I. Introduction        105

A. Alternation of generations        105

B. Some definitions        106

C. Classification- Gymnosperms and Angiosperms        106

II. Exercises        109

A. Gymnosperms        109

B. Angiosperms        112

Lab Report: Seed Plants        118

I. Introduction        118

C. Classification- Gymnosperms and Angiosperms        118

II. Exercises        118

A. Gymnosperms        118

B. Angiosperms        119

Lab Exercise: Animals I - Invertebrates        122

Objectives:        122

Introduction        122

Background        122

Some definitions        123

Classification        123

Invertebrates        127

Superphylum Lophotrochozoa        131

Superphylum Ecdysozoa        137

Superphylum Deuterostomia        141

Lab Report: Animals I – Invertebrates        143

Objectives:        143

I. Introduction        143

II. Invertebrates        144

A. Phylum: Porifera        144

B. Phylum: Cnidaria        146

Superphylum Lophotrochozoa        147

Superphylum Ecdysozoa        151

Superphylum Deuterostomia        153

Lab Exercise: Animals II - Vertebrates        155

Objectives:        155

I. Vertebrates        155

A.        Introduction        155

II. Vertebrate survey        157

i.        Overview and Hypothesis        157

Definitions        158

Clade: Myxini        158

Clade: Petromyzontida        160

Clade: Osteichthyes (Actinopterygii)        162

Clade: Amphibia        163

Clade: Reptilia        169

Clade: Aves        170

Clade: Mammalia        171

Lab Report: Animals II - Vertebrates        174

II. Vertebrate Survey        175

C. Clade: Myxini        176

D. Clade: Petromyzontida        176

D. Clade: Chondrichthyes        176

E. Clade: Osteichthyes (Actinopterygii)        177

F. Clade: Amphibia        177

G. Clade: Reptilia        182

H. Clade: Aves        183

I. Clade: Mammalia        183

Lab Exercise: Ecology I        185

Objectives:        185

I. Biomes        185

A. Introduction        185

B. Some key definitions        186

C. Activity        186

II. Population Growth        187

A. Introduction        187

B. Some key definitions        187

C. Activity        187

III. Predator-Prey Dynamics        188

A. Introduction        188

B. Some key definitions        188

C. Activity        188

Lab report: Ecology I        190

I. Biomes        190

C. Activity        190

II. Population Growth        192

C. Activity        192

III. Predator-Prey Dynamics        194

C. Activity        194

Lab Exercise: Ecology 2        196

Objectives:        196

I. Ecological Niche and the Effects of Competition        196

A. Introduction        196

B. Some key definitions        196

C. Activity        197

II. Keystone Species and Trophic Cascades        197

A. Introduction        198

B. Some key definitions        198

C. Activity        198

Lab Report: Ecology 2        200

I. Ecological Niche and the Effects of Competition        200

C. Activity        200

II. Keystone Species and Trophic Cascades        200

C. Activity        200

Lab Exercise: Evolution, Geological Time, Primate and Human Evolution, and Molecular

Farshad Tamari, Ph.D.

Figures and text are intended for OER

Objectives:

• Identify major geological and evolutionary events
• Create a scaled timeline of major evolutionary events and indicate the approximate date of each
• Calculate the proportion of earth’s history for which various groups of organisms have existed
• List derived characteristics of primates and humans
• Distinguish between primitive and advanced characteristics in primate facial and skull bones
• Analyze evolutionary relationships using molecular (DNA) evidence

I. Geological and Evolutionary Timeline

A. Geological Evolutionary Timeline

Earth is approximately 4600 million years old (equivalent to 4.6 billion years old). The major events of the evolution of life on earth are summarized in Table 1, and together with the exercise that follows are adopted (with modification), from Barrow 2016. For the full article entitled Picturing Evolution through Geologic Time click here.

Table 1: Major events throughout Earth’s history. Prepared by F. Tamari, adopted from Barrow 2016.

B. Exercise

Using string (thread) and a tape measure or a long ruler, measure a piece of string 4.6 meters long. Lay it flat on a surface. This represents a timeline of the Earth’s 4.6-billion-year history.

Calculate what each unit of the ruler represents in a number of years. For example:

If 4.6 m = 4.6 billion years, then 1 m = 1 billion years.

1 m = 1 billion years

1 cm = ________ years

1 mm = ________ years

Using masking tape, place a thin piece of tape on the string at the appropriate distance to indicate each evolutionary event from Table 1 on the string. On the tape indicate the date and the event.  Do this until you have marked all the events on the string. Please note that in order to mark the events to scale as required, you will need to use the conversion factors you calculated in the previous step. For example, if you are marking the origin of the prokaryotes to scale, you need to know how far 3400 million years would be from 4600 million years (the beginning of your timeline). You can solve it using algebra, like this:

3400 million      =     X

4600 million            4.6 m

You can solve for X algebraically as follows:

X = (3400 million * 4.6 m) / 4600 million

The units “million” cancel, and your final answer is in meters (m).

This tells you how many meters to measure from 0 (the end of your timeline, or the present day). That is the point where you will place the event “prokaryotes.” Calculate the other events in Table 1 in a similar manner.

Answer the following questions:

1. What patterns if any did you notice in the timeline? In other words, what did you notice or what surprised or interested you about the spacing of the events?

2. What proportion (in %) of Earth’s life history does each even represent?   Complete your work in table 2 below and show your calculations.

Table 2: Major events throughout Earth’s history exercise

II. Primate and Human Derived Characteristics

A. Primate Derived Characteristics 

Primates are a group of mammals that include all monkeys and apes. The apes include gorillas, orangutans, chimpanzees, and humans.

The following are five derived characteristics of all primates:

A. Hands and feet for grasping

B. Large brain short jaws

C. Forward-looking eyes

D. Complex social behavior and parental care

E. Opposable thumbs (monkeys and apes)

B. Human Derived Characteristics

The following are five derived characteristics of humans:

A. Upright posture

B. Bipedal locomotion (on two legs)

C. Large brain

D. Language

E. Symbolic thought

An indication of the larger brain size is evidence found from cranial bones in skull fossils of different taxa. Here are some representatives:

Figure 1: Cranial and facial bone replicas. Photos by D. Brogun, panel by F. Tamari.

C. Skull fossil exercise

Watch this short video by:

 American Museum of Natural History (https://www.youtube.com/watch?v=DZv8VyIQ7YU).

1. It is estimated that the chimpanzees diverged from the lineage that gave rise to the human lineage about 6 mya (including Australopithecines such as Lucy and later, other Homo species such as Homo neaderthalensis, the Neanderthals).  Modern humans (Homo sapiens) evolved approximately 200,000 years ago (0.2 mya).  What patterns do you notice with respect to the following cranial/facial features as we move from primitive (left) to advanced (right)?

1. Jaw length:
2. Cranium size:
3. Bridge of the nose:
4. Eyebrow ridge:
5. Teeth number and specialization:

III. Molecular Evolution

A. The use of molecular data

Molecular data can be used to delineate relationships among organisms. The more DNA identity (similarity) the more closely two species are (the more recent their ancestry and point of divergence). The more DNA difference (dissimilarity) the more distantly related two organisms are considered to be. It is, therefore, valuating to calculate how similar or dissimilar conserved sequences of DNA are.

For example, look at the following two sequences (N2 and N1), representing short DNA sequences belonging to two different species.  They have been aligned using bioinformatics software.  Consider the first 100 nucleotides.  How many of the nucleotides are identical between species N2 and N1?

Using the same software, the differences can be highlighted.  Here’s a snapshot of this simple analysis.

Figure 2: DNA alignment of two simulated DNA from two hypothetical species, N1 and N2. Alignment and figure by F. Tamari

Figure 3: DNA alignment of two simulated DNA from two hypothetical species, N1 and N2. Alignment and figure by F. Tamari

For the first 100 nucleotides:

 % difference        = {(100%)* (# changed nucleotides)}/total number of nucleotides considered

                =100%*3/100

                = 3%

% identity        = {(100%)* (# unchanged nucleotides)}/total number of nucleotides considered

                =100%*97/100

                = 97%

B. Molecular evolution exercise

1. Consider the following alignment for two species, N3 and N4.

Figure 4: DNA alignment of two simulated DNA from two hypothetical species, N3 and N4. Alignment and figure by F. Tamari.

Calculate both % difference and % identity. Show all your work.

% difference =

 % identity =

Lab Report: Evolution: Geological Time, Primate and Human Evolution, and Molecular

Farshad Tamari, Ph.D.

Figures and text are intended for OER

Click here to access a downloadable version of the lab report.

I. Geological and Evolutionary Timeline

B. Exercise

Using string (thread) and a tape measure or a long ruler, measure a piece of string 4.6 meters long. Lay it flat on a surface. This represents a timeline of the Earth’s 4.6 billion-year history.

Calculate what each unit of the ruler represents in a number of years. For example:

If 4.6 m = 4.6 billion years, then 1 m = 1 billion years.

1 m = 1 billion years

1 cm = ________ years

1 mm = ________ years

Now calculate the placement of each event in meters. Use the formula given in the lab instructions and record your answers in the table below.

Table 1: Major events throughout Earth’s history. Prepared by F. Tamari, adopted from Barrow 2016.

Answer the following questions:

1. What patterns if any did you notice in the timeline? In other words, what did you notice or what surprised or interested you about the spacing of the events?

2. What proportion (in %) of Earth’s life history does each even represent?   Complete your work in table 2 below and show your calculations.

Table 2: Major events throughout Earth’s history exercise.

II. Primate and Human Derived Characteristics

C. Skull fossil exercise

Watch these short videos by:

 Chris Stringer (https://www.cshl.edu/dnalcmedia/sorting-out-human-evolution-chris-stringer/) and the

 American Museum of Natural History (https://www.youtube.com/watch?v=DZv8VyIQ7YU).

1. It is estimated that the chimpanzees diverged from the lineage that gave rise to the human lineage about 6 mya (including Australopithecines such as Lucy and later, other Homo species such as Homo neaderthalensis, the Neanderthals).  Modern humans (Homo sapiens) evolved approximately 200,000 years ago (0.2 mya).  What patterns do you notice with respect to the following cranial/facial features as we move from primitive (left) to advanced (right)?

6. Jaw length:
7. Cranium size:
8. Bridge of the nose:
9. Eyebrow ridge:
10. Teeth number and specialization:

III. Molecular Evolution

B. Molecular evolution exercise

1. Consider the following alignment for two species, N3 and N4.

Figure 4: DNA alignment of two simulated DNA from two hypothetical species, N3 and N4. Alignment and figure by F. Tamari.

Calculate both % difference and % identity. Show all your work.

% difference =

 % identity =    

Lab Exercise: Evidence of Evolution and

Population Genetics

Farshad Tamari, Ph.D.

Figures and text are intended for OER

Objectives:

I. Hardy-Weinberg Theorem

• Describe evidence of evolution that is based on microevolution and population genetics
• Define and use the terminology of population genetics correctly
• Use the equations of the Hardy-Weinberg equilibrium to calculate allele and genotype frequencies
• Graph allele frequencies using Microsoft Excel and identify changes in allele frequencies
• Draw graphs and explain three types of selection

A. Introduction

Direct evidence of a population that is evolving is a change in allele frequencies.  The Hardy-Weinberg Theorem is one way to determine whether allele or genotype frequencies are changing from generation to generation. It states that under the conditions listed below, alleles and genotype frequencies do not change from generation to generation. As you know, most of the conditions (assumptions) are not met in real populations, and therefore, populations evolve.

Changes in allele frequencies can be calculated based on the following equations:

p + q = 1

    p = frequency of dominant alleles in a population

    q = frequency of recessive alleles in a population

Squaring both sides (p + q)2 = 12 will yield:

p2 + 2pq + q2 = 1        

p2 = frequency of homozygous dominant genotypes (individuals) in a population

q2 = frequency of homozygous recessive genotypes (individuals) in a population

2pq = frequency of heterozygous genotypes (individuals) in a population

The conditions (assumptions) of the Hardy-Weinberg Equilibrium are:

No mutations

Random mating

No natural selection

Extremely large population size

No gene flow

No genetic drift

B. Some definitions

C. Exercise 1: The Effect of Natural Selection

Helpful Videos:         Hardy-Weinberg problems        https://www.youtube.com/watch?v=xPkOAnK20kw

                        How to graph in Excel        https://www.youtube.com/watch?v=Xn7Sd5Uu42A

1. At-home activity: Selection of prey by a predator

Print the figure below, or draw forty small circles on a sheet of paper. Fill in 10 of the circles with black ink, fill 20 with a pencil (or a different color), and leave 10 of the circles white.  These circles represent the prey species, and their colors represent variations (such as the ability to hide or to run fast) that may help them avoid being hunted and eaten by a predator. There are two alleles for the color gene, white (p) and black (q). The gray individuals are heterozygous.

Now, you will act as the predator and “hunt” the circles by drawing an X through 3 white circles, 2 gray circles, and 1 black circle. Begin by setting 30 seconds on the timer on your phone, and then start the countdown and your hunt. After each X that you draw, count to 10 in your head (about 3 seconds) before drawing the next X. If you have crossed out 3 white, 2 gray, and 1 black and you still have time left on the clock, repeat the same pattern until your 30 seconds is up. But remember to leave a little time (3 seconds or so) between each kill, or you will run out of prey before the 30 seconds is up.

The number of each color that you hunt (3 white, 2 gray, 1 black) represents the advantage that each prey variation has. You can find and eat more white than black because white is less well adapted than black. Based on the instructions given here, which color do you think is best adapted to avoid predation? Write a hypothesis about what will happen to the frequency of each allele over time.

When 30 seconds are up, see how many survivors are left (circles not crossed out are survivors). Use the number of survivors to calculate p and q as instructed below. Once you have p and q, you will calculate p2, 2pq, and q2 and use those numbers to predict how many circles of each color there will be in the next generation, as instructed below.

Once you have the new generation, repeat the hunting and the calculations in the same manner (step-by-step instructions are below). You will do this 3 times for 3 generations in total, and then you will graph your results in Excel. KCC students have free access to a web version of Excel. Click here for details.

2. Calculations

In generation 1 (represented by the figure above), the allele and genotype frequencies are as follows:

CWCW = Homozygous (white)

CWCB =  Heterozygous (gray)

CBCB = Homozygous (black)

We will designate the white allele CW as p, and the black allele CB as q.  There are 40 individuals in the original population, which means there are 80 alleles total since the individuals are diploid.

10 white circles                20 gray circles                10 black circles

                p = [(2 x 10) + 20]/80 = 0.5

p + q =1, therefore, q = 0.5

If you are not sure why p and q are calculated in this way, please review the videos, the reading, or the PowerPoint for this week before continuing.

After the hunt, imagine that the following numbers of individuals survive. Record the number of survivors for generation 1 in the table in your lab report. Your actual number of survivors may be different from those below, but this example will show you how to do the calculations to figure out the proportions of colored circles for generation 2.

4 white circles                14 gray circles                8 black circles

Now we must recalculate new p and q values (see below). Remember that the white allele is p and the black allele is q.

                p = [(2 x 4) + 14]/52 = 0.423, and q = 1- 0.42 = 0.577

p2 = (0.423)2 = 0.18 (round to two decimal places)

q2= (0.577)2 = 0.33 (round to two decimal places)

2pq = 2 * 0.423 * 0.577 = 0.49 (round to two decimal places)

Now we construct a new population with 50 individuals using the above p2, 2pq, and q2 values. You will need to round up or down to the nearest whole number if the formula gives you a number such as 16.5.

9 white circles                           24 gray circles                        17 black circles

(50 x p2) = 9                (50 x 2pq) = 24 (round down)                (50 x q2) = 17 (round up)

You will calculate p, q, p2, 2pq, and q2 using the number of survivors in your own trial. Record the values in the table in your lab report.

Now draw generation 2 with the predicted values and do the hunt again. From the example above, we would use 9, 24, and 17 and draw the circles as shown below. Remember, you should use the numbers you calculated from your own hunt rather than these example numbers.

Now hunt again, as you did before, and record the number of survivors in your lab report. Then calculate new allele and genotype frequencies.

Based on the new allele and genotype frequencies you calculated, construct a new population (generation 3) of 50 individuals.  If you have time, repeat the experiment two more times for a total of 5 generations. It may be difficult to see a trend and draw any conclusions about your hypothesis with only 3 generations.

Using Microsoft Excel, plot allele frequencies (p and q) and copy and paste your graph into the lab report.  Your graph may look similar to the graph below.  In 3 or 4 sentences in your lab report, describe and interpret your graph. Do the results support your hypothesis?

D. Exercise 2: The Effect of Genetic Drift

Construct an original population as you did above using 40 circles (10 white, 20 gray, and 10 black) on a piece of paper.

Calculate the allele frequencies (p and q) for the original population as you did before.  They should be the same as before in generation 1.

Now, cut off a portion of the population due to genetic drift (bottleneck or founders effect). See the figure below as an example, but you can place the line anywhere you wish.  The line represents some kind of natural disaster that wipes out part of the population, and only the portion of the population on the right survives. Are the changes in allele frequencies related to the color of the circles, or not? Write a hypothesis in your lab report about the effect of the natural disaster on the frequencies of the black and white alleles. Calculate new allele frequencies for the survivors.  Are the new allele frequencies similar to those from the original population or do they differ significantly? In your lab report, predict how the allele frequencies would differ depending on where the line was placed. Is your hypothesis supported? How is this different from what happened in Exercise 1 above?

II. Types of Selection

Variation in a population is important for evolution.  Natural selection works on individuals’ phenotypes. In general, if there is selection, it may select for or against particular phenotypes. The figures below depict three possible scenarios. In your lab report, indicate which type of selection was at work in the activity you did for exercise 1.

1. Directional selection: A particular phenotype at an extreme end is being selected for, shifting the resulting population with respect to that particular phenotype to the right (below) or left (also possible).

2. Stabilizing selection: A particular intermediate phenotype is being selected for, resulting in a population with less frequency in extreme phenotypes and more of the intermediate phenotypes.

3. Disruptive selection: Phenotypes at the extremes are being selected for, and those that are intermediate are selected against; resulting in a population with more frequency in extreme phenotypes and less of the intermediate phenotypes.

Lab Report: Evidence of Evolution and Population Genetics

Farshad Tamari, Ph.D.

Figures and text are intended for OER

Click here to access a downloadable version of the lab report.

I. Hardy-Weinberg Theorem

C. Exercise 1: The Effect of Natural Selection

1. Write a hypothesis about what will happen to the frequency of each allele (black and white) over time.

2. Fill in the values for each generation.

*No data needed here, since we will not do the 6th generation

3. Copy and paste your graph here:

4. In 3 or 4 sentences, describe and interpret your graph. Do the results support your hypothesis?

D. Exercise 2: The Effect of Genetic Drift

1. Write a hypothesis about the effect of the natural disaster on the frequencies of the black and white alleles.1

2. Calculate new allele frequencies for the survivors.  

3. Are the new allele frequencies similar to those from the original population or do they differ significantly?

4. In 1-2 sentences, predict how the allele frequencies would differ depending on where the line was placed.

5. Was your hypothesis supported? How is this different from what happened in Exercise 1 above?

II. Types of Selection

After studying the graphs in your lab depicting directional, stabilizing, and disruptive selection, consider the results of exercise 1. In that exercise, was the selection directional, stabilizing, or disruptive? Explain in 1-2 sentences how you know.

Lab Exercise: Phylogenetics

Dmitry Y. Brogun, Ph.D.

* Figures and text are intended for OER

Objectives:

• Explain how the following evidence is used for phylogenetic reconstruction: the fossil record, DNA, and biogeography.
• Describe specific examples of phylogenetic reconstruction, such as the relationship of humans to other primates.
• Build and analyze a phylogenetic tree, identifying patterns of shared ancestry.
• Differentiate between the allopatric and sympatric modes of speciation.

I. Building the Phylogenetic Tree

A. Introduction

Taxonomy is the classification of organisms. Phylogenies represent the foundation in classifying organisms based on their evolutionary relationships. The phylogenetic tree, depicted on the next page in Figure 1, represents patterns of ancestry - an evolutionary relatedness. In order to reconstruct these relationships, biologists must collect and analyze evidence, which they use to form a hypothesis about how the organisms are related — a phylogeny. In laboratory one, you analyzed the fossil records as evidence to support your hypothesis. In this laboratory, you will be analyzing the DNA sequences to reconstruct human evolution.

To build a phylogenetic tree such as the one on the next page in Figure 1, biologists collect data about the characters of each organism they are interested in. (For the moment, don’t worry about trying to read the names of the organisms on each branch.) Characters are heritable traits that can be compared across organisms, such as physical characteristics (morphology), genetic sequences (DNA, RNA, and proteins), and behavioral traits. Biogeography (the distribution of organisms) can also be used as evidence in reconstructing phylogeny.

C. Understanding phylogenies

In this exercise, you will learn the terminology that phylogeneticists use when constructing or analyzing phylogenetic trees. It may help to watch this video first (strongly recommended):

Reading Phylogenetic Trees (13-minute video)

https://www.youtube.com/watch?v=wCRRhZ7rO7k

B. Definitions

Then, study Table 1 below to familiarize yourself with the scientific names of the organisms we will use in our sample tree.

Table 1.  Organisms used in this exercise. Prepared by D. Brogun.

Next, examine each image below, reading the explanations as you go. Figure 2 shows you the phylogenetic tree for the organisms in Table 1. Note that the left represents ancestors in the past, with the present-day species represented on the right. The length of each branch is proportional to the amount of time since the origin of each species from its common ancestor with the sister taxon next to it on the tree.

To start, we need to answer the question, what is a phylogenetic tree? First, for the evolutionary biologists, the phylogenetic tree represents a hypothesis that they use to test their predictions while reconstructing the ancestral relatedness.  Second, indeed, it is a geometrical structure that often uses complicated mathematical algorithms to decipher the actual patterns of evolutionary lineages.  With that in mind let’s dive into the phylogenetic tree terminology.   Like a living tree, a phylogenetic tree consists of branches that are connected to nodes. The external nodes, also called Operational Taxonomic Units (OTUs) can either represent organisms as depicted in figure 2 (confide by a larger red rectangle) or DNA and protein sequences.

 The internal nodes represent the ancestral lineages, and at the point, they join tree branches signifying speciation, depicted in figure 3 (confide by a black rectangle). Speciation can give rise to two or more daughter lineages, discussed below and figure 6.

Phylogenetic trees allow researchers to model common and unique evolutionary relationships between lineages. Since each lineage has the evolutionary changes that are specific to it alone and parts that are shared with other lineages.

In fact, each lineage has ancestors that are unique to that lineage and ancestors that are shared with other lineages through common ancestry.

A monophyletic group that includes all the descendants (living and extinct) of a common ancestry forms a clade. It is relatively easy to tell by just looking at the phylogenetic tree whether a monophyletic group of lineages forms a clade. You can ask yourself, what if I remove a single branch of the phylogenetic tree — will all of the organisms on that removed branch make up a clade? Thus, a monophyletic group includes all of the ancestors, whereas a non-monophyletic group, sometimes referred to as polyphyletic excludes some of them. Hint: follow the green-shaded and red-shaded rectangles in figure 6 below.

When reconstructing the evolutionary relatedness among organisms it is important to acknowledge the phylogenetic tree patterns of lineage branching might tell us the true phylogeny of the organisms being considered. Many of the phylogenies you encounter are groups of closely related species, but we can also use phylogeny to depict the relationships between all life forms.

Finally, there are many ways to depict the phylogenetic tree without changing its meaning. It is important to keep in mind that the tree branches can be rotated, and the choice of which clade goes to the right and which goes to the left does not change the relationships of the terminal nodes. Do the following phylogenetic trees below are equivalent?

Phylogeneticists often put the clade they are most interested in (whether that is Humans, chickens, or bacteria) on the right side of the phylogeny. You can read more about phylogenetic trees by following this link https://courses.lumenlearning.com/wm-biology2/chapter/phylogenetic-trees-2.

Exercise 1. Reconstructing primate evolution

Hypothesis: All primates share a common ancestry

The points described above can cause problems when it comes to human evolution because many of the related primate species are extinct (so-called “missing links” that make it more difficult to determine who our closest relatives are). The phylogeny of living species most closely related to us looks like this:

In your lab report, identify and label all parts of the phylogenetic tree depicted above (hint: check the definitions table in the introduction if you need help).

1. Root

2. Internal and External nodes

3. OTUs

4.  Redraw the tree to show the unique phylogeny of the Gorilla lineage with a dotted line. In other words, use a dotted line (or a different color, whatever you like) to indicate the shared lineage of the gorillas and the unique lineage of the gorillas (see Figure 4 for an example).

5. Do the Bonobos, Chimpanzees, and Humans form a clade? Is it monophyletic? Explain how you know.

Exercise 2. Building the Phylogenetic Tree

In this exercise, you will practice building your own phylogenetic tree. First, you will manually align DNA sequences from five primates, listed in Table 3 on p. 9. To do this, you will draw vertical lines between matching nucleotides and leave blank spaces between non-matching ones (as shown in Figure 9 on the next page). Your ultimate goal is to construct a phylogenetic tree based on your manual alignments.

You will use the human DNA sequence as your reference sequence during your experiment—meaning that all other sequences will be compared to the human sequence. On the next page, you will find an example of a manual alignment between Human and Bonobo nucleotide sequences (Figure 9).  These sequences are clearly similar yet are not identical. Your task is to identify which regions of the two sequences are homologous.

In the example below the two sequences are different at the sixth, seventh and eleventh positions. But with only 4 letters in the nucleotide code, you can imagine that it might be possible to align similar regions of two sequences in more than one way. More issues can arise the less related the two sequences are—if more time has passed since the common ancestor, then more mutations have probably accumulated, including deletions or insertions of larger sections of DNA that will affect alignment.  So, we need a way to determine which alignment is the most likely. We do this by calculating the “cost” of a particular alignment. The lower the “cost” of the alignment, the more likely it is that your alignment is correct.  

Using the formula D = s + wg, where D is the cost of alignment, s is the number of substitutions (the number of differences in the two sequences), g is the total length of any gaps, and w is the gap penalty, we can calculate the cost.  

Thus, the ‘cost’ of the alignment shown below is D = 3. This number was calculated based on the alignment analysis that there are no gaps, thus g = 0, and the gap penalty w = 1 (this will be a constant in our example since we will not be aligning any sequences with gaps).  There are only three nucleotide substitutions, thus s = 3.  So, when we plugged the numbers into the formula to calculate the cost of the alignment, D = 3 + 1 * 0, thus D = 3.   You need to repeat these calculations and manual alignments for the remaining primate species listed in Table 3 (on p. 9).  

After you have completed your manual alignments and calculated the costs, you must reconstruct the primate evolution in a phylogenetic tree in your lab report.  You will draw your nodes and connect your tree branches and list the OTUs.  Please, keep in mind that in your phylogenetic experiment the OTUs with the lowest alignment costs will be joined by the adjacent branches, i.e. forming a clade, as depicted below in figure 10. Will your phylogenetic trees resemble the phylogeny depicted in figure 8?

Below you will find a table summarizing the primate species and the DNA sequences that you will need to analyze during your phylogenetic experiment.

Table 3.  List of primates and the DNA sequences. Prepared by D. Brogun.

It is important to remember that:

1. Humans did not evolve from chimpanzees. Humans and chimpanzees are evolutionary cousins and share a recent common ancestor that was neither chimpanzee nor human.
2. Humans are not "higher" or "more evolved" than other living lineages. Since our lineages split, humans and chimpanzees have each evolved traits unique to their own lineages.

II. Types of Speciation

Biologists must answer the ultimate question in evolution - how a continuous process of evolutionary change can produce the discrete entities we recognize as different species? Could it be that specific gene changes are involved in reproductive isolation?   Below we describe the two types of population genetics of speciation. The figures below depict two possible scenarios:

A. Sympatric speciation:  During this speciation reproductive isolation takes place within a single population without geographical isolation. For example, an mtDNA phylogenetic tree of the cichlid fish species shows that they are monophyletic, suggesting that Lake 1 was colonized by the fish once, thus the speciation occurs without geographic isolation, phylogenetic tree branches depicted in red (Figure 11A).

   Lab Report: Phylogenetics

Dmitry Y. Brogun, Ph.D.

* Figures and text are intended for OER

Click here to access a downloadable version of the lab report.

Exercise 1.

Reading the phylogenetic tree.

Identify and label all parts of the phylogenetic tree depicted above (hint: check the definitions table in the introduction if you need help).

1. Root

2. Internal and External nodes

3. OTUs

4.  Redraw the tree to show the unique phylogeny of the Gorilla lineage with a dotted line. In other words, use a dotted line (or a different color, whatever you like) to indicate the shared lineage of the gorillas and the unique lineage of the gorillas (see Figure 4 for an example).

5. Do the Bonobos, Chimpanzees, and Human form a clade? Is it monophyletic?

Explain how you know.

Exercise 2.

Building the Phylogenetic Tree

In this exercise, you will practice building your own phylogenetic tree. First, you will manually align DNA sequences from five primates, listed in Table 3 on p. 9. To do this, you will draw vertical lines between matching nucleotides and leave blank spaces between non-matching ones (as shown in Figure 9 on the next page). Your ultimate goal is to construct a phylogenetic tree based on your manual alignments.

You will use the human DNA sequence as your reference sequence during your experiment—meaning that all other sequences will be compared to the human sequence. On the next page, you will find an example of a manual alignment between Human and Bonobo nucleotide sequences (Figure 9).  These sequences are clearly similar yet are not identical. Your task is to identify which regions of the two sequences are homologous.

In the example below the two sequences are different at the sixth, seventh and eleventh positions. But with only 4 letters in the nucleotide code, you can imagine that it might be possible to align similar regions of two sequences in more than one way. More issues can arise the less related the two sequences are—if more time has passed since the common ancestor, then more mutations have probably accumulated, including deletions or insertions of larger sections of DNA that will affect alignment.  So, we need a way to determine which alignment is the most likely. We do this by calculating the “cost” of a particular alignment. The lower the “cost” of the alignment, the more likely it is that your alignment is correct.  

Using the formula D = s + wg, where D is the cost of alignment, s is the number of substitutions (the number of differences in the two sequences), g is the total length of any gaps, and w is the gap penalty, we can calculate the cost.  

Thus, the ‘cost’ of the alignment shown below is D = 3. This number was calculated based on the alignment analysis that there are no gaps, thus g = 0, and the gap penalty w = 1 (this will be a constant in our example since we will not be aligning any sequences with gaps).  There are only three nucleotide substitutions, thus s = 3.  So, when we plugged the numbers into the formula to calculate the cost of the alignment, D = 3 + 1 * 0, thus D = 3.   You need to repeat these calculations and manual alignments for the remaining primate species listed in Table 3 (on p. 9).  

After you have completed your manual alignments and calculated the costs, you must reconstruct the primate evolution in a phylogenetic tree in your lab report.  You will draw your nodes and connect your tree branches and list the OTUs.  Please, keep in mind that in your phylogenetic experiment the OTUs with the lowest alignment costs will be joined by the adjacent branches, i.e. forming a clade, as depicted below in figure 10. Will your phylogenetic trees resemble the phylogeny depicted in figure 8?

Below you will find a table summarizing the primate species and the DNA sequences that you will need to analyze during your phylogenetic experiment.

Table 3.  List of primates and the DNA sequences. Prepared by D. Brogun.

II. Types of Speciation

Please, provide one example for each question below.

1. Biologists must answer the ultimate question in evolution - how a continuous process of evolutionary change can produce the discrete entities we recognize as different species?

2. Could it be that specific gene changes are involved in reproductive isolation?  

Lab Exercise: Bacteria

Kristin Polizzotto Ph.D., Dmitry Y. Brogun Ph.D., Farshad Tamari Ph.D.

* Figures and text are intended for OER

Objectives:

• Identify and define common bacterial shapes and features, including: cocci, bacilli, spirilli, pili, capsule, spore, fimbriae, flagella, plasmid, Gram-positive cell wall, Gram-negative cell wall.
• Describe various bacterial metabolic processes, including: photosynthesis, chemosynthesis, methanogenesis, nitrogen fixation.
• Identify and describe at least three vital roles that bacteria play in their ecosystems, such as primary production, decomposition, nitrogen fixation, and disease.

1. Three-Domain System

In a previous lab exercise, you learned about phylogenetics and the classification of organisms. In this laboratory, we will dive deeper into the phylogenetic tree, depicted below in figure 1, and learn about some of the organisms from the three Domains of Life. In 1760, Carolus Linnaeus grouped all living organisms into plants (non-motile) and animals (motile). It took almost two centuries to develop the more appropriate five-kingdom classification proposed by Whittaker and Lynn Margulis in 1968. The kingdoms proposed were Monera (unicellular prokaryotes including bacteria and cyanobacteria), Protista (unicellular eukaryotes including protozoa and unicellular algae, will be discussed in a future lab), Fungi (saprotrophs, will be discussed in a future lab), Plantae (autotrophs, will be discussed in future labs), and Animalia (heterotrophs, will be discussed in future labs). With modern DNA-based technology such as PCR and rDNA (ribosomal DNA) sequence analyses the living world is currently classified as a three-domain system with Bacteria, Archaea, and Eukarya; described by Carl Woese in 1990.

II. Overview of Bacteria

A. Introduction

The bacteria comprise one of the three domains of life and were among the first life on earth. In this lab activity, you will learn more about this fascinating group, without which no other life on earth would likely exist.

Before proceeding, you need to review the major concepts from this week relating to bacteria. You should watch this video, an entertaining and informative (7 min 31 sec) video from the Amoeba Sisters, and read this short article. These two resources will give you an excellent overview of the concepts you will need for this lab activity. You may also find the definitions below useful.

B. Some definitions

C. Photosynthetic Bacteria

Cyanobacteria have their ecological importance in the global carbon, oxygen, and nitrogen cycles, as well as their evolutionary significance in relationship to plants. Chloroplasts (plastids) are formed by symbiogenesis of cyanobacteria in plants: land plants, green algae (e.g., Chlamydomonas), and red algae. Of the 5 major groups of photosynthetic bacteria, only cyanobacteria are oxygenic during photosynthesis. They are often called blue-green algae, even though not all members are blue-green in color. Most cyanobacteria have a slimy sheath, or coating, which is often deeply pigmented, particularly in species that occur in terrestrial habitats. Cyanobacteria utilize chlorophyll a, which is responsible for their green coloration. In addition, they have unusual accessory pigments called phycobilins, which absorb wavelengths of light for photosynthesis that are missed by chlorophyll and the carotenoids, thus giving blue-green or reddish color to cyanobacteria.

Most cyanobacteria have a Gram-negative type cell wall that consists of an outer membrane component, even though they may show a distant phylogenetic relationship with certain Gram-positive bacteria.

3. Exercises

Exercise 1: Prokaryotic survey 1

In this exercise, you will examine cyanobacteria for structural and functional organization.

Exercise 2: Prokaryotic survey 2

This exercise is designed to familiarize you with different types of bacteria.

Preliminary Hypothesis: Bacteria will show diversity due to their morphology

The Gram stain technique is used to differentiate bacteria based on the biochemical composition and structure of their cell wall. After staining Gram-positive bacteria appear purple and Gram-negative bacteria appear pink. Identifying an unknown bacterial sample as Gram-negative or Gram-positive tells the investigator a good deal about the structure of the bacterial cell envelope. In clinical settings, knowing whether a patient is suffering from a Gram-negative or Gram-positive infection will influence the selection of antibiotics used to treat the infection.

Procedure:

Analyze images of prepared Gram stain slides of each bacterium provided below.

Examine the pictures of the bacteria provided below and note the cell morphology of each bacterial culture.

Staphylococcus aureus: Staphylococcus spp. are Gram-positive cocci arranged in

irregular, often grape-like, clusters.

Fill out the Prokaryotic survey table below based on your exploratory observations of figures 3-5, by recording the cell shape and Gram-stain.

 Table 2.  Prokaryotic survey.

Please, continue with a Prokaryotic survey Part 2 located on the next page.

Question:

1) Please draw one bacterium in each empty circle from the four different types of bacteria depicted in the top row.

2)What type of morphology, cellular structures, or arrangement do you observe in these samples presented above?  Please, draw your exploratory observations. For better viewing, please, enlarge the images on your screen.

Exercise 3. Bacteria versus conditions.

 Some prokaryotes belong to the domain Archaea.  Many are extremophiles and thrive in harsh conditions.  Here are a few groups:

Methanogens

Sulfur-loving

Thermophiles

Hyperthermophiles

Psychrophiles

Acidophiles

Halophiles

Which specific conditions do you think each group thrives in?

Exercise 4: Adopt a Bacterium

In this exercise, you will choose a specific bacterium and produce a short report on its structure and physiology. Use the lab report page (provided separately) to record specific information about your bacterium. The goal of this investigation is to gain a better understanding of the relationship between specific bacterial structures and their functions.

Here is a list of some of the features you may include in your report. Of course, you will include only those that relate to your chosen bacterial species.  Use this list for ideas of what to include.

Bacterial shape

Coccus

Bacillus

Spirillum

Vibrio

Bacterial arrangement

Diplo

Staphylo

Strepto

Bacterial structures

        Flagella

        Pili or fimbriae

        Endospore (capability to form)

        Plasmids

        Capsule

        Gram positive cell wall

        Gram negative cell wall

        Gram variable cell wall

Bacterial metabolism

        Photosynthesis

        Chemosynthesis

        Methanogenesis

        Extremophile

        Aerobic respiration

        Anaerobic respiration

                Fermentation

                Methanogenesis

        Nitrogen fixation

And if you need help finding an interesting species, here are some websites to check out (although you may certainly find a species on your own).

http://www.bbc.com/earth/story/20150730-ten-bacteria-with-superpowers

https://www.inverse.com/article/11386-ranking-the-top-10-best-bacteria-on-earth

https://www.discovermagazine.com/planet-earth/top-ten-bacteria

Lab Report: Bacteria

Kristin Polizzotto Ph.D., Dmitry Y. Brogun Ph.D., Farshad Tamari Ph.D.

* Figures and text are intended for OER

Click here to access a downloadable version of the lab report.

Exercises

Exercise 1: Prokaryotic survey 1

In this exercise, you will examine cyanobacteria for structural and functional organization.

Exercise 2: Prokaryotic survey 2

This exercise is designed to familiarize you with different types of bacteria.

Please, write your Preliminary Hypothesis:

Procedure:

Analyze images of prepared Gram stain slides of each bacterium provided below.

Examine the pictures of the bacteria provided below and note the cell morphology of each bacterial culture in Table 2.

Staphylococcus aureus: Staphylococcus spp. are Gram-positive cocci arranged in irregular, often grape-like, clusters.

Fill out the Prokaryotic survey table below based on your exploratory observations of figures 3-5,

by recording the cell shape and Gram-stain.

 Table 2.  Prokaryotic survey.

Question:

1) Please draw one bacterium in each empty circle from the four different types of bacteria depicted in the top row.

2)What type of morphology, cellular structures, or arrangement do you observe in these samples presented above?  Please, draw your exploratory observations. For better viewing, please, enlarge the images on your screen.

Exercise 3. Bacteria versus conditions.

 Some prokaryotes belong to the domain Archaea.  Many are extremophiles and thrive in harsh conditions.  Here are a few groups:

Methanogens

Sulfur-loving

Thermophiles

Hyperthermophiles

Psychrophiles

Acidophiles

Halophiles

Which specific conditions do you think each group thrives in?

Exercise 4: Adopt a Bacterium

In this exercise, you will choose a specific bacterium and produce a short report on its structure and physiology. Use the lab report page (provided separately) to record specific information about your bacterium. The goal of this investigation is to gain a better understanding of the relationship between specific bacterial structures and their functions.

Here is a list of some of the features you may include in your report. Of course, you will include only those that relate to your chosen bacterial species.  Use this list for ideas of what to include.

Bacterial shape

Coccus

Bacillus

Spirillum

Vibrio

Bacterial arrangement

Diplo

Staphylo

Strepto

Bacterial structures

        Flagella

        Pili or fimbriae

        Endospore (capability to form)

        Plasmids

        Capsule

        Gram positive cell wall

        Gram negative cell wall

        Gram variable cell wall

Bacterial metabolism

        Photosynthesis

        Chemosynthesis

        Methanogenesis

        Extremophile

        Aerobic respiration

        Anaerobic respiration

                Fermentation

                Methanogenesis

        Nitrogen fixation

And if you need help finding an interesting species, here are some websites to check out (although you may certainly find a species on your own).

http://www.bbc.com/earth/story/20150730-ten-bacteria-with-superpowers

https://www.inverse.com/article/11386-ranking-the-top-10-best-bacteria-on-earth

https://www.discovermagazine.com/planet-earth/top-ten-bacteria

Lab Exercise: Protista

Kristin Polizzotto Ph.D., Dmitry Y. Brogun Ph.D., Farshad Tamari Ph.D.

* Figures and text are intended for OER

Objectives:

• Define the term “protist” and explain why this is not a monophyletic group.
• Identify representatives from each supergroup Excavata, ”SAR” clade, Archaeplastida, and Unikonta.
• Draw a phylogenetic tree for the eukaryotes and explain why the eukaryotic supergroups form a polytomy.
• Indicate the position of plants, animals, and fungi on the eukaryote tree, and identify the group of protists most closely related to each.
• Give examples of protist species from each eukaryotic supergroup.
• Give two examples of the significant impact of specific protists on their ecosystems.

1. Eukaryotic Survey

1. Introduction: Protists - Who are they?

Protists are a diverse collection of eukaryotic organisms, while exceptions are made, they are fundamentally unicellular, found free-living in all kinds of habitats. The cells are complexly organized with organelles and nuclear envelope membranes. Most protists possess a single nucleus, but some species may have two or more. Despite drastic differences in plant-like and animal-like organelles, they share key features with many organisms that allow them to thrive in reproduction, growth, and survival with common traits found in many complex organisms. They usually reproduce asexually, but sexual reproductive processes are also known to occur between two different mating types mt+ and mt-. However, protists display certain characteristics that allow them to be placed under a different category based on the organelle structure and locomotion (motility).  Amoeboids use cytoplasmic projections called pseudopodia; flagellates use flagella; ciliates use cilia, or sporozoans if they lack any motility structures. Understanding the complex history of protists and their evolutionary process is essential for our scientific discourse and discovery.  In this part of the lab, we will analyze different protists to identify characteristics correlated with the cell-structure and movement.  But there is little else that unifies this group—it is a term used to designate all eukaryotes that are not classified in one of the monophyletic eukaryotic kingdoms. You might ask why biologists have not created monophyletic groups for the protists. This is in fact, the main question that this lab exercise will focus on. As you work through the activities, you will discover for yourself the reasons!

Watch this video on the Paramecium movement: https://youtu.be/mh7KOtQTXrw

Hypothesis: Protists will show diversity due to its morphology and locomotion

From your previous courses, list as many characteristics of eukaryotes as you can in the space below:

2. Some key definitions

3. Classification and Phylogeny of Protists - Four Eukaryotic Supergroups

The reconstruction of evolution and phylogenetic relationships within the four Eukaryotic supergroups is still an ongoing process.  With the new Whole Genome Sequencing (WGS) technologies along with the sequencing of the complete 18S rRNA markers, new members are being added to the supergroups.  Not all of the clades within the supergroups are monophyletic, and an example of it is the Stramenopiles.  Most of the members have arisen from the primary and secondary endosymbiosis, highlighting the fusion of the photosynthetic bacteria and heterotrophic eukaryotes.  The following figure summarizes the phylogeny of Protists depicting the Four Eukaryotic Supergroups (Figure 1).

2. Four Eukaryotic Supergroups- Excavata,”SAR” clade, Archaeplastida, and Unikonta

1. Supergroup Excavata

This supergroup consists of several major groups, such as: Diplomonads and Parabasalids, with modified mitochondria, and Euglenozoans including Kinetoplastids and Euglenids, containing spiral or rod inside flagella.  Some of the examples of the Diplomonads and Parabasalids include Giardia and Trichomonas. Examples of the Kinetoplastids and Euglenids include Trypanosoma and Euglena.

Example: Euglena & Trypanosoma

Euglena is a flagellated protozoan with a single flagellum used for movement. It strives in nutrient-rich freshwater, except for a few marine species, and it can be found in sewage systems.  It is unique like a plant carrying out photosynthesis and if sunlight is not available, it can absorb nutrients from decayed organic material.  As in higher plants the chloroplast contains chlorophylls (a and b). There are usually many bright green chloroplasts, although some species are colorless. Euglena is unusual for an alga in that it can lose its chloroplasts when kept in the dark or treated with the antibiotic streptomycin, but it is able to survive as a heterotroph. Thus, this organism has an affinity for both algae and protozoa. For this reason, Euglena is claimed by botanists as an alga, but as a protozoan by zoologists.

Trypanosoma is another example of flagellated Euglenozoa. Trichomonas vaginalis is an example causing vaginal infection and more potent trichomoniasis. Trypanosomes are microscopic, one-celled protozoans of the genus Trypanosoma, of which hundreds of species are known. A trypanosome is long, pointed, and possesses a flagellum. The flagellum arises at the front, or anterior end of the parasite and curves back to form the edge of a long undulating membrane used in locomotion. T. gambiense and T. rhodesiense cause African sleeping sickness and both are transmitted by tsetse flies.

1. Supergroup “SAR” Clade (Stramenopiles, Alveolates & Rhizarians)

This supergroup consists of several major groups, such as: Stramenopiles with hairy and smooth flagella, Alveolates containing membrane-enclosed sacs (alveoli) beneath the plasma membrane, and Rhizarians with threadlike pseudopodia.  Some of the examples of the Stramenopiles include diatoms, golden and brown algae, and Trichomonas. Examples of the Alveolates include dinoflagellates, Apicomplexans, and Ciliates. Examples of the Rhizarians include Foraminifera and Cercozoans.

1. Stramenopiles.

Example Diatoms

Diatoms form a large group known as heterokonts, some are autotrophs (e.g., golden algae, kelp) and the rest are heterotrophs (e.g., water molds). There are more than 200 genera of living diatoms, and it is estimated that there are approximately 100,000 extant species. Diatoms form two hard cell walls (called frustules) composed of silicon oxide. Their chloroplasts contain pigments such as fucoxanthin which gives them yellowish-brown color. Diatoms can be found in saline and hypersaline environments, in freshwater, in soils, and on damp surfaces. Most live-in open water, although some live as surface films at the water-sediment interface, or even under damp atmospheric conditions. They are especially important in oceans, where they are estimated to contribute up to 45% of the total oceanic primary production.

2. Alveolates

Example: Apicomplexa, Ciliophora & Tetrahymena

Sporozoans lack locomotion structures. An example is Plasmodium, the genus responsible for malaria (caused by Plasmodium falciparum transmitted via a female Anopheles mosquito vector) in humans and other animals. In humans, the parasite is found intracellularly in red blood cells and is used as a diagnosis of malaria. Another member of this group includes ciliated protozoan Ciliophora that possesses numerous cilia for locomotion. And Paramecium is a unicellular organism found in freshwater throughout the world. It has a stiff outer covering that gives it a permanent slipper shape. It swims rapidly by coordinated wavelike beats of its many cilia: short, hair-like projections of the cell. The paramecium has an external oral groove lined with cilia and leading to a mouth pore and gullet; food is digested in food vacuoles. Paramecium can divide asexually by cell division called “binary fission”. Tetrahymena is a ciliated protozoan with an oral apparatus used for feeding on bacteria. The organism swims by means of rows of cilia arranged longitudinally over the surface of the organism. The organism has two nuclei in the cell that perform different functions. Several discoveries in cellular physiology were established by studying this organism.

3. Rhizarians

The widespread foraminiferans and radiolarians are amoeboid protozoa, both striving in the marine environment.  They contribute to the ocean sediments significantly from their external shells made up of either CaCO3 or silica, respectively. They possess slender, thread-like pseudopodia for locomotion. Members of the phylum Actinopoda also use slender pseudopodia for locomotion. These organisms differ from the Foraminifera in the composition of the shells that surround them. In the Actinopoda, the shells are made of silica, the same material in glass.

2. Supergroup Archaeplastida

This supergroup includes a large group of algae, specifically the green algae from which higher plants have emerged. This supergroup consists of several major groups, such as red algae that contain phycoerythrin (photosynthetic pigment), green algae with plant-type chloroplasts, and land plants (discussed in later labs). The microscopic green algae include unicellular as well as various colonial, coccoid, and filamentous forms of flagellates (usually with two flagella per cell), that all contain chloroplasts. Genera within the green algae include Euglena, Chlamydomonas, Volvox, Ulothrix, and Spirogyra.

There are about 6000 species of green algae; many species live most of their lives as single-cells, other species form colonies or long filaments. Algae employ simple reproductive structures and lack the extensive vascular structures characteristic of higher plants. Eukaryote organisms are capable of oxygenic photosynthesis. They are classified into different groups on the basis of morphology, types of chlorophylls, carbon reserve storage materials, cell wall composition, and habitat. Although many algae, like the higher plants, are non-motile, they may have motile reproductive cells.  

This organism usually exists in a haploid stage, having just one copy of each chromosome (like a mammalian gamete). When mating type plus (mt+) and minus (mt-) meet (Figure 6A), flagellar adhesion and gamete activation are initiated (Figure 6B). They join their cytoplasm (plasmogamy) and their nuclei fuse (karyogamy) to form a diploid zygote (Figure 6C). The zygote is the only diploid cell stage in the life cycle, and it eventually undergoes meiosis to form a tetrad, germinating to form four new Chlamydomonas cells, two mt+ and two mt- following the Mendelian genetics. (Figure 6D). When vegetative cells are grown in a nitrogen-free growth medium, Chlamydomonas cells undergo gametogenesis and develop into gametes (Figure 6A).

3. Supergroup Unikonta

This supergroup consists of several major groups, such as Amoebozoans, with the lobe-shaped or tube-shaped pseudopodia, and highly variable Opisthokonts.  Some examples of the Amoebozoans include Amoeba and Dictyostelium (slime molds). Examples of the Opisthokonts include choanoflagellates, animals, and fungi (discussed in future labs). This group consists of amoeboid protozoa that use pseudopodia for locomotion. Examples include amoebae (Entamoeba histolytica is the cause of amoebiasis) that do not have an outer covering on the pseudopodia (naked) or have a protein or mineral coating over the pseudopodia.

3. Exercises

1. Eukaryote phylogeny

1. As you have learned, a group must be monophyletic in order to be considered a kingdom. Watch the video “Kingdom of Protista” from the beginning to 1:10. Based on the tree shown (0:23-0:42), do you think that protists are truly a kingdom? Explain how you know. Write your answers in the lab report.

2. Watch the video “Eukaryotic Supergroups” and answer the questions below in your lab report as you go. Use the timestamps given to help you find the answers. The video is just under 9 minutes long.

This video explains that each domain can be divided into subgroups (0:42). Ideally, the next level after the domain is kingdoms, but since these have not been worked out for many eukaryotes, biologists use “supergroups” while the work of classification is in progress.

1:24        This video uses the classification system with five supergroups of eukaryotes, each of which is assumed to form a clade. What does the word “clade” mean?

2:39        Do you agree with the definition “protists are unicellular eukaryotes”? Why or why not? (see also 3:23-3:50 in the video)

4:55        Different biologists make different phylogenetic trees and different supergroups for eukaryotes. Why do you think this is so? (see also 5:11)

5:43        What is the definition of polytomy? (watch until 6:21) Why must the eukaryote tree include polytomy?

6:37        Why is it easier to figure out the evolutionary relationships for the more specific subgroups towards the right in the diagram?

7:30        How can you differentiate among the various supergroups—in other words, how can you tell them apart, according to this video?

8:00        Do you think the method in the previous question will work well for classifying an unknown protist? Why or why not?

8:16        Why does the narrator say that the term algae are not useful? In what way?

2. Classifying protists

1. Visit the PondLife website and skim the page Microbes in Motion, which contains 13 short videos. Twelve of these are common protists collected from ponds and marshes right here in NYC. Choose three of these protists (don’t choose the bacteria!). Read the caption and watch the video for each of the three you selected. Then write in your lab report the following for each:

Scientific name:         Use the name given on the web page

Classification:         Use the tree below, and write the group to which your species belongs

How did you know:         List the clues from the caption or video that helped you figure out to which group this species belongs?

2. Now let’s bump it up a notch! Watch the video “Meet the Microcosmos” with particular attention to the section on protists (4:34-6:45). Then complete the activities below.

Choose one species from the video (make sure it’s a protist!) and then find out what supergroup it belongs to and why it belongs to that group (in other words, what traits does it have that place it in that group?). Write these answers in your lab report. To find the correct supergroup, you can try to google the name of the protist along with the word “classification” (for example, “Euglena classification”). If that doesn’t work, go to the Tree of Life website and enter the protist’s name into the search box in the upper right corner. If this does not immediately bring up the page for your taxon, click on the name that is closest to your protist on the results page. You can also try clicking on the links for “containing group” on the right-hand side of the page, which will tell you the higher classification for the taxon you are viewing.

Now, answer these two questions in your lab report using what you learned in the video at the indicated timestamps:

• What would be the impact on the world if diatoms suddenly went extinct? (6:10)
• What is the difference between protists and microscopic animals? How can you tell them apart? (6:46) Hint: it’s not size, and it’s not multicellularity. If you need another hint, go back to the first video in today’s lab (Kingdom of Protista) and watch 0:55-1:04 again.

3. Now that you have a better idea of what a protist is and how to differentiate it from microscopic animals, let’s test your expertise. Watch the video “Introduction to the Protists” and see if you can find at least two places where the filmmaker mistook an animal for a protist. Can you catch his mistakes? One caveat: I do not mean the animals you see during the first minute of the video (bird, lizard, snake, fish, and humans). The animals that appear later on are tricky—they are microscopic, like many protists, which is why the filmmaker may not have known that they are animals. When you see an organism that you think is an animal, write down the timestamp in your lab report.

Was this easy or difficult? What made it easy or difficult for you? Write your answer in the lab report.

3. Conclusion

In your lab report, summarize what you have learned by answering the following questions.

1. How is the classification of protists different from that of other eukaryotes?
2. What does the current status of protist classification tell you about their evolution?
3. What can you conclude about diversity in the protists? (Is it greater or less than other eukaryotes, for example?)
4. What have you learned about the importance of protists in their ecosystems? What important roles do they play?

Lab Report: Protista

Kristin Polizzotto Ph.D., Dmitry Y. Brogun Ph.D., Farshad Tamari Ph.D.

* Figures and text are intended for OER

Click here to access a downloadable version of the lab report.

4. Exercises

1. Eukaryote phylogeny

3. As you have learned, a group must be monophyletic in order to be considered a kingdom. Watch the video “Kingdom of Protista” from the beginning to 1:10. Based on the tree shown (0:23-0:42), do you think that protists are truly a kingdom? Explain how you know. Write your answers in the lab report.

4. Watch the video “Eukaryotic Supergroups” and answer the questions below in your lab report as you go. Use the timestamps given to help you find the answers. The video is just under 9 minutes long.

This video explains that each domain can be divided into subgroups (0:42). Ideally, the next level after the domain is kingdoms, but since these have not been worked out for many eukaryotes, biologists use “supergroups” while the work of classification is in progress.

1:24        This video uses the classification system with five supergroups of eukaryotes, each of which is assumed to form a clade. What does the word “clade” mean?

2:39        Do you agree with the definition “protists are unicellular eukaryotes”? Why or why not? (see also 3:23-3:50 in the video)

4:55        Different biologists make different phylogenetic trees and different supergroups for eukaryotes. Why do you think this is so? (see also 5:11)

5:43        What is the definition of polytomy? (watch until 6:21) Why must the eukaryote tree include polytomy?

6:37        Why is it easier to figure out the evolutionary relationships for the more specific subgroups towards the right in the diagram?

7:30        How can you differentiate among the various supergroups—in other words, how can you tell them apart, according to this video?

8:00        Do you think the method in the previous question will work well for classifying an unknown protist? Why or why not?

8:16        Why does the narrator say that the term algae is not useful? In what way?

2. Classifying protists

4. Visit the PondLife website and skim the page Microbes in Motion, which contains 13 short videos. Twelve of these are common protists collected from ponds and marshes right here in NYC. Choose three of these protists (don’t choose the bacteria!). Read the caption and watch the video for each of the three you selected. Then write in your lab report the following for each:

Scientific name:         Use the name given on the web page

Classification:         Use the tree below, and write the group to which your species belongs

How did you know:         List the clues from the caption or video that helped you figure out to which group this species belongs?

5. Now let’s bump it up a notch! Watch the video “Meet the Microcosmos” with particular attention to the section on protists (4:34-6:45). Then complete the activities below.

Choose one species from the video (make sure it’s a protist!) and then find out what supergroup it belongs to and why it belongs to that group (in other words, what traits does it have that place it in that group?). Write these answers in your lab report. To find the correct supergroup, you can try to google the name of the protist along with the word “classification” (for example, “Euglena classification”). If that doesn’t work, go to the Tree of Life website and enter the protist’s name into the search box in the upper right corner. If this does not immediately bring up the page for your taxon, click on the name that is closest to your protist on the results page. You can also try clicking on the links for “containing group” on the right-hand side of the page, which will tell you the higher classification for the taxon you are viewing.

Now, answer these two questions in your lab report using what you learned in the video at the indicated timestamps:

• What would be the impact on the world if diatoms suddenly went extinct? (6:10)
• What is the difference between protists and microscopic animals? How can you tell them apart? (6:46) Hint: it’s not size, and it’s not multicellularity. If you need another hint, go back to the first video in today’s lab (Kingdom of Protista) and watch 0:55-1:04 again.

6. Now that you have a better idea of what a protist is and how to differentiate it from microscopic animals, let’s test your expertise. Watch the video “Introduction to the Protists” and see if you can find at least two places where the filmmaker mistook an animal for a protist. Can you catch his mistakes? One caveat: I do not mean the animals you see during the first minute of the video (bird, lizard, snake, fish, and humans). The animals that appear later on are tricky—they are microscopic, like many protists, which is why the filmmaker may not have known that they are animals. When you see an organism that you think is an animal, write down the timestamp in your lab report.

Was this easy or difficult? What made it easy or difficult for you? Write your answer in the lab report.

7. From your previous courses, list as many characteristics of eukaryotes as you can in the space below.

3. Conclusion

In your lab report, summarize what you have learned by answering the following questions.

5. How is the classification of protists different from that of other eukaryotes?
6. What does the current status of protist classification tell you about their evolution?
7. What can you conclude about diversity in the protists? (Is it greater or less than other eukaryotes, for example?)
8. What have you learned about the importance of protists in their ecosystems? What important roles do they play?

Lab Exercise: Fungi

Farshad Tamari, Ph.D.

Figures and text are intended for OER

Objectives:

• Describe fungal classification into phyla, and provide a phylogeny of Kingdom Fungi.
• In a sentence or two, describe the characteristics of the three largest phyla in the Kingdom Fungi (Zygomycota, Ascomycota, Basidiomycota).
• Using images, explain the life cycle of typical multicellular fungi.
• Give three examples of how humans benefit from specific uses of fungi.

I. Fungal Classification and Phylogeny

A. Introduction

The study of fungi is termed mycology. Members of the Kingdom Fungi are eukaryotic organisms, which have characteristics that distinguish them from prokaryotes and from the rest of the eukaryotes. For example, fungi possess nuclei with a nuclear envelope and an endomembranous system that includes the endoplasmic reticulum and Golgi bodies. Fungi exhibit diverse morphological features that have been used in their classification, but all members do share some characteristics. For example, fungi have a cell wall composed of a durable polysaccharide called chitin (also found in arthropod exoskeletons).  If not pathogenic, fungi can form symbiotic relationships or act as decomposers.  Fungal species may be unicellular (e.g., yeast) or multicellular (e.g., mushrooms). Human fungal infections are called mycoses. Reproduction in fungi may include both sexual and asexual reproduction, although some species reproduce strictly asexually.

For more information, watch the following videos (optional):        

Thirty-minute PowerPoint lecture on fungi

Nine-and-a-half minute overview of fungi

B. Some definitions

C. Classification/Phylogeny of Fungi

The phylogenetic relationships between the fungal classes are still under investigation.  With the sequencing of complete rRNA of many different species, the classification is ever-changing and new phylogenetic trees have been generated based on this data (Tedersoo et al., 2018).  The general consensus among phylogeneticists that study Kingdom Fungi is that they form a monophyletic group. The ancestor of the fungi was likely an aquatic organism that resembled a flagellated single-celled protist belonging to the group we currently know as Opisthokonta (Unikonta).  Figure 1 summarizes the phylogeny of Kingdom Fungi.

Here are some highlights from each group:

D. Life cycle of Fungi

All unicellular fungi (such as yeast) reproduce by asexual, mitotic cell division. Multicellular fungi can reproduce via a combination of sexual and asexual reproduction (Figure 2).  The sexual portion of the life cycle begins with two different parent mycelia, which grow from spores. A mycelium is a collection of structures called hyphae (long, branching filaments that increase surface area). In fungi, there are no males and females—only genetically distinct mating types (often designated + and -). Two cells from the different mating types join together, but their nuclei remain separated, each still enclosed by its nuclear envelope. This fusion of two-parent cells is called plasmogamy and results in a cell with two genetically different nuclei.  Such cells are called heterokaryotic and may continue to divide and produce daughter cells that also contain two nuclei each. Some fungi remain in this heterokaryotic stage for a long time, but eventually, the nuclei combine to form diploid cells with a set of chromosomes from each parent mycelium.  This fusion of the heterokaryotic nuclei into diploid nuclei is called karyogamy and produces a zygote. The zygote undergoes meiosis shortly after karyogamy, forming haploid spores which can then disperse and germinate into new mycelia to continue the life cycle. The asexual part of the life cycle results in the production of haploid spores by mitosis (Figure 2). Approximately 20,000 species of fungi, collectively known as the Deuteromycota, reproduce strictly asexually. Deuteromycota contains species from both Ascomycota and Basidiomycota.

II. Exercises: Fungal Diversity

A. Zygomycota         

Rhizopus is a member of the Zygomycota. They are mainly saprotrophic, meaning that they feed on dead organisms, and therefore play a major role in decomposition. Look at the images of Rhizopus below.  

1. In figure 3, what do you think the black granulation (dots) represent?

2. Lookup a common Rhizopus species.  What does it grow on?

3. What are the functions of each structure in Figure 5?

1. Sporangium:

2. Stolon:

3. Rhizoid:

Watch this optional video on Zygomycota.

B. Ascomycota         

Examine the images of Peziza and other ascomycetes below.

4. What are the small, oval, green/black structures called in figure 8? (consult the caption for help)

5. What is the name of the structure that contains the small, oval, green/black structures?

Some Ascomycota species are opportunistic pathogens of humans. For example, candidiasis (thrush), is caused by the fungus Candida albicans.

Watch the following interesting video on Candidiasis infections and treatment.

Here’s one example of how thrush may look:

C. Basidiomycota

Basidiomycota is the second-largest class of fungi. Collectively they are referred to as the club fungi. Observe examples in the images below.

6. Many basidiomycetes are useful to humans as food, but fungi from this phylum and the other phyla have many other uses that are beneficial to humans.  Look around your house and find an example of a product that could not be made without a fungus (don’t use mushrooms or other food—find something else!). If you need some ideas, watch this video from National Geographic (3:30) and this video on the uses of fungi (2:21). It’s ok if the fungus you choose is not from Phylum Basidiomycota. Take a photo of the product, copy it into your lab report, and explain how fungi were involved in the item’s production.

D. Imperfect Fungi (Deuteromycota)

Deuteromycota is composed of approximately 20,000 species belonging to the Ascomycota and the Basidiomycota.  Observe the images of Penicillium and Alternaria conidia below.

7. What is the significance of Penicillium?

E. Mutualistic Fungi

Fungi can form mutualistic relationships with unicellular photosynthetic organisms. These are generally called lichens.  Observe the lichens in the figures below:

8. Name two types of photosynthetic organisms that can form mutualistic relationships with a fungus to form lichens:

9. What does the fungus bring to this mutualistic relationship?

10. What does the photosynthetic organism bring to this relationship?

Lab Report: Fungi

Farshad Tamari, Ph.D.

Figures and text are intended for OER        

Click here to access a downloadable version of the lab report.

Complete the report by filling in the information below. When you are done, submit your report as a Word doc or pdf (or, if you have printed the document and filled it in by hand, you may take a picture and submit it as a png, jpg, or pdf).

II. Exercises: Fungal Diversity

A. Zygomycota                 

11. In figure 3 of your lab instructions, what do you think the black granulation (dots) represent?

12. Lookup a common Rhizopus species.  What does it grow on?

13. What are the functions of each structure in Figure 5?

4. Sporangium:

5. Stolon:

6. Rhizoid:

B. Ascomycota        

4. What are the smaller oval (green/black) structures called in figure 8 of your lab instructions? (consult the caption for help)

5. What is the name of the thin, transparent structure that contains the smaller oval (green/black) structures?

C. Basidiomycota

6. Look around your house and find an example of a product that could not be made without a fungus (don’t use mushrooms or other food—find something else!). If you need some ideas, watch this video from National Geographic (3:30) and this video on the uses of fungi (2:21). It’s ok if the fungus you choose is not from Phylum Basidiomycota. Take a photo of the product, copy it below, and explain how fungi were involved in the item’s production.

D. Imperfect Fungi (Deuteromycota)

7. What is the significance of Penicillium?

E. Mutualistic Fungi

8. Name two types of photosynthetic organisms that can form mutualistic relationships with a fungus to form lichens:

9. What does the fungus bring to this mutualistic relationship?

10. What does the photosynthetic organism bring to this relationship?

Lab Exercise: Nonvascular Plants and Seedless Vascular Plants

Kristin Polizzotto, Ph.D.

Figures and text are intended for OER

Objectives:

• Distinguish members of Kingdom Plantae from their nearest relatives (charophyte algae)
• Draw a basic phylogeny for Kingdom Plantae
• Diagram and explain the life cycle of plants (alteration of generations)
• Differentiate the characteristics of nonvascular plants versus seedless vascular plants
• Using images, explain the life cycles of moss (a nonvascular plant) and ferns (a seedless vascular plant)