Island Biogeography and Evolution: Solving a
Phylogenetic Puzzle With Molecular Genetics

Target age or ability group:	9-12 college prep biology or honors biology.
Class time required:	Three days: first day, students can work independently, second day students work in teams of four, and the third day should be a small group or class discussion.
Materials and equipment:	Scissors and glue, an atlas would be a desirable option.
Summary of activity:	This activity is a logic problem that is based on real organisms and real data. The problem is to develop phylogenies for seven related populations of lizards living on the Canary Islands. Three phylogenetic charts will be constructed, each using different forms of data, geography, geology, morphology, and molecular genetics.
Prior knowledge, concepts or vocabulary necessary to complete activity:	This activity would be best used near the end of an evolution unit. Knowledge of the DNA code is needed and a basic understanding of biotechnologies such as gel electrophoresis, and DNA finger printing would be desirable. Students must know the difference between a population and a species. Students should be aware of the conditions of speciation including isolation, genetic divergence, and reproductive isolation.

Teacher Instructions:

You may give this assignment out in pieces so as not to give students too much information on determining phylogenies. Students should not be given Figure 1 and Map 2 until completing Part I. This could be done at home as homework. Part IV is the most difficult and most logistically demanding part of this investigation. Students will be comparing the DNA sequences for cytochrome b from seven populations. Only the first population has the complete sequence. All others listed below it have listed only what is different. However, each population must be compared with each other population. This makes for twenty-one pairings, a formidable task even for the teacher. The strategy is to share the work. All pairing combinations are given in the student instructions. Basically students work in teams of four, each student counts differences for five pairings. Teams compare results to check for accuracy. Students will cut the DNA sequence pages into strips and paste the matching ends together to produce seven sequences. These can be easily manipulated for pair comparisons.

The final activity will be to use the results from the pairings and compare the differences and use this information to develop a final phylogeny chart. The solutions are provided. The basic scheme to remember is that low numbers of base-pair differences imply closer evolutionary relationships. The phylogeny charts are intended to stimulate student thinking about the problems of understanding past and future evolution. There are many variations to phylogenies students can come up with, some are better than others. The criteria should really be: Can the solution be logically explained and justified? Only the last phylogeny based on molecular genetics has fewer variations and needs some serious discussion to close the subject. Finally, most questions on this assignment require student explanation. You should emphasize that answers may vary, but logic is required for all solutions.

References:

Thorpe, R.S., et al. (1994). "DNA Evolution and Colonization Sequence of Island Lizards in Relation to Geological History," Evolution, 48: .230-240.

Anguita, F., et al. (1986) "Geochronology of some Canarian dike swarms: Contribution to the volcano-tectonic evolution of the archipelago, Journal of Volcanology and Geothermal Research, 30: 155-162.

Table 2 Solution:

Phylogenetic Solutions: Below is one possible solution based on geographical distance and island hopping. It does not take into account actual currents. There can be other reasonable solutions. The idea here is to get the student thinking about the logic of the problem, not its ultimate answer.

Below is a possible solution using island distribution and morphology. In using body size, one is tempted to guess that medium lizards of Palma could have been the immediate ancestors to Gomera and the small lizards of Gomera are ancestors to the small lizards of Hierro. This could contradict the argument based on distance. Again there is no one perfect answer. Ecologists and geneticists have debated several hypotheses for years.

The solution below is based on DNA evidence to indicate genetic distance. It is considered the most relevant criterion to establish who is most closely related to whom since all base pairs have an equal chance for mutation and that mutation rate is relatively constant even if evolution rate is not. Note that north and south Tenerife populations are listed as #3 but each has probably been a source for new colonization. The real surprise here is not the evolution of the smaller morphs. The surprise is that stehlini on Gran Canaria appears to have the oldest ancestor although atlantica is actually closer to Africa. It would be interesting to know more about the actual ocean currents affecting this island group. It could provide an answer to this unexpected surprise.

Ever since Charles Darwin formulated his hypothesis on how the finches of the Galapagos Islands evolved into 13 species, islands have been a prime target for the study of evolution. By their very nature, islands are isolated and are essentially a living laboratory of evolution. In this investigation you should be familiar with the terms speciation, geographic isolation, gene flow, gene pool, and reproductive isolation. You will work with real data from real populations. The data will include observations of lizard morphology (body form), geological age estimates of various islands in the Canary Island Archipelago, geographic distances, and genetic distances based on nucleotide base differences in DNA between different populations of lizards.

Background

There are many populations of lizards living on the Canary Islands, and photographs of various examples can be found in Thorpe (1989). The Canary Islands form an archipelago of seven volcanic islands just west of the African continent (Map 1). The island chain starts about 85 km (50 miles) from the continent following a fault line of the Atlas Mountain in northern Africa. Geologists theorize that a geologic hot spot of upwelling magma has been drifting westward for the past 20 million years gradually forming the islands as it moves. Thus, the furthest east island, Lanzarote is oldest while the smaller western island, Hierro is the youngest, about 0.5 million years old. Volcanic islands are particularly good laboratories for evolutionary science because they can be dated accurately using radioactive isotope decay and because they start out as lifeless masses of rock emerging from the sea.

The development of ecosystems on volcanic islands is somewhat unpredictable. However, ecological succession does occur first with pioneer organisms that gradually alter the environment until a stable climax community is established. What is unpredictable is: What plant and animal species will colonize these new environments? Much of this is left to climate, proximity to other land masses, and of course, chance. This investigation deals with three species of lizards of the genus Gallotia, and within one of these species, Gallotia galloti, four separate island populations. The arrival of the Gallotia lizards was probably by rafting (See Map 1), that is, rafts of natural vegetation are often washed out to sea when high river levels cause river banks to collapse, carrying away both plants and clinging animals alike. Colonization by airborne organisms such as insects and birds usually occurred during storms. In any case, there are some general principles of island colonization which follow:

1) The closer the island is to another land mass, the higher the probability of colonization.

2) The older the island, the more likely it will be colonized.

3) The larger the island, the more species are likely to be established.

4) The geographic isolation reduces gene flow between populations.

5) Over time, colonial populations become genetically divergent from their parent population due to natural selection, mutation, and/or genetic drift.

Problem: Evolution biologist have been faced with an interesting problem. What is the phylogenetic history of the three species and seven populations of Gallotia lizards on the islands? Does the presence of four morphologically different populations of G. galloti on the four western most islands (Map 2) imply continuing evolution? In this investigation, you will use data from geography, geological history, morphology (body size), and molecular genetics to develop answers to these questions.

Procedure

Part I: Phylogeny Based On Geographic Distance

Using Map 1, measure the distances in kilometers of each island to the mainland (Africa). List these distances on a separate page. Include the following islands: Lanzarote, Fuerteventura, Gran Canaria, Tenerife, Gomera Palma, and Hierro.

1. Which island would have been colonized first and which last? Explain why you think so.

2. Using Map 2 and your geographic reasoning, diagram on a separate page a hypothetical phylogenetic (family) tree of the three species and three additional populations of G. galloti. Your teacher will demonstrate. Label your end branches with the following population names:

atlantica	stehlini	galloti	galloti	galloti	galloti
		Tenerife	Palma	Gomera	Hierro

Map 2. On the map above, three species of lizards are shown, Gallotia atlantica, G. stehlini, and G. galloti. G. galloti has colonized the four western most islands and each population is morphologically distinct from the other. (Redrawn from R.S. Thorpe, 1993.)

Part II: Phylogeny Based on Geological History

Check your hypothetical phylogenetic tree against the geological data in the Table 1. The maximum age of each island was estimated by sampling volcanic rocks found on all islands. The ratio of radioactive potassium to its breakdown product argon was used to estimate the age of the rocks

Table 1. Maximum age in millions of years for the Canary Islands (from F. Anguita, 1986).

1. Explain how the data in Table 1. Support your phylogeny diagram? Or, what changes should you make and why?

Part III: Phylogeny Based On Morphology

Study the drawings from each lizard population in Figure 1 and compare and contrast their body size with the distribution on Map 2. To be sure differences are genetic, not ecological, researchers collected individuals from all island populations and bred and raised them in captivity. Their offspring still displayed differences according to their parental characteristics. Draw a new phylogeny chart based on morphological similarities and differences.

1. Compare your two phylogeny charts. Describe how they are different.

Figure 1. The relative sizes of typical lizards from each population are shown. Lizards on Lanzarote and Fuerteventura are essentially the same. (Redrawn from R.S. Thorpe, 1994.)

Part IV: Phylogeny Based On Molecular Genetics

Recent studies by R. S. Thorpe (1993, 1994) have attempted to support various phylogenetic hypotheses by comparing genetic differences among the populations of the Gallotia lizards on the Canary Islands. The gene for cytochrome b which is coded by DNA found in every cell's mitochondria was used in this study along with DNA from other genes. Cytochrome b is an important substance for cell metabolism and has probably been around since the first prokaryotes. Changes in its nucleotide base sequence (A, T, C, and G) that do not disrupt the gene's function provides us with a kind of evolutionary clock. The rate of mutational changes due to pairing errors is relatively constant. The chances for such mutations are the same for any of these bases. This means that the more time, the more changes. When two populations are isolated and gene flow between them is restricted, the mutational differences accumulate over time. The longer the isolation the greater the difference.

Thorpe and his colleagues used restriction enzymes to cut the DNA and gel electrophoresis to separate the fragments. Radioisotope tagging eventually led to the sequencing of the samples of DNA for each of the seven populations. Thorpe tested two populations on Tenerife to see if ecological differences were part of the story. He felt that because Tenerife is moist and lush in the north while arid and barren in the south, populations on that island might have some genetic differences. Also, he wondered if Tenerife was supplying colonizing lizards from two different directions. The results for Thorpe's tests appear on the last two pages of this investigation.

Your task is to count the differences between all pairings of the seven populations and use that data to construct a final phylogenetic tree based of genetic similarities and differences.

Procedure For Part IV:

There are twenty-one different pair combinations possible using seven populations. You should work in a team of four and each person will be responsible for counting all of the base differences for five of the twenty pairs. The pairings are listed on Table 2. Note that the first pairing has been counted for you. Record your results in Table 2. When all teams are done, the data will be checked for agreement. The easiest way to make accurate counts is to cut out each sequence and tape together in the correct order, end to end. Each strip will consist of four pieces that you will glue by matching ends. You will then compare pairs of strips side by side to count the differences.

	1. G.stehlini	2. G. atlantica	3. G. galloti P.	4. G. galloti N. T.	5. G. galloti S.T.	6. G. galloti G.	7. G. galloti H
1.G. stehlini
2. G. atlantica	36
3. G. galloti Palma	41	25
4. G. galloti N. Tenerife	40	21	8
5. G. galloti S. Tenerife	40	19	10	6
6. G. galloti Gomera	45	22	13	19	15
7. G. galloti Hiero	49	28	17	21		4

There are 21 possible pairings; each team member should select five pairings other than 1/2.

Student #1 1/3 1/7 2/6 3/6 4/7

Student #2 1/4 2/3 2/7 3/7 5/6

Student #3 1/5 2/4 3/4 4/5 5/7

Student #4 1/6 2/5 3/5 4/6 6/7

Once your data table is complete, look for low numbers which express more genetic similarity and imply more recent common ancestry. Pairs that produce high numbers are said to have greater genetic distance between them. In other words, large numbers imply they are less genetically alike and have more distant ancestry. On a phylogenetic tree, distant ancestry is expressed by low branches while more recently evolved and more recent ancestry are on the higher branches.

Interpretations and Conclusions

1. On Table 2, large numbers imply that pairs of populations have more distant ancestry. Why is this?

2. How many base pair differences do you think separates any two species of these lizards? Give an example to support your answer.

3. Which two populations are most closely related? Justify your answer.

4. Why should you expect the populations S. Tenerife (ST) and N. Tenerife (NT) to have fewer differences than other pairings?

5. Which population is least related to the rest? Why do you say so?

6. Draw a phylogeny chart using genetic similarities and differences found in Table 2. Compare it to the phylogeny based on the geologic age of the islands.

7. What difference is there between the two phylogenies?

8. Would you say that G. stehlini is the ancestor of G. atlantica or vice versa or are they ancestors at all? Explain you reasoning.

9. Predict what is likely to happen to the four populations of G. galloti on the four western islands and identify what conditions will support your predictions.

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DNA Simulation with Paper Chromatography
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