Evolution of Coloration and Biology of the Ibiza Wall LizardCase Study Authors:Nathan Dappen, Ph.D., Olga Korotkova, Ph.D., James Wilson, Ph.D.

Case Study Editors:Jane Indorf, Ph.D., S. Barry Issenberg, M.D., FACP, Alex J. Mechaber, M.D., FACP

Case Study Objectives: 1. Apply basic knowledge of evolutionary biology to understand color evolution in a lizard species. 2. Interpret data to draw conclusions about the outcome of an experiment. 3. Use simple mathematics to solve basic population biology questions. 4. Apply basic knowledge of the physics of light to understand color in nature. 5. Utilize spectrographic figures to answer questions about color in nature. 6. Apply understanding of light to the evolution of color in a lizard species.

Part 1. IntroductionNate Dappen is a graduate student working on his Ph.D. in evolutionary biology. He studies the evolution of color in a lizard species endemic to the Pityusic Archipelago in the Spanish Mediterranean. He is here in these Spanish islands with a handful of students collecting data on basic lizard biology (densities, behaviors, diet) and trying to understand why there is so much color diversity among island populations of this species.

The Ibiza Wall Lizard: The Ibiza wall lizard (Podarcis pityusensis) is a diurnal terrestrial lizard species endemic to the Pityusic Archipelago in the Spanish Mediterranean. This archipelago consists of Ibiza, Formentera and about 40 small islands and islets. This species exhibits striking color diversity across its geographic range – perhaps the highest color diversity of any reptile. On most islands lizards are some shade of green or blue, but lizard color varies dramatically within and among populations. On some islands lizards are brown, yellow or orange, or even black. On other populations lizards are a combination of these colors (Figure 1).

Figure 1. A map of the Pityusic Archipelago in Spain’s Mediterranean Sea and examples of male Ibiza Wall Lizards from various islands. This archipelago makes up the southernmost tip of the Balearic Islands. Ibiza and Formentera are the largest of the 42 islands and islets in the Pityuses that are home to populations of P. pityusensis. There is tremendous color diversity across this species’ range.Males from: (a) Bledes Plano; (b) Es Vedra; (c) Formentera, Es Trocadores; (d) Formentera, Cap de Barbaria; (e) Tagomago; (f) Ibiza; (g) Islas Negras; and (h) Formentera, central. In general, males are more colorful than females, but female color covaries with male color among island populations.

To get an introduction to this system, students can watch this video: http://vimeo.com/27056680

Questions 1. Name and describe five phenomena that can contribute to a population’s evolution.

2. Darwin described how organisms can evolve by means of natural selection. These ideas can be distilled into four main tenets. Name and describe these four tenets.

3. What is the difference between natural selection and sexual selection?

4. In sexual selection, what are the two ways that one sex can compete for mating access to the other sex? Give examples of these two forms of sexual selection in humans.

Part 2. Spectral Background InformationNate decided to manipulate the color of lizards by artificially coloring their skin. In order to create the tattoo ink mixtures that Nate used to manipulate lizard color in the color manipulation experiment (described above), he needed to make sure that the tattoo ink mixture realistically mimicked the coloration of colorful lizards. To do this, he used a spectrophotometer to measure the spectral reflectance of real lizards’ skin and compared these measurements to the spectral reflectance of the tattoo ink mixtures that he created (Figure 2).

Questions5. What range of wavelengths of light are visible to humans (human visible spectrum)?

6. For humans, which color corresponds to which wavelength(s)? red blue yellow violet

Which of these has the highest energy?

7. What possible optical mechanisms could contribute to the unusual, bright coloring of this lizard species?

Figure 2. Spectral reflectance curves of lizard scales reflecting ultraviolet (UV), blue, and brown.

8. Which curve corresponds to which color lizard scale? UV – Blue – Brown –

9. Judging from the curves in Figure 2, do you suspect that lizards might not have the same visual spectrum as humans? Explain.

10. If you put a bright blue lizard in a room that was completely devoid of all light, what color would it be?

Part 3. Estimating Population Density: Lincoln-Petersen IndexBefore Nate could perform the color-manipulation experiment described above, he needed to know how many lizards he could put into outdoor enclosures to mimic natural lizard densities. A commonly used method to estimate populations for which counting every member is not realistic is the capture-mark-release-recapture method. The Lincoln-Petersen Index can be used to estimate a total population size (N), based on the number of marked animals captured, marked, and then recaptured in a second capture event.

N = (total # tagged individuals) x (total # of individuals captured in second trial)(total # of recaptured individuals in second trial)

Nate put out 50 lizard traps and left them out for five hours. He permanently marked each of the captured lizards with a unique combination of beads, and then released the lizards back into the wild.

On the island of Espardell, which has an area of 500 m2, Nate captured and beaded 136 lizards in his 50 traps in five hours. A week later, he returned to Espardell and set up his 50 traps again. This time, he caught 121 lizards in five hours. Only 35 of these lizards had the permanent bead tags.

11. Estimate the population density of lizards on Espardell.

Part 4. The Color Manipulation Experiment

Most populations have lizards that are shades of green or blue. On the islands with blue or green lizards, conspicuous color increases in both saturation and coverage area on male lizards’ bodies as they increase in age and size: the most conspicuously colored male lizards are also the largest (Figure 3).

Nate suspects that conspicuous male coloration is the result of sexual selection, but he doesn’t know whether this sexual selection is from intrasexual selection or intersexual selection. Nate sets up an experiment to test whether color is subject to sexual selection and whether that sexual selection is driven by male-contest or female choice.

Figure 2. Examples of male P. pityusensis that vary in age, size and color expression. Conspicuous green and blue colors increase in both saturation and coverage area as lizards increase in size and age. Thus, the largest lizards are generally the most conspicuously colored lizards. (SVL=snout-vent length is the distance from the tip of the snout to the end of the cloaca (vent), just cranial to the tail.)

The Color-Manipulation ExperimentOne of the best ways to figure out the function of a particular trait (such as conspicuous color) is to manipulate that trait (e.g., increase or decrease the intensity of that trait) and see how that manipulation influences the ability of an organism to survive or reproduce. This is exactly what Nate did.

Nate and his assistants captured 40 male and 40 female lizards on the island of Formentera (where lizards range in color from brown to extremely blue). Using precise mixtures of human tattoo inks, Nate painted half of the males to look lijke the most colorful male lizards on Formentera. The remaining males were painted with water as controls (the color was not manipulated, but they underwent similar handling).

Nate then placed all the lizards into an 80 m2 semi-natural outdoor enclosure so that 20 color-enhanced and 20 control males could interact with each other and with the 40 female lizards. At the height of the reproductive season, Nate and his field assistants recorded and compared

1) the outcome of all male aggressive interactions involving color-enhanced males fighting control males2) the number of times either color-enhanced or control males courted females3) the number of times courtship attempts by color-enhanced or control males ended in copulation.

Some of the results of the color manipulation experiment appear in Figure 4.

Figure 4. In order, the histograms show 1) the outcome of all male aggressive interactions involving color-enhanced males fighting control males2) the number of times either color-enhanced or control males courted females3) the number of times courtship attempts by color-enhanced or control males ended in copulation.

Questions12. Do the results in Figure 4 indicate that color acts as a signal for intrasexual selection? Which

experiment(s), and why?

13. Do the results in Figure 4 indicate that color acts as a signal for intersexual selection? Which experiment(s), and why?

Part 5. Black Lizards, Orange LizardsA mutation in the MC1R - melanocorticoid receptor 1- gene causes an increased production of the black pigment melanin. Lizards expressing this mutation are black, or melanistic.

On the larger islands (Ibiza and Formentera) approximately 1 in every 1000 lizards is melanistic. However, on a small neighboring island called Bledes, virtually every lizard is melanistic (see Figure 1a).

14. List three potential explanations for the high abundance of black lizards on Bledes.

Nate collected tissue samples from individuals on these islands and conducted some genetic experiments. He found that individuals on Bledes are highly related and the population has significantly lower genetic diversity compared to other islands of similar size and lizard population densities.

15. Given this new information, which of your explanations from #14 is most likely?

On the island of Islas Negras, lizards feed on the flowers of the native shrub, Flavo virgultum, which is

contains the carotenoid pigment, -zeacarotene. Lizards acquire an orange coloration due to their diet of these carotenoid pigments.

On the neighboring island of Penjats, Flavo virgultum is slowly being replaced by Flos sanguine, introduced by flower farmers. This plant which contains the carotenoid pigment -carotene instead of -zeacarotene. The structures and absorption maxima of these compounds are shown in Figure 5.

Figure 5. -zeacarotene structure and maximal spectral absorbance (max) vs. -carotene structure and maximal spectral absorbance (max).

16. If the lizards eating Flos sanguine also incorporate the carotenoids into their skin pigmentation, do you expect their coloration to change with the new diet? In what way?

17. Could a change in male coloration affect sexual selection in the populations colored by their diets? Discuss.

Part 6. Evolution of Coloration: Multiple considerationsThe evolution of color patterns in animals can be complicated. In the Ibiza Wall Lizard, sexual selection is not the only factor that affects coloration. Other selective forces are at work.

For example, colorful male lizards may have higher mating success, but colorful males are also more conspicuous to predators. The phenotypes we see in nature are the result of a compromise between selective forces that favor and disfavor a particular trait.

Natural and sexual selection often influence individuals of different ages or sexes quite differently.Recall that color brightness and saturation increases with increase in lizard size.

18. If colorful male lizards are more successful, why aren’t all the male lizards colorful?

19. What mechanisms could maintain variation for traits that are subject to strong directional selection? List them here.

20. Would color evolve differently if the genes responsible for color were on the autosomal genome versus the sex chromosomes? Why?

21. If bright, saturated color increases male reproductive success, why do young lizards not fully express

this trait as early in life as possible? (These lizards can be sexually mature at a relatively small size.)

Discussion Questions1. Why are all male lizards not equally colorful (i.e. what mechanisms might maintain color variation)?

2. How might selection on color differ among individuals of different age groups or between the sexes of the same age groups?

3. Female lizards are also colorful. Why do think some females are as colorful as males? What could explain their male-typical coloration?

4. How might color evolve differently if the genes responsible for color were on sex chromosomes as opposed to the autosomal chromosomes?

Final ThoughtsThis case study was based on the doctoral research of Nathan Dappen, PhD. For his dissertation, he studied the evolution of color in both sexes and among age groups of the Ibiza Wall Lizard.

He found evidence suggesting that color evolves in male lizards as the result of sexual selection in male-male contest competition. In females, conspicuous color does not appear to have an adaptive function, and may be expressed as the result of sharing the genes for color with males (i.e. color genes are probably autosomal , not sex-linked).

He also found evidence suggesting juvenile lizards generally do not express full coloration because the costs associated with being colorful (increased predation, cannibalism, metabolic costs) are much higher for juveniles who do not get any of the reproductive benefits that color affords adult males. As a result, these lizards undergo a dramatic ontogenetic color change. Expression of the color genes is age-dependent.

Much research is still needed to understand why there are populations of orange, black, brown and yellow lizards. Perhaps one of you can help figure that out!