STUDENT’S GUIDE Case Study - DNA to Darwin · Case Studes /M_Q ?`aPUQ_ Case Study Evolution of colour ... green and blue receptors, ... MWSLWS MWSLWS MWSLWS. 12

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Case Study

Evolution of colour vision in primates

Version 1.3

Jarosław BrykMax Planck Institute for Evolutionary Biology, Plön

STUDENT’S GUIDE

Copyright © Jarosław Bryk, 2011

evolution of colour vision in primates

www.dnadarwin.org2

Evolution of colour vision in primates In this activity you will explore the molecular basis and evolutionary origin of trichromatic (red/green/blue) colour vision in humans and our close evolutionary relatives using nucleic acid sequences of opsins, key proteins involved in the process.

Colour vision evolved relatively early: most bony fish, reptiles and birds have excellent colour vision. The first mammals, however, are thought to have been largely nocturnal species. For them, the ability to see in low-light conditions was at a premium, rather than the ability to see in colour. The ancestors of modern mammals consequently lost colour vision at the time of the dinosaurs. Some primates, including humans of course, have re-evolved colour vision. This ability applies mainly to Old World species. In contrast, with the exception of Howler monkeys, all male and some female New World primates lack colour vision.

Most — 80% according to some estimates — of the information our brains receive about their surroundings is transmitted via the sense of sight. Seeing is such a good way of experiencing the environment that camera-like eyes have evolved independently more than 50 times. In mammals, light is focused by the lens on a layer of specialised cells (cones and rods) in the retina that contain visual pigments. These pigments are proteins called opsins or rhodopsins. Each cone cell contains one of three sorts of opsins. The different opsins are each sensitive to a different range of wavelengths of light. Sometimes these are referred to as red, green and blue receptors, although their peak sensitivities are violet, green and yellow-green.

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Section through the retina. Notice how badly ‘designed’ the mammalian eye is: the photoreceptors (the rods and cones) face away from the light source.

Nocturnal South American ‘night monkeys’ (Autus spp.), like most New World primates, lack full-colour vision.

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Opsins are evolutionarily ancient proteins that take part in the process of colour vision, while rhodopsins are involved in dim light vision. In this exercise, we will concentrate on the opsins.

Each opsin is bound to 11-cis-retinal, a derivative of vitamin A. The 11-cis-retinal is the light-sensitive molecule, but the way it reacts to light depends on the opsin molecule it is attached to. Different opsins absorb different wavelengths of light, and each type has evolved so that it is sensitive to a specific, narrow range of wavelengths. This reaction can be studied experimentally: opsin proteins can be synthesised and/or mutated in cells on a Petri dish, isolated and then mixed with 11-cis-retinal to obtain visual pigments. The absorption spectra of these pigments can be then measured in a spectrophotometer to identify the wavelengths of light they absorb. Thus it is possible to compare properties of different visual pigments and identify features responsible for their fine-tuning.

Humans are trichromats: we have three distinct opsins encoded by three genes, named after the range of wavelengths they absorb: SWS absorbs short-wavelength light, MWS, medium-wavelength and LWS, long-wavelength light. The SWS opsin is encoded by a gene on chromosome 7 and is sensitive to blue/violet light of 430 nm. The MWS and LWS opsins, subjects of the following exercises, are both located on the X chromosome and are sensitive to 530 nm (green) and 560 nm (green/yellow) light, respectively.

Structure of an opsin. Opsins are trans-membrane proteins. There are seven helices in the protein which are anchored in the cell membrane. The 11-cis-retinal (shown in red) lies near the centre, within the helices.

Let’s start by comparing the nucleic acid* sequences of human MWS and LWS opsins to see exactly how different they are.

* the sequences are mRNA sequences in which the uracil (U) of RNA has been replaced by thymine (T). Hence they look like DNA. RNA sequences are stored in GenBank (an online database of sequence data) in this way.

Simplified human cone response curves. SWS= short-wavelength-sensitive opsin; MWS= medium-wavelength-sensitive opsin; LWS = low-wavelength-sensitive opsin.

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Comparing human MWS or LWS opsins

1. Double-click on the human_green_red.geneious document. This will start the Geneious software and load two files of nucleic acid sequence data into the programme. Note: If a box appears over the Geneious start-up screen, saying that your trial of the ‘Pro’ version has ended, click on Use Geneious Basic.

2. The main Geneious window will now show the protein-coding part of the mRNA sequences of human MWS (green-light-sensitive) and LWS (red-light-sensitive) opsins. Remember, these will look like DNA because the ‘U’ (uracil) of RNA has been replaced by a ‘T’ (thymine).

3. Ensure that the two files are highlighted in the upper Geneious window, then click the Alignment button. Click OK to accept the default Geneious settings. The programme will now align both of the sequences, and a new file of the aligned sequences will appear in the top panel and be displayed in the main Geneious window:

RNA sequencesNames

Zoom buttons

Alignment button

Selecting ‘Wrap’ here means you can scroll down through the sequences.



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4. Use the magnifying glass buttons to zoom in on the data. Quickly scroll through the data, looking for any differences between the two sequences. Don’t spend too long on this — you just want to obtain a general impression of the data.

Translate button

Questions

a. Do you think there are many differences between the two sequences? What is the sequence similarity between the two genes? Hint: use the Statistics panel in Geneious to obtain this information (the % tab on the lower right of the screen).

b. Which of the differences between the sequences might be responsible for the different properties of the proteins? Can you tell from the RNA sequences themselves? Do you think that looking at the amino acid sequences might be more helpful? Hint: click on the Translate button to see the amino acids encoded by the mRNA.

c. How might a scientist be able to test whether the differences in the proteins influence their sensitivity to different wavelengths?

The MWS and LWS opsin genes are very similar. In fact, their DNA sequences are almost 98 % identical, even including the non-coding sequences of their introns. There are only 24 nucleotides that differentiate the two coding sequences, which corresponds to 15 amino acid changes between the proteins encoded by these genes (why not 24 amino acid changes?).

Each of these amino acid changes influences the sensitivity of an opsin to certain wavelengths of light. Thanks to experimental work on opsins in vitro we now know that the most critical changes are in amino acid positions 180, 277 and 285. If you know which amino acids are in these three positions, it is possible to predict which wavelength of light a particular opsin will be sensitive to.

Amino acid positionOpsin Max. absorption (nm) 180 277 285

Human MWS (green) 561 S (serine) Y (tyrosine) T (threonine)

Human LWS (red) 530 A (alanine) F (phenylalanine) A (alanine)

The Geneious statistics panel



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Both the MWS and LWS genes are positioned very close to each other and in a head-to-tail fashion on chromosome X in all species of Old World monkeys, which, in addition to the high sequence similarity, suggests that they originated by duplication. This is one of the major ways new genes can emerge, and we can use their sequences to explore when the duplication occurred and to work out which sequence is likely to be the ancestral one.

Question

d. How might you check whether the MWS and LWS duplications occurred independently within humans and other species of Old World primates or whether they have had a common origin before the Old World monkeys evolved?

The origin of MWS and LWS opsins

We can answer the question above by comparing pairs of sequences (of MWS and LWS opsin genes) from several species of Old World primates.

If all of the MWS and LWS sequences are descendants of an ancient duplication, then the MWS sequences should be more similar to one another than they are to the LWS sequences. This would give a tree similar to Possibility One, below.

If, however, the duplications occurred independently in each of the species, the MWS and LWS sequences should be most similar within each species, corresponding to Possibility Two, below.

Let’s investigate these two possibilities using mRNA sequences from a few species of Old World monkeys.

Outgroup

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POSSIBILITY ONEAncient duplication of opsin gene(Duplication events marked with a blue dot)

POSSIBILITY TWOIndependent duplication of opsin gene(Duplication events marked with a blue dot)

X chromosomewith MWS and LWS genes

MultipleMWS genes

SingleLWS gene

Chromosome 7with SWS gene

Location of the opsin genes in humans. The genes on the X chromosome are arranged head-to-tail.



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5. Double click on the origin_green_red.geneious document. This will load a file of 13 ready-aligned mRNA sequences into the programme.

6. The main Geneious window will show the mRNA sequences of fragments of the MWS and LWS opsins from a human, a chimpanzee, a gorilla, a macaque, a diana monkey and a talapoin monkey. There is also sequence of chicken iodopsin to be used as an outgroup, to which all other sequences will be related to in a phylogenetic tree.

7. Click the Tree button to open tree settings panel. Select Tamura-Nei as the Genetic Distance Model, Neighbor-Joining as the Tree build Method and use Chicken iodopsin as an outgroup. Select the Resample tree option and change the Number of replicates to 1000 and Support Threshold % to 5. Ensure that Create Consensus Tree is selected. Leave all the other options at the default values and click OK to build the tree.

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www.dnadarwin.org8

Questions

e. How are the MWS opsins and LWS opsins grouped on the tree?f. Which of the two possible patterns of evolution suggested in the two

diagrams on page 6 does your tree support? (Independent duplication the opsin in each species, or duplication in an ancestor of all the species?)

Now let’s see what the tree would look like if we add MWS and LWS opsins from a more distantly-related, New World species — a howler monkey.

8. Double-click on the origin_green_red_howler.geneious document. This will load some aligned mRNA sequence data into the programme.

9. The main Geneious window will now show the mRNA sequences of part of the MWS and LWS opsins from a human, a chimpanzee, a gorilla, a diana monkey, a talapoin monkey, a macaque (all Old World primates) plus a New World howler monkey. As before, there is also the sequence of chicken iodopsin, to act as an outgroup.

10. Click the Tree button to open tree settings panel. Select Tamura-Nei as the Genetic Distance Model, Neighbor-Joining as the Tree build Method and use Chicken iodopsin as an outgroup. Select the Resample tree option and change the Number of replicates to 1000 and Support Threshold % to 5. Ensure that Create Consensus Tree is selected. Leave all the other options at the default values and click OK to build the tree. Red howler monkey

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Questions

Study the tree you have just produced.

g. Howler monkeys can see in full colour: they are, like most humans and Old World monkeys, trichromatic. What can you tell about the origin of trichromatic colour vision in the howler monkey from this tree?

h. Can you tell which of the sequences — MWS or LWS — is ancestral (in other words, which gene was duplicated: MWS or LWS?)

i. How do we know that howler monkeys did have a gene duplication and not just different alleles of the same gene?

Different ways of evolving trichromatic colour vision

The howler monkey is a unique New World species that has acquired trichromatic colour vision by gene duplication, similar to the one that happened in Old World monkeys. All other New World monkey species that have evolved trichromatic vision have done so without gene duplication.

Question

j. Could you suggest how it would be possible to achieve trichromatic colour vision with just two genes? Would it be possible with just one?

Almost all species of New World monkeys have trichromatic colour vision based on only two genes. One of these genes (short-wavelength sensitive, which is sensitive to blue light), similarly to Old World monkeys, is located on an autosome. The other opsin gene is on the X chromosome. In contrast to the Old World species, the gene on the X chromosome did not undergo a duplication but it exists as different alleles in the population. This means that alleles of MWS gene located on each of the X chromosomes may have different sequences and thus, the proteins encoded by these alleles may have different absorption spectra. And because one of the genes is on the X chromosome, it also implies that the colour-sensing abilities of the two sexes may be different.

Questions

k. What might the differences be between male and female New World monkeys? Compare and contrast the situation in New World monkeys with humans suffering from colour blindness.

l. Trick question: Do you think that different alleles of MWS and LWS genes exist in humans? If you think they do, how might this affect the colour vision abilities of humans? If you think we don’t have different alleles, why not?

Colour blindness

Colour blindness, sometimes called ‘Daltonism’ after the English chemist who first described it in detail, is most commonly inherited from mutations on the X chromosome. The human genome project has shown, however, that mutations capable of causing colour blindness originate from at least 19 different chromosomes and 56 different genes.

Brown howler monkey

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www.dnadarwin.org10

In New World monkeys, with the exception of the howler monkey, males are dichromatic: they only have an SWS (blue-light-sensitive) opsin on their autosome and an MWS (green-light-sensitive) opsin on their X chromosome. But because the MWS opsin gene is very polymorphic (there are many alleles in the population) and proteins encoded by the different variants of the gene may be sensitive different wavelengths, individual males may see the world differently. In fact, in New World monkeys scientists do not call the opsins MWS or LWS, but simply give them numbers indicating the wavelength with the maximum absorption of light for that variant of the opsin. In any case, New World monkey males are all colour blind, but each individual may be blind to a different colour.

New World monkey females, in contrast, can be trichromats if the two alleles on their two X chromosomes produce proteins sensitive to different wavelengths. If the females have identical alleles on both X chromosomes, then they are dichromats and, just like the males, they can be colour blind to different colours.

Colour blindness

The inability to distinguish red and green colours is the most common form of colour blindness in humans, affecting up to 10% of population. It is caused by missing or mutated MWS or LWS opsin genes.

Question

m. Why are men colour blind much more often than women are?

Male New World monkey

The chemist John Dalton was the first person to describe red-green colour blindness (his brothers’ and his own) in detail. He requested that his eyes were dissected after his death because he suspected that the reason for his condition was blue fluid in his eyeballs. After his death this was shown to be false, but some 150 years later DNA from his preserved eyeballs (shown on the left) was analysed and it showed that Dalton had had deuteranopia: his MWS gene was missing.Ja

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Autosomewith SWS gene

X chromosomewith opsin allele

DichromatColour blind

Female New World monkeys

DichromatColour blind

TrichromatColour vision



www.dnadarwin.org11

Let’s now compare opsin sequences from the Old and New World primates to see how they are related.

11. Double click on the origin_new_old_world.geneious document. This will load a file of 26 aligned mRNA sequences into the programme. The main Geneious window will show the mRNA sequences of part of the MWS and LWS opsins from the Old World primates we used before and, in addition, some New World monkeys: a spider monkey, a squirrel monkey, a weeper capuchin and a marmoset. As before, there is also a sequence of chicken iodopsin to be used as an outgroup, to which all other sequences will be related in a phylogenetic tree.

12. Click the Tree button to open tree settings panel. Select Tamura-Nei as the Genetic Distance Model, Neighbor-Joining as the Tree build Method and use Chicken iodopsin as an outgroup. Select the Resample tree option and change the Number of replicates to 1000 and Support Threshold % to 5. Ensure that Create Consensus Tree is selected. Leave all the other options at the default values and click OK to produce the tree.

Questions

n. Identify which species on your tree are Old World primates, and which are New World primates.

o. Describe how the MWS and LWS opsins of the Old World primates are distributed on the tree.

p. Some of the New World monkeys have their opsins described as a number. The number is the wavelength the opsin is sensitive to. Describe how the New World monkey’s opsins are arranged on the tree.

q. Do you think the New World opsins are more closely related to the MWS or the LWS opsins of Old World primates?

Convergent evolution of colour vision in Old and New World monkeys

One of the possible explanations for this pattern of variation is that is it a result of convergent evolution. In many different species, a similar sensitivity of opsins is achieved with the same mutations, thus making even unrelated sequences quite similar, confusing the phylogenetic relationships between species. As you have seen in the first exercise, there are 24 mutations differentiating MWS from LWS opsin genes in humans, but only three of these mutations are responsible for majority of the sensitivity shift. Let’s compare sequences from other species that have similar sensitivity to human MWS and LWS opsins to see what mutations are responsible for their properties.

Spider monkey

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13. Double click on the convergence561.geneious document. This will load the file of ready-aligned mRNA sequences into the programme.

14. The main Geneious window will now show part of the protein-coding mRNA sequences of pairs opsins from a chimpanzee, howler monkey, a weeper capuchin monkey and a squirrel monkey.

15. Use the Translate button to translate the nucleotide sequence into protein sequence.

16. Ensure that the sequences are selected in the top window, then click on the Alignment button. When the panel appears, click OK to accept the Geneious default settings. The programme will now align the amino acid sequences.

17. Use the magnifying glass buttons to zoom in on the data.

18. Scroll through the data, and look at positions 34, 89 and 97 (they are equivalent to positions 180, 277 and 285 in the full-length opsin sequence). Compare what you find with the data in the table on page 5.

All of these species achieve the 561 sensitivity of their opsins by identical key mutations as in Old World monkeys, even though these sequences are distantly related. Indeed, if we compared the whole sequences of many opsins sensitive to certain wavelengths, we would find out that apart from those key mutations they are otherwise different, thus emphasising the importance and uniqueness of the key amino acid positions in the opsins.

Questions

r. Can you think of the reasons why there seems to be a limited number of ways to evolve opsins sensitive to certain wavelengths?

s. Last but not least: why do you think colour vision evolved in the first place?

Further readingThe making of the fittest. DNA and the ultimate forensic record of evolution by Sean B. Carroll (2009) Quercus Books (Paperback) ISBN: 978 1847247247. A popular account of some of the molecular evidence for evolution. Chapters four and six cover the evolution of colour vision in primates.

Additional references are given in the Teacher’s guide.

Documents

STUDENT’S GUIDE Case Study - DNA to Darwin · Case Studes /M_Q ?`aPUQ_ Case Study Evolution of colour ... green and blue receptors, ... MWSLWS MWSLWS MWSLWS. 12