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The Effect of Opsin DNA Sequences on Color Vision in Humans
and Other Mammals
By Brian A. Johnson
The Eberly College of Science at The Pennsylvania State University
Abstract: This paper analyzes the relatedness between the different mammalian opsin
gene sequences to determine the phylogenetic relationship between them and their rates
of mutation in humans. Pairwise distance matrices were made using the Jukes-Cantor
model and were in turn used to create a Neighbor-Joining and Maximum Parsimony tree
for each of the three types of opsins. A relative rate test was done on human opsin to
determine the presence of the molecular clock, which did exist between red and green
opsins. The results of this research infer that these opsins evolve in order to adapt to the
environment that their species is faced with, and that the red and green opsin genes
evolve at the same rate due to their proximity on the X chromosome.
Introduction
Vision comprises the majority of sensory information that our body takes in to
assess its surroundings. Without it, individuals would need to rely on other senses that do
not offer the same amount of detail that sight provides. Humans typically maintain a form
of trichromatic vision, a type of sight that utilizes three different types of retina cone cell
receptors. For humans, these are blue opsin (short wavelength receptors), green opsin
(medium wavelength receptors), and red opsin (long wavelength receptors). Any
abnormalities in these cone cell receptors can reduce the amount of sensory information
that will be sent along the optic nerve to the brain’s visual cortex within the occipital
lobe. This entails the loss of color sensation for the particular colors that each opsin
receptor codes for. For example, a mutation in red opsin in cone cells of the retina would
make the eye unable to sense the longer wavelengths of visible light that a human’s brain
interprets as the color red.
Most mammals maintain dichromatic vision, with the exception of humans and
other closely related primates that are trichromats. Some marine mammals have even
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evolved monochromatic vision where they only have one type of light wavelength
receptor.5 The evolution and phylogeny of vertebrates has been determined such that life
essentially migrated from water, for fish and amphibians, to land, for reptiles and birds.
Birds in particular utilize tetrachromatic vision, which later went through selection to
become dichromatic vision in early placental mammals. This dichromatic vision once
again evolved in humans and closely related primates to finally achieve trichromatic
vision.6 This evolutionary change between different species of mammals that all
developed from a common ancestor makes a point that each species needed to adapt to its
own unique environment in order to survive and have the maximum amount of
reproductive fitness possible. Since marine mammals live in water for their entire lives,
they really would only need to sense light wavelengths that are visible in water for
survival, while terrestrial animals typically require a greater sensation of color to survive
in environments where there exists an increase amount of different types of light
wavelengths available.
Because of this seemingly related adaptation of opsin to accommodate different
environments, the goal of my research was to analyze the homologous opsin nucleotide
sequences between several mammalian species to determine the phylogenetic relationship
behind how these opsins in the retina changed from one another to create the unique
opsin sequences per each species. I compared the relatedness between the different
mammalian opsin gene sequences to determine the phylogenetic relationship between
them, as previously stated. Additionally, I looked to see how the three opsins were related
within humans, looking at how they evolved in relation to which chromosome they are
located on and using the relative rate test to determine if there was a molecular clock
between the human red and green opsin genes.
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There have been several studies conducted to support the theory that mammalian
species opsin sequences diverged from one another in response to their environments.
First, a study conducted on the blue opsin gene in nocturnal lemurs searched to discover
the selection of the number of short wavelength cones present in the lemurs’ retinas. The
results of the study found that lemurs living in areas of low light, where less short
wavelengths of light exist, had undergone selection to reduce both the number and
functionality of the blue opsin receptors. This was seen as a result of the lack of light in
their home environments, since lemurs that lived in high levels of light had the opposite
type of selection that promoted the number and function of the blue opsin receptors.7
The second form of support for this change comes from another study that
focused on the evolution of color vision in humans and their closely related primates. The
findings of the study showed that some of the evolution and selection relating to opsins in
these species depended on the psychological development of these species in response to
their environments and social surroundings. It provided an example of the red opsin
sequences evolving in humans and primates to permit these beings to examine the
feelings of other beings in their surroundings, as well as to exert their own feelings by
changing their own bodily coloration. The red opsin was particularly important,
according to the study, in the detection of anger and competition among humans and
primates. Being able to visualize the red color of competitors in their respective
environments allowed these mammals to process the possible threat facing them, since
red is the color expressed by the skin when one emotes anger or competition. This gave
humans and primates that could sense red an advantage for survival, and therefore was
selected for to fix the trait in the genome coded for by the red opsin gene.8
I chose to analyze these particular sequences of DNA because I am highly
interested in becoming an optometrist following my graduation from Penn State. The way
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that vision has changed throughout time fascinates me, and I believe it can give us clues
as to how to improve techniques for correcting vision problems in humans. My research
takes a look at how these homologous opsin sequences relate across several mammalian
species, while simultaneously comparing the three opsin sequences that code for human
color vision, providing a dual outlook at how and why vision has evolved.
Materials & Methods
For my analysis, I collected a total of 20 nucleotide sequences from GenBank.
Table 1, found two pages ahead, indicates the common name of the sequence, GenBank
accession number, and the origin of the data for each opsin sequence I used for my
research. I utilized the BLAST function to search for homologous genes of the human
opsin sequences, and selected sequences relating to other mammals that scored closely to
the human genes.
I analyzed the nucleotide data using MEGA software to create phylogenetic trees
with bootstrap values, calculated using 1000 replications for each tree, to assess the
relatedness of the homologous opsin sequences among species. I first needed to align the
sequences in their FASTA formats using the CLUSTALW2 online software, which then
provided me with a file that I could format in MEGA. I then calculated pairwise distances
using the Jukes-Cantor model to determine a distance matrix for each type of opsin
consisting of sequences from many species, as well as for the three types of human
opsins. I used these distances to construct two types of phylogenetic trees, NeighborJoining and Maximum Parsimony, using 1000 bootstrap replications for each of the three
distance matrices I formatted for the three types of opsin across mammalian species. I
made these two types of trees in order to get two variations of the relatedness of my data
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since the Neighbor-Joining method seeks to have the shortest tree length, while the
Maximum Parsimony method creates a tree that supposes the least evolutionary change.
I then analyzed the bootstrap values of these trees to determine the relatedness of
the species opsin sequences. I used the Jukes-Cantor substitution model since it is the
simplest model that only contains one parameter, the overall substitution rate, and does
not account for transitions and transversions between the sequences. I did not account for
the identity of these changes because my research was more concerned with the overall
picture of the evolution between the opsin sequences, and not how individual nucleotide
substitutions affected the selection process.
For the comparison of only the human opsin sequences, I created a Neighbor
Joining tree without bootstrap values. The main reason I created this tree was not only to
distinguish a relationship between the human opsin sequences, but also to provide a
visual alongside my calculation of the existence of a molecular clock between the red and
green human opsin sequences. I conducted the relative rate test to determine the existence
of the molecular clock, hence providing me information about whether or not the
sequences evolved at a similar rate. I used the results of this test to determine how the
chromosomal positioning of the green and red opsin, both located on the long arm of the
X chromosome,2, 3 affected these two sequences divergence from the blue opsin, located
on chromosome 7.4
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Table 1: Opsin Nucleotide Sequence Information
Species
Type of Opsin
GenBank Accession #
African Bush
Elephant
Crab Eating
Macaque
Florida Manatee
Blue
XM_003407257
Source of Data
(PubMed #)
NCBI RefSeq
Blue
AF158977
(11809481)
Blue
XM_004382643
NCBI RefSeq
Humans
Blue
NM_001708
(20801516)
Northern White
Cheeked Gibbon
Olive Baboon
Blue
XM_003261297
NCBI RefSeq
Blue
XM_003896561
NCBI RefSeq
Sumatran Orangutan
Blue
XM_002818421
NCBI RefSeq
Western Lowland
Gorilla
Crab Eating
Macaque
Humans
Blue
XM_004046176
NCBI RefSeq
Green
AF158975
(11809481)
Green
NM_000513
(23139274)
Northern White
Cheeked Gibbon
Olive Baboon
Green
XM_003279299
NCBI RefSeq
Green
NM_001168739
Sumatran Orangutan
Green
XM_002832301
Inferred RefSeq from
DP000488.1
NCBI RefSeq
Western Lowland
Gorilla
African Bush
Elephant
Crab Eating
Macaque
Humans
Green
XM_004065128
NCBI RefSeq
Red
XM_003421718
NCBI RefSeq
Red
AF158968
(11809481)
Red
NM_020061
(23139274)
Killer Whale
Red
XM_004286442
NCBI RefSeq
Olive Baboon
Red
NM_001168797
West Indian Manatee
Red
XM_004390580
Inferred RefSeq from
DP000488.1
NCBI RefSeq
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Results
The results of my research have provided me with three solid sets of trees to
analyze between the three opsins, and a conclusive relative rate test to determine the
existence of a molecular clock between human red and green opsins. The figures on the
following pages depict the phylogenetic trees and calculations of my research.
Figure 1 depicts the trees for blue opsin among different mammalian species. It
can be seen in each tree that the human blue opsin relates strongly, bootstrap values over
90 for both trees, to gorilla blue opsin. Other pairs of strong relations according to high
bootstrap values in each tree include the olive baboon and crab eating macaque
relationship and the Florida manatee and African bush elephant relationship. The
northern white cheeked gibbon has a weak relationship with the human/gorilla branch in
each tree, while the Sumatran orangutan seems to be an out group for the trees. The weak
relationship of the gibbon points to the possibility that it also could be used as an out
group in the tree, however it is more related to the other sequences than the orangutan’s.
The fact that these two primate species could be used as out groups allows for the
inference that they have retained the most primitive sequences amongst these mammals,
and therefore their sequences could be used as bases for analysis of the changes that have
occurred in the other mammalian sequences.
Figure 2 depicts the trees for green opsin among different mammalian species.
The bootstrap value for the olive baboon and the crab eating macaque is once again very
high, showing a direct relationship. The two other relationships (human/orangutan and
gibbon/gorilla), however, both have weak bootstrap values that do not support these
groups relatedness. Another factor that makes these trees only able to determine the
baboon/macaque relationship is that the Neighbor-Joining tree and Maximum Parsimony
tree place the two weakly related pairs in different positions juxtaposed to the
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baboon/macaque relationship. Even though the Maximum Parsimony tree contains higher
bootstrap values, it is still an insufficient measure to accurately relate the different
groups. This is peculiar that these trees prove somewhat ineffective as RepeatMasker
revealed a simple repeat with the nucleotide sequence of CAACAC that was present in all
six of the green opsin sequences.
Figure 3 depicts the trees for red opsin among different mammalian species. It is
interesting to note that these trees are identical for both the Neighbor-Joining and
Maximum Parsimony methods, with the exception of the pairing of the manatee with the
elephant in the Neighbor-Joining tree. These trees continue the trend of the strong
baboon/macaque relationship with bootstrap values of 100 in each tree. In fact, both of
the trees for red opsin have bootstrap values that all equal 100, exemplifying a complete
relatedness between the species that are paired with one another. These trees are possibly
the most revealing of the three opsins as it gives a definite and complete picture of the
relationship between species. The pairing of the manatee and elephant in the NJ tree can
be inferred to be a result of that specific method, as it aims to reduce tree length. The
Maximum Parsimony tree, however, aims for the arrangement with the least possible
amount of evolution, which allows one to infer that the manatee, a marine mammal,
should be used as an out group to all of the land mammals, including the elephant.
Figure 4 depicts the comparison of the three types of opsin within humans.
Shown below the tree is the distance matrix used to build the tree that was also used in
the relative rate test to determine the presence of a molecular clock. It was determined
that there is a molecular clock between red and green opsins in humans, which is most
likely due to the fact that these two sequences are located extremely close to one another
on the X chromosome and would assumingly have similar rates of evolution.
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Neighbor-Joining
Figure 1: Blue Opsin Neighbor-Joining and Maximum Parsimony Trees with Bootstrap
99
46
Western Lowland Gorilla Blue O
Humans Blue Opsin
98
Northern White Cheeked Gibbon
Sumatran Orangutan Blue Opsin
Olive Baboon Blue Opsin
100
Crab Eating Macaque Blue Opsin
Florida Manatee Blue Opsin
100
Max Parsimony
100
African Bush Elephant Blue Ops
Olive Baboon Blue Opsin
Crab Eating Macaque Blue Opsin
98
Florida Manatee Blue Opsin
100
African Bush Elephant Blue Ops
Northern White Cheeked Gibbon
Western Lowland Gorilla Blue O
59
93
Humans Blue Opsin
Sumatran Orangutan Blue Opsin
Max Parsimony
Neighbor-Joining
Figure 2: Green Opsin Neighbor-Joining and Maximum Parsimony Trees with Bootstrap
38
Northern White Cheeked Gibbon
Western Lowland Gorilla Green
Humans Green Opsin
59
Sumatran Orangutan Green Opsin
Olive Baboon Green Opsin
100
Crab Eating Macaque Green Opsi
100
Olive Baboon Green Opsin
Crab Eating Macaque Green Opsi
Northern White Cheeked Gibbon
44
Western Lowland Gorilla Green
Humans Green Opsin
63
Sumatran Orangutan Green Opsin
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Neighbor-Joining
Figure 3: Red Opsin Neighbor-Joining and Maximum Parsimony Trees with Bootstrap
Olive Baboon Red Opsin
100
100
Crab Eating Macaque Red Opsin
Humans Red Opsin
Killer Whale Red Opsin
African Bush Elephant Red Opsi
100
West Indian Manatee Red Opsin
Olive Baboon Red Opsin
Max Parsimony
100
100
Crab Eating Macaque Red Opsin
100
Humans Red Opsin
Killer Whale Red Opsin
African Bush Elephant Red Opsi
West Indian Manatee Red Opsin
Figure 4: Human Opsin Neighbor-Joining Tree and Distance Matrix
with Relative Rate Test Data
Human Opsin
Green
Red
Blue
Green
Red
0.022
0.521
0.527
Relative Rate Test for Molecular Clock: Green = A
Red = B
Dac = 0.521 Dbc = 0.527 D = -0.006
D ~ 0 = Molecular Clock
SD = 0.003
Blue
Blue = C
Absolute Value of D < 2 X SD  Therefore statistically insignificant, and unable to
reject the null hypothesis.
Conclusion: Molecular Clock exists between Human Red and Green Opsins.
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Discussion
The data resulting from my research shows that opsins evolve in order to adapt to
the environment that their species is faced with. The red and blue opsin trees, which
contained mammalian species that were not all primates, placed the two and three, red
and blue tree respectively, non-primate species apart from the primates. This would make
sense as these non-primate species, the elephant, manatee, and killer whale, have
different environments that they live in. Open space on the savannah, for the elephant,
and water, for the manatee and killer whale, provide completely different survival
challenges compared to the normally forested, plant-infested environments of the
primates. These challenges require different sensory information to be obtained by the
species brain in order to survive achieve the highest amount of reproductive fitness.
The green opsin sequences in my research came solely from primates, which
continues to strongly support my theory that opsin sequences develop depending on the
species environment. Since these green opsins detect the color green in the primates, it
makes perfect sense as to why primates would need these opsins, and other mammalian
species would not. The forested environment of primates requires that they be able to see
the green of the plants they are surrounded by. If the primates did not acquire this green
opsin, it would be likely that predators would be able to hide amongst the foliage, putting
the primates at great risk for predation, and thus a lower reproductive fitness level. The
common repeat that was found amongst these green opsin sequences further cements the
fact that the primates evolved the sequence from a common ancestor, as each primate has
the identical CAACAC repeat.
Finally, the red and green opsin genes in humans were determined to have similar
evolution rates. The main reason for this molecular clock, tested for by the relative rate
test, is most likely due to their proximity on the X chromosome. Another reason that
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accounts for this similar rate between red and green opsins in humans, is supported by
other literature about color blindness. It has been found that various forms of color
blindness, caused by the loss of one of the three opsins resulting in dichromacy, are the
result of X-linked cone dysfunctions.1 The majority of color blindness is red-green color
blindness, or the inability to decipher between the two, and previous research seems to
indicate that the red opsin in humans is normally the cause of this abnormality. Since
both the red and green opsin are located very closely on the X chromosome it would
make sense that these two colors are the main ones that humans have trouble seeing. The
molecular clock that exists for red and green opsin is the cause for the red and green color
confusion in the majority of color blindness. Because both of these opsins evolve at the
same rate, and on the same chromosome, the sensory interpretation of each is similar,
therefore making it easy for someone with defective opsins to confuse the two colors
since they have similar genetic coding.
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References
1) McClements, M., W. Davies, M. Michaelides, T. Young, M. Neitz, R. MacLaren, A.
Moore, and D. Hunt. "Variations in Opsin Coding Sequences Cause X-linked Cone
Dysfunction Syndrome with Myopia and Dichromacy." Investigative Ophthalmology
& Visual Science 54.2 (2013): 1361-369. Print.
2) "OPN1LW." Genetics Home Reference. N.p., 16 Apr. 2013. Web.
3) "OPN1MW." Genetics Home Reference. N.p., 16 Apr. 2013. Web.
4) "OPN1SW." Genetics Home Reference. N.p., 16 Apr. 2013. Web.
5) Rowe, Michael H. "Trichromatic Color Vision in Primates." Physiology 17.3 (2002):
93-98. Print.
6) Strauss, James A. "Mini Review of Vertebrate Evolution and Phylogeny." BIOL 411
Class Notes. State College: ProCopy, 2013. 39. Print.
7) Veilleux, C., E. Louis, and D. Bolnick. "Nocturnal Light Environments Influence
Color Vision and Signatures of Selection on the OPN1SW Opsin Gene in Nocturnal
Lemurs." Molecular Biology and Evolution (2013): n. pag. 21 Mar. 2013. Web.
8) Weidemann, D., R. Barton, and R. Hill. "Chapter 18: Evolutionary Perspectives on
Sport and Competition." Applied Evolutionary Psychology (2011): 1-29. Print.