Introduction
The melanocortin 1 receptor (mc1R) is a gene that has been implicated in the wide
variety of colors that exist in nature. It is responsible for hair and skin color in humans and the
various colors that can be seen in animals across the globe. The extent of the importance of the
gene is continually undergoing research, but is already known to be the only gene that controls
the observed variations in human pigmentation (Rees 200). One way to determine the importance
of a gene is to observe the evolution of it between and among different species. Canis lupus
familiaris, or the dog, is an ideal species to use due to the wide variety of colors that exist among
the different breeds. However, humans have played an important role in the domestication of the
present day dog, and artificial selection may sway the results. However, by comparing many
breeds of dogs to other carnivores and omnivores, both closely and distantly related, and using
the results to observe the evolution of mc1R across species, we will determine the importance of
the gene.
It is well known that the wolf is the closest relative of the dog. We will use the sequences
for the mc1R gene from multiple wolf breeds to compare the various dog breeds to determine
which wolf breed is closest related to the dog. We will also use the mc1R sequences from other
carnivores and omnivores to create trees that will tell us how well the gene has been conserved
across species. This will tell us how important the gene is, has been, and can be predicted to be
throughout the years.
The species that were used were all either omnivores or carnivores, the majority of which
are also land mammals. The dog breed sequences that were used consisted of a Boxer,
Greyhound, Eurasier, Siberian husky, Chowchow, Tibetan Mastiff, and the Alaskan Malamute.
The dog breeds were then compared to three different breeds of wolf; the Eurasian wolf, Arctic
wolf, and Gray wolf. The other land mammals that were used were a bear, fox, Arctic fox,
raccoon dog, human, chimpanzee, Fisher, jaguarundi, Tasmanian devil, gopher, and mouse. Most
of the species used are very well known. However, the raccoon dog, fisher, jaguarundi, and
Tasmanian devil are a little less talked about. The raccoon dog (Nyctereutes procyonoides) is a
type of wild dog, about the size of a fox, which has markings that resemble a raccoon. Its habitat
and many of its behaviors are very similar to that of a wolf or dog (WAZA 2014). It was
included in the analysis due to its similarities between both the wolf and the dog. The fisher
(Martes pennanti) is a carnivorous member of the marten family that resides in the forests of
North America (Kyle et al. 2002). The jaguarundi (Puma yagouaroundi) is a close relative to the
puma and lives in regions of Atlantic Rainforest and eucalypt plantations of southeastern Brazil
(Tofoli et al. 2009). It was included in the analysis to provide multiple comparisons between
different members of the feline family. The Tasmanian devil (Sarcophilus harrisii) is a
carnivorous marsupial that is endemic to the Australian island of Tasmania. It was included in
the data analysis because it is unique since it is the largest carnivorous marsupial, but also
possesses some qualities that are similar to a dog or a wolf (Jones et al. 2008). Finally, a
Weddell seal, alligator, and killer whale were also included.
The hypothesis is that there will be positive or diversifying selection among the different
dog breeds due to the influence of humans and artificial selection. Artificial selection would
allow humans to select for more appealing colors and phenotypes that would alter natural
evolution of the gene (Lindblad-Toh et al. 2005). However, across species, the hypothesis is that
there will be purifying selection that will confirm the importance and conservation of the mc1R
gene. It can also be assumed that the dog breeds will be closest related to the wolf breeds.
Materials and Methods
The data analysis consisted of the alignment of the various sequences, the production of
phylogenetic trees, and calculations to determine what, if any, kind of selection was present
between the various dog breeds. The sequences were found using the gene search for mc1R on
the National Center for Biotechnology Information website. The accession numbers for each of
the sequences can be found in Appendix A. The first step was to align the sequences using
CLUSTALW. Once the sequences were aligned, the next step was to create distance matrices
and phylogenetic trees. Two distance matrices were created; one using the Jukes and Cantor
model and one using Kimura’s model. This was accomplished using the program for Molecular
Evolutionary Genetics Analysis, or MEGA. Using MEGA, UPGMA (unweighted pair group
method with arithmetic mean), maximum likelihood, and maximum parsimony trees were
created. The UPGMA and maximum likelihood trees were created using both the Jukes Cantor
model and Kimura model, resulting in two of each of the two kinds of trees. Each tree also used
the bootstrap method with 1000 replicas. For both UPGMA and maximum likelihood trees, a
bootstrap cutoff value of 50 was applied. Multiple bootstrap trees were also created for the
maximum parsimony tree; one with 30% certainty and one with a 90% certainty. The various
trees with different certainties provide insight into the probably relationships between the
sequences, and then a description of how likely the relationships really are. After producing the
distance matrices and then the phylogenetic trees, we then analyzed a few of the different
relationships among breeds of dog using Tajima’s D test for neutral selection, also in MEGA.
We tested the relationship between the various dog sequences to determine which, if any, type of
selection was acting between the various breeds. Along with the selection test between dogs, we
also ran the Tajima D test between the three subspecies of wolves. Using all of the data from
MEGA, we were able to visualize and quantify the relationships between the various species and
breeds to answer our questions and determine the accuracy of our hypotheses.
Results
41
34
40
ArcticWolf
ArcticWolf
Greyhound
Greyhound
EurasianWolf
EurasianWolf
GrayWolf
44
GrayWolf
100
TibetanMastiff
100
33
69
TibetanMastiff
Boxer
Boxer
Eurasier
Eurasier
Chowchow
100
Chowchow
100
SiberianHusky
85
97
AlaskanMalamute
SiberianHusky
97
RacoonDog
60
83
57
33
85
RacoonDog
Fox
70
93
Fox
ArcticFox
ArcticFox
93
Bear
Bear
WedellSeal
WedellSeal
Jaguarundi
Jaguarundi
Fisher
Fisher
KillerWhale
100
KillerWhale
Human
100
Chimpanzee
100
TasmanianDevil
Alligator
100
Gopher
100
Mouse
Image 1. Maximum Parsimony Tree with Bootstrap Cutoff at 30%
Human
Chimpanzee
TasmanianDevil
61
AlaskanMalamute
Alligator
Gopher
100
Mouse
Image 2. Maximum Parsimony Tree with Bootstrap Cutoff at 90%
These trees show the results of a maximum parsimony analysis with 1000 bootstrap
replicates. Image 1 represents a condensed tree with a 30% certainty value while Image 2 is a
condensed tree cutting off the bootstrap to include only taxa separation above 90. The numbers
indicate the percentage of data sets that group these taxa together. This tree shows the MC1R
gene in the seven breeds of dogs being the most closely related to the three different species of
wolves. Along this branch of the tree is another subtree shows the relationship between the
Siberian husky and the Alaskan malamute. The next closest related species to the dogs and
wolves are the raccoon dog, artic fox and fox. Without the 30% cutoff, the tree shows a subtree
that relates the gene in the fox to the Arctic fox. The 30% tree shows that the bear and Weddell
seal have a similar form of MC1R but because this number is quite low, we refer to the second
tree with the cutoff showing that they are just a related to each other as they are to the
jaguarundi, fisher, killer whale and chimpanzee. One surprising result is the relationship between
the Tasmanian devil and the alligator. All 1000 bootstrap replicates showed a further degree of
similarity between the Tasmanian devil and alligator than to anything else. This subtree is also
connected to a group containing the human and chimpanzee in 61% of the bootstrap replicates.
TibetanMastiff
TibetanMastiff
Greyhound
Greyhound
EurasianWolf
EurasianWolf
ArcticWolf
99
ArcticWolf
99
GrayWolf
GrayWolf
Boxer
Boxer
59
62
Eurasier
Chowchow
100
91
SiberianHusky
74
89
AlaskanMalamute
RacoonDog
68
Chowchow
100
SiberianHusky
74
86
Fox
54
Arctic
Bear
69
Jaguarundi
96
Fisher
65
90
WedellSeal
91
Jaguarundi
94
Fisher
KillerWhale
KillerWhale
Gopher
100
100
Chimpanzee
Gopher
100
Alligator
Image 3. Jukes and Cantor Model Maximum Likelihood Tree
Mouse
TasmanianDevil
TasmanianDevil
100
Human
Chimpanzee
Mouse
Human
100
Arctic
Bear
64
WedellSeal
84
AlaskanMalamute
RacoonDog
67
Fox
51
Eurasier
100
Alligator
Image 4. Kimura Model Maximum Likelihood Tree
Image 3 and 4 are both maximum likelihood trees one run using the Jukes and Cantor
model and the other using Kimura’s model. The trees were run with 1000 bootstrap samples and
have a cutoff value of 50. Both trees group together dogs, wolves, foxes and the raccoon dog.
The bear, Weddell seal, jaguarundi, fisher, and killer whale are all on separate branches while the
chimpanzee and human are on a separate branch, with the gopher, and mouse sharing their own
as well. Like maximum parsimony, these trees also show in all the bootstrap replicates, that the
Tasmanian devil and the alligator share a distant common form of the MC1R gene as well as the
human and chimpanzee. A deeper look at the dog and wolf branch shows a close relation
between the Siberian husky and the Alaskan malamute just as the maximum parsimony tree
showed us. The bootstrap numbers indicate that 89 percent and 91 percent of the replicates
placed these two breeds on a separate branch for Kimura model and Jukes and Cantor model
respectively. The next closest related to these would be the chowchow however only 74 percent
of bootstrap replicates showed this. Another subtree shows the Boxer and Eurasier resulting from
a common structure of MC1R in about 600 of the bootstrap replicates.
EurasianWolf
TibetanMastiff
TibetanMastiff
57
Greyhound
59
Greyhound
GrayWolf
56
ArcticWolf
EurasianWolf
Boxer
89
Boxer
90
Eurasier
53
Chowchow
100
91
SiberianHusky
57
AlaskanMalamute
90
RacoonDog
93
64
Fox
54
Arctic
67
Jaguarundi
92
65
99
Bear
60
WedellSeal
68
99
KillerWhale
Human
100
KillerWhale
Human
100
Chimpanzee
100
Gopher
85
Image 5. Jukes and Cantor Model UPGMA Tree
WedellSeal
Fisher
Fisher
100
Arctic
Jaguarundi
89
Bear
61
AlaskanMalamute
RacoonDog
92
Fox
51
Eurasier
Chowchow
100
SiberianHusky
53
ArcticWolf
GrayWolf
64
Chimpanzee
Gopher
Mouse
82
Mouse
TasmanianDevil
TasmanianDevil
Alligator
Alligator
Image 6. Kimura Model UPGMA Tree
Tree 5 and 6 above are UPGMA trees based on Jukes and Cantor and Kimura’s models
respectively. Both trees were condensed for bootstrap values lower than 50. The bootstrap
numbers correspond to percentages of 1000 bootstrap replicates. The results of the UPGMA trees
are very similar to both the maximum parsimony and maximum likelihood trees. Dog MC1R
genes show a high resemblance to those of wolves with the Siberian husky and Alaskan
malamute showing a high similarity to each other. The bootstrap values for the relationship
between Boxer and Eurasier are lower than the maximum likelihood trees at only 44 and 52 for
the Jukes and Cantor and Kimura models respectively. A subtree shows the jaguarundi, bear and
Weddell seal having similar versions of the MC1R gene however only 60% of the bootstrap
replicates showed this making it not very significant. Unlike both maximum parsimony and
maximum likelihood, the UPGMA trees do not show the Tasmanian devil and the alligator on
their own subtree.
Table 1. Jukes And Cantor Model Distance Matrix
Table 2. Kimura Model Distance Matrix
Table 1 and 2 are distance matrices computed using the Jukes and Cantor Model and
Kimura’s Model respectively. While the trees show the evolutionary relationship of MC1R in
different species, the distance matrices show how similar the gene coding sequences are. Higher
numbers correspond to larger difference between the sequences. The upper left section of the
matrices shows relatively low numbers which indicate a high similarity between the breeds of
dogs and species of wolves. The comparison between the Arctic wolf, Eurasian wolf, Tibetan
Mastiff and the Greyhound displays all zeros indicating that these sequences are identical.
Moving further down the table the numbers elevate slightly but not extremely. The distances
between the dogs, wolves, foxes, bear, seal, and jaguarundi are all around a distance of 0.1 or
below. The rest of the species have distances that are not only higher compared to the species
mentioned above but also between themselves particularly the Tasmanian devil and the alligator.
However, the human and the chimpanzee mc1R genes have a low distance between them.
m
n
S
π
D
7
942
5
0.002831
1.519165
Table 3. Results from Tajima’s Neutrality Test on Dogs
m
n
S
π
D
3
942
0
0
n/a
Table 4. Results from Tajima’s Neutrality Test on Wolves
Table 3 and 4 show the results of Tajima’s Neutrality test. The symbols indicate as
follows: m- Number of sequences, n- number of sites, S- number of segregating sites, πnucleotide diversity and D- Tajima Test Statistic. The seven sequences that were run in table 3
were from all seven breeds of dogs; Siberian husky, Tibetan Mastiff, chowchow, Boxer, Alaskan
malamute, Eurasier, and Greyhound. In table 4, the three different sequences were the gray wolf,
Arctic wolf and the Eurasian wolf. All three of the wolf sequences were identical leading to a
result that was not able to be calculated.
Conclusions
The mc1R Gene is Conserved across a Variety of Species
Looking at the results from all three tree types we can confirm prior knowledge that dogs
and wolves are most similar to each other. The next closest species are the fox, artic fox and the
raccoon dog. Across the trees the trend showed large sub grouping of species and within those
subtrees, particularly the dog and wolf subtree, the bootstrap values were quite low. The data
from the wolf Tajima D test shows a complete conservation of the gene across three subspecies.
Adding in data from the distance matrices backs up the close relationship between MC1R in
dogs and wolves. These distances between species are also low when also considering the foxes,
bear, seal, and jaguarundi. Drawing from this data we can conclude that the MC1R gene is
conserved across carnivores with the exception of the fisher, alligator and the Tasmanian devil.
As we see from the trees, the alligator and Tasmanian devil resulted from a common ancestor.
The Greyhound and Tibetan Mastiff are the Closest Dog Breeds to Wolves
Both the Jukes and Cantor and the Kimura model distance matrices show a section in
which there is a complete match between 5 sequences of mc1R. These sequences are from the
gray wolf, Arctic wolf, Eurasian wolf, Greyhound and the Tibetan Mastiff. This data along with
the grouping of the taxa on the 6 different trees shows that the Greyhound and Tibetan Mastiff
are the closest breeds of dogs compared to the wolf.
mc1R is Under Positive Selection in Dog Breeds
Based on the results from Tajima’s D test for selection, we see that the mc1R gene in
dogs is under positive selection. The result from the test showed a D value of 1.519 which is
above 1. Values above 1 are considered to show positive selection for the species. Due to mc1R
controlling coat color in dogs we predict that this positive selection may be due to human
interaction in selecting the preferable coat color.
Appendix A
Species Dog Eurasian Wolf Arctic Wolf Gray Wolf Bear Fox Raccoon Dog Human Cat Chimpanzee Fisher Aortic Fox Weddell Seal Jaguarundi Tasmanian Devil Alligator Killer Whale Gopher Mouse Dog-­‐Boxer Dog-­‐ Greyhound Dog-­‐ Eurasier Dog-­‐ Siberian Husky Dog-­‐ Chowchow Dog-­‐ Tibetan Mastiff Dog-­‐ Alaskan Malamute Accession Number KC332684 JX273639 JX273636 JX273640 JN575070 JX083391 JX083393 AY363625 JF501545 AJ245705 AB725425 AJ786718 XM_006746161 AY237399 XM_003759223 XM_006269398 FJ773303 EF488834 FJ389441 JX273625 JX273612 JX273609 JX273579 JX273599 JX273619 JX273591 References
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