Journal of Heredity 2009:100(6):709714 The American Genetic Association. 2009. All rights reserved.

doi:10.1093/jhered/esp059 For permissions, please email: journals.permissions@oxfordjournals.org.

Advance Access publication July 30, 2009

The Genetic Basis of Melanism in the

Gray Squirrel (Sciurus carolinensis)
HELEN MCROBIE, ALISON THOMAS, AND JO KELLY
From the Department of Life Sciences, Anglia Ruskin University, Cambridge CB1 1PT, UK (McRobie) and the Department
of Life Sciences, Anglia Ruskin University, Cambridge CB1 1PT, UK (Kelly and Thomas).

Address correspondence to Helen McRobie at the address above, or e-mail: helen.mcrobie@anglia.ac.uk.

Abstract
The black squirrel is a melanic variant of the gray squirrel (Sciurus carolinensis). We found 3 coat color variants in the gray
squirrel: the wild-type gray, a jet-black, and a brownblack phenotype. These 3 morphs are due to varying distributions of
eumelanin and phaeomelanin pigment in hairs. The melanocortin 1 receptor (MC1R) plays a central role in regulating
eumelanin and phaeomelanin production. We sequenced the MC1R gene for all 3 coat color phenotypes and found
a 24 base-pair deletion. The gray phenotype was homozygous for the wild-type allele E, the jet-black phenotype was
homozygous for the MC1R-D24 allele EB, and the brownblack phenotype was heterozygous for the E and EB alleles. We
conclude that melanism in gray squirrels is associated with the MC1R-D24 EB allele at amino acid positions 8794 and that
this allele is incompletely dominant to the wild-type allele. We predict that the MC1R-D24 EB allele encodes a constitutively
active or hyperactive receptor.
Key words: gray squirrel, MC1R, melanocortin 1 receptor, melanism, Sciurus carolinensis

Introduction 1993), rock pocket mice (Nachman et al. 2003), horses

A native of North America, the gray squirrel (Sciurus
carolinensis), inhabits deciduous woodland feeding on nuts,
seeds, and berries. Introduced to Britain in captivity in the
late 19th century, the gray squirrel has repeatedly escaped
into the wild and has subsequently become a successful
invader all but outcompeting the native red squirrel (Sciurus
vulgaris). Melanic variants of the gray squirrel are common in
North America, but the first sighting reported in Britain was
in the early 20th century. These black squirrels are now
a common sight in Bedfordshire, Cambridgeshire, and
Hertfordshire where they live in mixed populations with the
gray squirrels (Thomas and Pankhurst 2005). Although exact
numbers remain unknown, it is clear that the black squirrel
population is increasing in both size and geographic range.
The first stage in investigating whether this increase and
spread might be due to a selective advantage associated with
melanism in the gray squirrel is to identify the gene
responsible for the change in pigment color.
More than 100 loci have been associated with vertebrate
pigmentation (reviewed by Lin and Fisher 2007). Two
critical loci are the extension locus (E) that encodes the
melanocortin 1 receptor (MC1R) and the agouti locus (A)
that encodes agouti signaling protein (ASIP). Mutations of
the MC1R gene have been found to be associated with coat
color changes in many vertebrates: mice (Robbins et al.
(Marklund et al. 1996), pigs (Kijas et al. 1998), fox (Vage
et al. 1997), cattle (Klungland et al. 1995; Theron et al.
2001), dogs (Everts et al. 2000), chicken (Takeuchi
et al. 1996), bananaquit (Theron et al. 2001), as well as
hair color in humans (Valverde et al. 1995). The MC1R gene
encodes a 7-transmembrane, G-proteincoupled receptor
which is expressed in melanocytes (Donatien et al. 1992;
Mountjoy et al. 1992; Robbins et al. 1993).
Mammalian melanocytes produce 2 distinct forms of
melanin: eumelanin which is a dark brown/black pigment
and phaeomelanin which is a paler red/yellow pigment.
Coat color depends on the distribution and relative amounts
of these pigments in the hairs, and the MC1R plays an
essential role in regulating these amounts. The MC1R is
activated by its agonist alpha melanocyte-stimulating
hormone (a MSH). When MC1R is bound by a MSH,
eumelanin is produced. However, if MC1R is bound by its
competitive antagonist ASIP, a MSH binding is blocked and
phaeomelanin is produced (Abdel-Malek et al. 2001 and
reviewed by Garcia-Borron et al. 2005).
The gray squirrel, like many wild mammals, has color-
banded hairs on the dorsum. These bands of different color
are caused by pulses of ASIP expression during hair growth.
The result is a hair with bands of phaeomelanin and
eumelanin. This banding can only happen if the MC1R is
functioning as a switch, with a MSH binding representing

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Figure 1. Diagrammatic representation of hair types from different parts of the body for the wild-type gray, jet-black, and
brownblack phenotypes of the gray squirrel, Sciurus carolinensis. White indicates little or no pigment (white hair), gray indicates
phaeomelanin (gray hair), and black indicates eumelanin (black hair).

on and ASIP binding representing off. Mutations in the with melanism include the L99P substitution in cattle and pigs
MC1R can affect its fundamental ability to function as
a switch (reviewed by Garcia-Borron et al. 2005).
A number of MC1R mutations have been identified that
result in the constitutive activation of the receptor leading to
the production of eumelanin even in the absence of a ligand
(Robbins et al. 1993). These mutations, leading to melanism in
the mouse, chicken, fox, and sheep, have been pharmacolog-
ically investigated and found to be constitutively active:
specifically, the E92K mutation in mice with the sombre allele
(Robbins et al. 1993), the E92K mutation in chickens with the
E and ER alleles (Ling et al. 2003), the C125R mutation in the
fox with the EA allele (Vage et al. 1997), and the D119N
mutation in the sheep (Lu et al. 1998). The E92K mutation is
also associated with melanism in the bananaquit, Coereba flaveola
(Theron et al. 2001), and the Japanese quail, Coturnix japonica
(Nadeau et al. 2006), but has not been pharmacologically
investigated in these 2 species. Other mutations associated
Table 1. Frequencies of the E and the EB alleles from the 3
different phenotypes of Sciurus carolinensis showing complete
concordance between the presence of the EB allele and the
melanic phenotype
Alleles/phenotype Gray Brownblack Jet black Total
E 32 16 0 48
EB 0 16 4 20
(Klungland et al. 1995; Kijas et al. 1998), the MC1R-D15 in the
jaguar, Panthera onca, and MC1R-D24 in the jaguarundis,
Herpailurus yaguarondi (Eizirik et al. 2003). In contrast, the S69L
mutation in the tobacco mouse leads to increased activation
when bound by its ligand (Robbins et al. 1993).
Many mutations of the MC1R associated with melanism
are located around the boundary of the second trans-
membrane domain and the second extracellular loop or the
third transmembrane domain and third extracellular loop
(Robbins et al. 1993; Eizirik et al. 2003; Mundy 2005).
Studies of 3-dimensional models of the MC1R and its ligand
suggest that a MSH could bind to a pocket below the
plasma membrane between the second, third, and sixth
transmembrane domains on the MC1R. This is an acidic
domain where there is likely to be an interaction with the
arginine in the a MSH (Prusis et al. 1995; Lu et al. 1998 and
reviewed by Garcia-Borron et al. 2005). Replacement of the
acidic residues with basic residues as in the E92, D119, and
C125 mutations leads to constitutive activation where the
mutation is thought to have the effect of mimicking ligand
binding (Lu et al. 1998). These gain of function mutations
result in increased eumelanogenesis and are dominant.
Other mutations, which result in loss of function, lead to
increased phaeomelanogenesis and are recessive (Robbins
et al. 1993). Melanism can also be the result of mutations in
the ASIP gene (Vrieling et al. 1994).

710

The spread of the black squirrel across the counties of
Bedfordshire, Cambridgeshire, and Hertfordshire in the UK
has inspired much general interest. Considering the critical
role of the MC1R in coat color variation in so many species,
this gene appeared to be the ideal candidate to begin
investigating the genetic basis of melanism in the gray
squirrel (S. carolinensis).
Materials and Methods
Thirty-four squirrels (mostly from Cambridgeshire) were
sampled comprising 16 gray and 18 melanic (16 brown
black and 2 jet black) squirrels. Hairs removed from the
dorsum, flank, and belly of the squirrels were examined in
detail under a microscope. Genomic DNA was extracted
from skeletal muscle of the hind leg using a commercial kit
(Qiagen Tissue kit). The primers mshr4f (5#-TGC TTC
CTG GAC AGG ACT ATG-3#) and mc1r11r (5#-TCG
TGT CGT YGT GRA GGA AC-3#) were used to amplify
99% of the MC1R gene. The polymerase chain reaction
(PCR) product from this reaction was then used to perform
nested PCR using the mshr4f and mc1r3r primers (5#-GGC
AAG CAT GTG GAT GTA GA-3#). This enabled the first
627 base pairs of the gene to be sequenced. The second
section of the gene was amplified using the primers mc1r10f
(5#-CAG CCT RGG GCT GGT GAG-3#) and mc1rer5
(5#-CAC AGG ATG CAG GCC ACT-3#). The PCR
product from this reaction was then used to perform nested
PCR using the primers mc1r2f (5#-GAC CG (GC) TAC
ATC TCC ATC TTC-3#) and mc1rer2 (5#-ACT GTC ACC
CTC TNC CCA GN-3#). This allowed the last 533 base
pairs to be sequenced. All PCR reactions were carried out
Figure 2. Diagrammatic representation of the MC1R protein of Sciurus carolinensis showing the 314 amino acids. Dark gray circles
indicate the deleted amino acids of the EB allele. Information for the predicted sequence of the MC1R protein was obtained from
Robbins et al. (1993) and Mundy (2005).
McRobie et al.  Melanism in the Gray Squirrel (Sciurus carolinensis)
on a DNA thermocycler (Techne touchgene gradient) in
a total volume of 25 ll using approximately 25 ng template
DNA, 1
Bioline PCR buffer, 3 mM Bioline MgCl2,
0.2 mM dNTPs, 0.4 lM primers, and 0.1 ll Bioline Taq
polymerase using the following PCR parameters: initial
denaturation 94 C for 2 min followed by 35 cycles of 94 C
for 30 s, 60.2 C for 45 s, and 72 C for 1 min. The final
extension was 72 C for 5 min. PCR products were
sequenced using the ABI Prism 3130 Genetic Analyzer, and
sequences were inspected manually with particular attention
paid to the heterozygotes and aligned with the CLUSTAL W
program.
Results and Discussion
We identified 3 phenotypes of gray squirrel. These
phenotypes are completely distinct and readily identifiable
as wild-type gray, brownblack, and jet black. Microscopic
inspection of hairs removed from the dorsum, flank, and
belly revealed that the wild-type gray had 6 distinct hair
types, compared with 4 from the brownblack and only
1 from the jet-black squirrel. Figure 1 summarizes the hair
types found in each phenotype. These different hair types
give the gray an overall grizzled appearance with a white
underbelly, the brownblack an overall dark brown
appearance with an orange underbelly, and the jet black
a uniform black appearance.
Analysis of the wild-type gray and melanic squirrel
sequences revealed a 24 base-pair in-frame deletion (MC1R-
D24) in all the melanic squirrels at amino acid positions
8794. We have named the wild-type allele E and the
melanic allele EB. The complete sequence for both alleles
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Journal of Heredity 2009:100(6)

Figure 3. Amino acid alignments of MC1R variants in the squirrel, pig, mouse, cattle, jaguar, jaguarundis, rabbit, bananaquit,
chicken, and Japanese quail. Numbering is according to the human MC1R. The wild type (wt) of each species is shown in contrast
to the melanistic variant underneath. Dashes indicate agreement with the consensus sequence used (squirrel-E). Bold letters with
asterisks indicate substitutions associated with melanism, and triangles indicate deletions. Transmembrane domains are indicated by
boxes. The second transmembrane domain begins at amino acid position 71 and ends at position 99. Sequences were obtained
from GenBank using the following accession numbers: pig-MC1R*1 AF082487, pig-MC1R*2 AF082488, jaguar-wt AY237396,
jaguar-mel AY237397, jaguarundis-red AY237399, jaguarundis-dark AY 237398, rabbit-wt AM180878, rabbit-ED AM180880,
bananaquit-Y AF362600, bananaquit-M AF362601, chicken-wt DQ395092, chicken-B AB201631, Japanese quail-wt AB201633,
and Japanese quail-black AB201635. Sequence information was also obtained from Kijas et al. (1998).

can be found on GenBank accession numbers EU604830
and EU604831. The gray squirrel was found to be
homozygous for the E allele, the brownblack heterozy-
gous for the E and EB alleles, and the jet black
homozygous for the EB allele. Table 1 summarizes the
complete concordance between the presence of the EB allele
and the melanic phenotype.
Considering the intermediate coloring and heterozygous
genotype of these brownblack squirrels, we conclude that
the EB allele is incompletely dominant to the E allele.
Incomplete dominance is also observed in melanism in the
jaguarundis, which is associated with a 24 base-pair deletion
in a similar position in the MC1R gene as shown in Figure 3
(Eizirik et al. 2003).
The MC1R-D24 falls in the second transmembrane
domain of the encoded protein. Figure 2 shows positions of
the amino acids and the deletion on the MC1R of S. carolinensis.
The deletion is close to many other mutations associated with
melanism. Figure 3 shows a selection of amino acid sequence
alignments, illustrating how many of these mutations are

712

clustered around the boundary of the second transmembrane
domain and the second extracellular loop. The 24 base-pair
deletion in the jet-black squirrel corresponds to 8 amino acids:
serine (polar uncharged), asparagine (polar uncharged),
alanine (hydrophobic), leucine (hydrophobic), glutamic acid
(negatively charged), threonine (polar uncharged), and iso-
leucine (hydrophobic). The absence of glutamic acid is
particularly critical as this is also absent in the melanistic
rabbit, mouse, chicken, bananaquit, and Japanese quail. The
loss of this acidic residue in the mouse and chicken is known
to lead to constitutive activation. Models of the MC1R suggest
that the negatively charged glutamic acid is part of an acidic
domain that interacts with the positively charged arginine of
the a MSH (reviewed by Garcia-Borron et al. 2005). Removal
of this acidic residue is thought to mimic ligand binding
leaving the receptor constitutively active in the absence of the
ligand. We predict that the loss of glutamic acid in the MC1R
of the squirrel leads either to constitutive activation or
hyperactivity of the receptor resulting in eumelanogenesis and
melanism. This is supported by observations of the under-
bellies of each phenotype: the gray has a white underbelly
where the wild-type MC1R is bound by ASIP, the brown
black has an orange underbelly where half of the MC1R
molecules are bound by ASIP and the other half are
hyperactive or constitutively active, and the jet black has
a black underbelly where all MC1R molecules are active.
The results observed in this study do not rule out the
possibility that other genes may also contribute to melanism
in the gray squirrel. The observations and predictions
presented here merit further investigation into the
intracellular effects of the MC1R-D24.
Conclusion
This study has revealed that a 24 base-pair deletion in the
MC1R is associated with melanism in the gray squirrel. The
deletion is at amino acid positions 8794 and has been
named the MC1R-D24 EB allele. We conclude that the
wild-type gray phenotype is homozygous for the wild-type
allele E, the jet-black phenotype is homozygous for the
MC1R-D24 allele EB, and the brownblack phenotype is
heterozygous for the MC1R-D24 EB and wild-type E
alleles. Further, we conclude that the MC1R-D24 EB allele
is incompletely dominant to the wild-type E allele and
predict that the MC1R-D24 EB allele encodes a constitutively
active or hyperactive receptor.
Funding
Department of Life Sciences, Anglia Ruskin University.
Acknowledgments
The authors would like to thank Shelia Parkhurst, Andrew Lancaster,
Monera Alrukeyes, Mado Maniotti, and Jienian Wosley for invaluable
contributions to the project.
McRobie et al.  Melanism in the Gray Squirrel (Sciurus carolinensis)
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Received June 28, 2009; Revised June 28, 2009;
Accepted June 29, 2009
Corresponding Editor: William Modi