A Novel Role for Mc1r in the Parallel Evolution of
Depigmentation in Independent Populations of the
Cavefish Astyanax mexicanus
Joshua B. Gross1, Richard Borowsky2, Clifford J. Tabin1*
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America, 2Cave Biology Research Group, Department of Biology, New York
University, New York, New York, United States of America
The evolution of degenerate characteristics remains a poorly understood phenomenon. Only recently has the identification
of mutations underlying regressive phenotypes become accessible through the use of genetic analyses. Focusing on the
Mexican cave tetra Astyanax mexicanus, we describe, here, an analysis of the brown mutation, which was first described in
the literature nearly 40 years ago. This phenotype causes reduced melanin content, decreased melanophore number, and
brownish eyes in convergent cave forms of A. mexicanus. Crosses demonstrate non-complementation of the brown
phenotype in F2individuals derived from two independent cave populations: Pacho ´n and the linked Yerbaniz and Japone ´s
caves, indicating the same locus is responsible for reduced pigmentation in these fish. While the brown mutant phenotype
arose prior to the fixation of albinism in Pacho ´n cave individuals, it is unclear whether the brown mutation arose before or
after the fixation of albinism in the linked Yerbaniz/Japone ´s caves. Using a QTL approach combined with sequence and
functional analyses, we have discovered that two distinct genetic alterations in the coding sequence of the gene Mc1r cause
reduced pigmentation associated with the brown mutant phenotype in these caves. Our analysis identifies a novel role for
Mc1r in the evolution of degenerative phenotypes in blind Mexican cavefish. Further, the brown phenotype has arisen
independently in geographically separate caves, mediated through different mutations of the same gene. This example of
parallelism indicates that certain genes are frequent targets of mutation in the repeated evolution of regressive phenotypes
in cave-adapted species.
Citation: Gross JB, Borowsky R, Tabin CJ (2009) A Novel Role for Mc1r in the Parallel Evolution of Depigmentation in Independent Populations of the Cavefish
Astyanax mexicanus. PLoS Genet 5(1): e1000326. doi:10.1371/journal.pgen.1000326
Editor: Gregory S. Barsh, Stanford University School of Medicine, United States of America
Received July 16, 2008; Accepted December 2, 2008; Published January 2, 2009
Copyright: ? 2009 Gross et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Post-doctoral fellowship support was provided to JBG through an NIH-NRSA Kirschstein award (1F32 GM081439-01). This work was supported by a
collaborative grant from the NSF (IOS-0821982) to CJT and RB and an NIH grant to RB (R03EY016783-01).
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The blind Mexican cave tetra, Astyanax mexicanus, is a troglobitic
characin fish exhibiting a variety of cave-specialized traits. In
general, the cave ecosystem supports the evolution of some traits
that are enhanced or increased over time (i.e., ‘‘constructive’’ traits),
as well as some traits that decrease or degenerate over time (i.e.,
‘‘regressive’’ traits) [1,2]. It is important to note that the term
‘‘regressive’’ does not connote anything about whether the trait in
question ismore adaptiveorwhetheritslossisselected,onlythat itis
lost. Examples of constructive traits include enhanced chemosen-
sory reception, e.g., increased number of taste buds and organs of
the lateral line system.Alternatively, examplesof regressive traits
include reduction of eye size and depigmentation [4–6].
At least 29 different cave populations from northeastern Mexico
have been described, with Pacho ´n cavefish being geographically
isolated and cave-specialized (Figure 1) [7–9]. Members of each
cave can be crossed with the Surface, sighted ancestral form to
create viable F1hybrids . Crosses and trait distribution analyses
in F2individuals have demonstrated that several regressive traits,
e.g. eye loss and depigmentation are polygenic [4,11–15].
Among the most notable traits characterizing these fish is the
marked reduction in skin pigmentation [4,16], occurring indepen-
dently in multiple cave forms . While broadly defined,
pigmentation in Astyanax is polygenic; some particular aspects of
pigmentation are inherited in a monogenic, recessive fashion[4,18].
As an example, albinism was recently discovered to be a monogenic
trait caused by loss-of-function alleles of Oca2, this gene having been
independently mutated in three different cave forms .
An additional simple trait affecting body pigmentation, termed
the brown mutation, was described in the late 1960’s as being
present in several caves (Figure 1). The brown phenotype, affecting
eye color as well as the number and size of melanophores on the
body , was observed in the wild in fish from the Chica, Pacho ´n
and Sabinos caves. In addition, complementation test crosses
carried out between F1 individuals derived from surface and
various cave populations showed that the same locus was
responsible for the brown phenotype in the Curva, Pacho ´n,
Piedras and Yerbaniz caves . Three cave populations have
been reported to harbor albinism mutations, including individuals
in the Molino, Pacho ´n and the inter-connected Yerbaniz and
Japone ´s caves [1,4,16,19]. As noted above, the brown phenotype
also is found in two of these cave systems.
Further, in contrast to the Pacho ´n cave, where the brown
phenotype has been observed in individuals that do not carry the
albino mutation, there is no published evidence that fish from the
PLoS Genetics | www.plosgenetics.org1 January 2009 | Volume 5 | Issue 1 | e1000326
linked Yerbaniz/Japone ´s populations ever display the brown
phenotype in nature. Therefore, it is not clear whether the brown
mutation arose prior to the evolution of albinism in this population
or, alternatively, if the brown mutation became fixed following the
presence of epistatic albino mutations. Therefore, the cave
populations that exhibit the brown mutation in nature, based on
published data and/or inference through lack of albinism in these
caves, include the Chica, Curva, Pacho ´n, Piedras and Sabinos
populations (Figure 1; green).
Laboratory crosses have been used to examine the inheritance
of the brown phenotype. Segregation was analyzed in fish
descended from a Surface6Pacho ´n cave cross by scoring eye
color of seven-day-old F2larvae (derived from a cross of F1hybrids
of Surface and Pacho ´n cavefish) as black, brown or pink (i.e.,
albino). When controlling for albinism, the frequency of
individuals demonstrating the brown phenotype strongly predicted
the participation of a single, recessive allele (black-eyed frequen-
cy=0.73, brown-eyed frequency=0.27, N=5094) .
In this report, we investigate the genetic basis for the brown
(Surface6Pacho ´n cave hybrids) to that used in the original
descriptions of this mutant . We screened a pedigree of 488
individuals with 262 microsatellite markers, expanding upon
pedigrees previously described [1,2]. Consistent with other studies,
our linkage analysis revealed a single, strong QTL influencing
melanophore number in the post-optic region of the head and the
dorsal flank in individuals derived from the Surface6Pacho ´n cross
number in a Molino cave6Surface cross no statistically significant
QTL were obtained. This is consistent with the reported absence of
the brown mutation in this particular cave population (Figure 1) .
Using a candidate gene approach, we cloned and characterized the
Astyanax form of the gene, melanocortin type 1 receptor (Mc1r), as the likely
locus controlling this trait. Sequence analyses of the open reading
frame (ORF) of Mc1r in Pacho ´n individuals revealed a 2-base-pair
deletion in the extreme 59 end of the coding sequence, corresponding
to the N-terminal domain of Mc1r (Figure 3A).
The Mc1r protein is a member of the GPCR superfamily of
genes, comprised of an N-terminal domain, seven hydrophobic
transmembrane domains, and a carboxy terminal domain .
One of the primary functions of Mc1r is to activate adenylyl
cyclase in response to ligand binding, resulting in an intracellular
increase in cAMP levels . Mc1r binding leads to activation of
downstream effectors in the pigmentation pathway, including the
target gene mitf, which is transcriptionally upregulated by cAMP
signaling in melanocytes . Coding mutations in this gene have
been described in model systems, including the classical ‘extension’
locus mouse mutant, which lacks normal functioning of Mc1r .
Coding sequence alterations are also known from natural
populations, associating strongly with distinct coat and plumage
color morphs in a variety of mammals and birds, respectively .
Depigmentation has arisen multiple times in different caves;
therefore we extended our search for variant alleles to twelve other
caves. We found an additional, independent mutation in the
Yerbaniz cave (known to harbor the brown mutation) as well as
Japone ´s cave individuals (Figure 3B,C). The point mutation
present in these caves, C490T, alters an arginine residue
homologous to that identified in certain human individuals with
the red hair color (RHC) phenotype [25–28].
This analysis identifies a novel role for Mc1r in the evolution of
degenerative phenotypes in blind Mexican cavefish. Further, we
demonstrate that the brown phenotype has arisen independently
in multiple forms of cavefish, mediated through different
mutations of the same gene. This example of parallelism is
consistent with other recent studies suggesting that certain genes
may be frequent targets of mutation in the repeated evolution of
Crosses, Genotyping, and QTL Mapping
We used a previously described Pacho ´n F2pedigree obtained
from a cross between the Surface6Pacho ´n cave morphs of Astyanax
mexicanus [1,2]. Briefly, a Pacho ´n individual was crossed to a
Surface individual, and two sibling F1individuals were crossed to
produce 539 F2 individuals. 488 of these individuals were
genotyped for Mc1r. All F2 progeny were raised with two
individuals per tank (to control for size variation) and euthanized
at 7 months of age. We performed an additional analysis of the
melanophore number trait in a Molino backcross comprised of
111 individuals. This cross was performed by crossing a Molino
cavefish to a Surface fish, and then mating an F1individual to a
second Molino fish. This backcross progeny set was reared in
group tanks, euthanized at 14 months and fixed in 4%
paraformaldehyde. Fin clips were collected from all individuals
to isolate genomic DNA for subsequent genotyping. All Astyanax
animal care protocols were approved by the NYU/University
Animal Welfare Committee. Each individual was genotyped with
262 microsatellites using PCR reactions carried out in a 10 ml
volume containing: 0.1 mM MgCl2, 6 mM Tris-HCl, pH 8.3,
30 mM KCl, 0.006% glycerol, 0.25 mM dNTP mix (Roche),
0.06% Tween, 0.06% Nonidet P-40, 0.25 units of Taq DNA
polymerase (Roche), 5 nM forward primer, 200 nM reverse
primer and 200 nM of the fluorescent tag primer: 59-CAC-
GACGTTGTAAAACGAC-39 labeled with one of two phosphor-
amidite conjugates (Hex and Fam) and amplified using the PCR
program previously described .
QTL mapping was carried out using the interval mapping
function of MapQTL (version 4.0) to determine the LOD scores
and percent variance explained (PVE) at the melanophore number
locus using a permutation test, as previously reported . Mc1r
As we approach the 150th year since publication of On the
Origin of Species, understanding the genetic architecture
underlying evolutionary change remains an important
challenge. When an organism enters a completely new
environment or ecological niche, certain traits are no
longer necessary for survival, while other new traits
become critical for maintaining fitness. An example of
such a transition is provided by cave animals. Many
disparate taxa (e.g., crustaceans, salamanders, fish) have
colonized caves, presumably to escape predation or
expand populations into an unexploited niche. Strikingly,
similar traits evolve convergently despite significant
phylogenetic distance between these organisms. Caves
provide a unique environment including the absence of
light, few predators, few sources of food, etc. Under these
conditions, one observes striking changes in morphology
including reduction in eyes, expansion of non-visual
sensory systems, and a suite of metabolic and behavioral
changes. To understand the genetic underpinnings of
these shifts, we have established the blind Mexican cave
tetra, A. mexicanus, as a genetic system. In this paper, we
use this system to investigate a classic morphological
feature in these animals, depigmentation. We identify the
gene Mc1r as being responsible for reduction in melanin
content in multiple caves.
Depigmentation in the Blind Mexican Cave Tetra
PLoS Genetics | www.plosgenetics.org2 January 2009 | Volume 5 | Issue 1 | e1000326
, [reviewed in ]. Genome-wide analyses, however, failed to
find strong evidence for selection at Mc1r locus in humans .
Evolution of Pigmentation Alterations in Teleost Fish
Regressive phenotypes have long been of interest to evolution-
ary biologists, given the many examples of regressive (degenerate)
phenotypic variation when comparing humans to their primitive
ape-like ancestors, e.g., hair loss . While many theories seek to
explain the process of phenotypic degeneration, genetic insights
into the underlying bases of these traits has remained elusive.
Here, we describe the genetic basis of one of the earliest described
genetic traits in the blind Mexican cave tetra, the brown mutation.
We have found coding sequence modifications in two independent
cave forms, the Pacho ´n and Yerbaniz/Japone ´s caves, that explain
the reduced function of the 7-transmembrane domain receptor
Complementation tests indicate that other cave populations of
Astyanax, including Piedras and Curva, have brown phenotypes
caused by mutations at the same locus as in Pacho ´n and Yerbaniz.
We failed to find any coding alterations in Mc1r sequence from fish
collected from these other caves, suggesting that these populations
likely carry regulatory mutations leading to a decrease or loss of
Mc1r activity (Figure 1).
The genetics of pigmentation have been explored in several
model teleost fish including zebrafish [81,82], medaka  and
fugu . More recently, the evolution of pigment variation in
natural populations of sticklebacks has been explored using linkage
analysis . These authors discovered the relevant locus
controlling lighter pigmentation to be due to cis-regulatory
changes affecting expression of the gene, kit ligand (kitl). Further,
it was discovered that evolution in the cis-regulatory regions of the
kitl locus similarly appears to control the evolution of lighter skin in
recent humans [85,86]. Interestingly, another genetic locus
examined in the zebrafish golden mutant (SLC24A5) appears to
also play a role in pigmentation evolution in recent humans
[86,87]. Mutations in Mc1r likewise are found in humans in
addition to Astyanax and other species. Thus, there seems to be a
common tool-kit of pigmentation genes used to modify coloration
during evolution of widely divergent taxa.
The participation of Mc1r in the evolution of Astyanax cavefish
depigmentation clarifies the essential role of this gene in
vertebrates. Its role in the regressive evolution of pigmentation
in independently derived cavefish indicates that certain genes may
be important loci for convergent evolution of specialized traits of
The authors wish to thank Paul E. Scheid, Jenna Galloway, Artie
McCollum, Diana Esshaki, Maria Samson and members of the Cepko,
Tabin, Dymecki and Zon labs for indispensable technical advice,
suggestions and assistance. The authors are grateful to Rusty Wessel for
generously providing DNA samples of surface individuals drawn from the
Mosquito Coast, Jutiapa, Carolina and Pantepec localities; Jake Schaefer
for generously providing DNA samples of surface individuals drawn from
the Honduras locality; and Horst Wilkens for providing individuals from an
inbred strain of Piedras cavefish. The authors also wish to thank Meredith
Protas, Gregory Barsh, Hopi Hoekstra and two anonymous reviewers for
helpful comments and advice. Maria Ericcson and Louise Trakimas (HMS
Electron Microscopy core facility) provided invaluable assistance with EM
tissue preparation and imaging.
Conceived and designed the experiments: JBG RB CJT. Performed the
experiments: JBG RB. Analyzed the data: JBG RB CJT. Contributed
reagents/materials/analysis tools: JBG RB. Wrote the paper: JBG RB
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Depigmentation in the Blind Mexican Cave Tetra
PLoS Genetics | www.plosgenetics.org14 January 2009 | Volume 5 | Issue 1 | e1000326