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) 
. It is important to note that the term “regressive” does not connote anything about whether the trait in question is more adaptive or whether its loss is selected, only that it is lost. Examples of constructive traits include enhanced chemosensory reception, e.g., increased number of taste buds and organs of the lateral line system 
. Alternatively, examples of regressive traits include reduction of eye size and depigmentation 
At least 29 different cave populations from northeastern Mexico have been described, with Pachón cavefish being geographically isolated and cave-specialized () 
. Members of each cave can be crossed with the Surface, sighted ancestral form to create viable F1
. Crosses and trait distribution analyses in F2
individuals have demonstrated that several regressive traits, e.g. eye loss and depigmentation are polygenic 
Schematic map and localities of cave and Surface populations of the Mexican tetra, Astyanax mexicanus.
Among the most notable traits characterizing these fish is the marked reduction in skin pigmentation 
, occurring independently in multiple cave forms 
. While broadly defined, pigmentation in Astyanax
is polygenic; some particular aspects of pigmentation are inherited in a monogenic, recessive fashion 
. 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 (). 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, Pachón 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, Pachón, Piedras and Yerbaniz caves 
. Three cave populations have been reported to harbor albinism mutations, including individuals in the Molino, Pachón and the inter-connected Yerbaniz and Japonés caves 
. As noted above, the brown phenotype also is found in two of these cave systems.
Further, in contrast to the Pachón 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 linked Yerbaniz/Japonés 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, Pachón, Piedras and Sabinos populations (; green).
Laboratory crosses have been used to examine the inheritance of the brown phenotype. Segregation was analyzed in fish descended from a Surface×Pachón cave cross by scoring eye color of seven-day-old F2
larvae (derived from a cross of F1
hybrids of Surface and Pachón 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 frequency
0.73, brown-eyed frequency
In this report, we investigate the genetic basis for the brown mutation by screening F2
individuals derived from an equivalent cross (Surface×Pachón 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 
. 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 Surface×Pachón cross (). When we used the same criteria for mapping melanophore number in a Molino cave×Surface cross no statistically significant QTL were obtained. This is consistent with the reported absence of the brown mutation in this particular cave population () 
. Using a candidate gene approach, we cloned and characterized the Astyanax
form of the gene, melanocortin type 1 receptor
), as the likely locus controlling this trait. Sequence analyses of the open reading frame (ORF) of Mc1r
in Pachón individuals revealed a 2-base-pair deletion in the extreme 5′ end of the coding sequence, corresponding to the N-terminal domain of Mc1r
Astyanax linkage group P09 anchors strongly to Danio chromosome 18.
Sequence analyses of Mc1r open reading frame in Surface, Pachón, Yerbaniz and Japonés cavefish of Astyanax mexicanus reveal three coding mutations.
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 Japonés cave individuals (). 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 
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 similar phenotypes.