Subterranean Fishes of the World

3204

2013

AIM 2013

Pigmentation reduction and loss is remarkably common among animals that colonize the cave microenvironment. Genetic analyses in the model system Astyanax mexicanus have revealed the identity of causative genes leading to two Mendelian traits (albinism and brown) that evolved in cave-adapted fish within this species. Both genes harbor coding sequence mutations in multiple, independently-derived cave populations. It remains unclear why these particular genes (Oca2 and Mc1r) are repeatedly implicated in the convergent evolution of reduced pigmentation. In 2001, a new population of cave-adapted Astyanax aeneus was reported from the Granadas cave locality in southern México. These cave-adapted fish represent fish of the same genus, which have independently colonized the subterranean environment. These fish harbor classic troglomorphic phenotypes such as reduced eye size and pigmentation, albeit in a variable fashion at the population level. To determine if Mc1r is widely vulnerable to the accumulation of coding sequence mutations, we investigated the coding sequence structure of Mc1r in both depigmented cave forms (n=11) and surface-dwelling forms (n=12). We found that full-length sequence primers that successfully amplified Mc1r in mexicanus failed to amplify the entire open reading frame in aeneus. This implies sequence divergence between the two species in the 5’ or 3’ regions of the Mc1r open reading frame (ORF). A second approach using degenerate PCR primers successfully amplified a significant region of the ORF of Mc1r in all individuals. We observed a low level of spurious amplification, which often resulted in amplification of a fragment of the Mc4r gene. However, we also observed significant sequence variation, including a variety of SNPs, which were shared broadly among cave and surface-dwelling morphs. One individual harbored a deletion that is predicted to cause loss-of-function of the Mc1r gene product. Interestingly, this deletion is different from the 2-bp deletion that was originally identified from the northern Astyanax mexicanus Pachón cave population. We did not observe a correlation between Mc1r genotypes and reduced pigmentation phenotypes, implying that this gene may not mediate reduced melanic phenotypes in aeneus cavefish. Moreover, the unexpectedly high level of sequence and allelic variation may indicate copy number variation of the Mc1r locus in the southern Astyanax aeneus population.

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