We investigated differentiation processes in the Neotropical fish Astyanax that represents a model system for examining adaptation to caves, including regressive evolution. In particular, we analyzed microsatellite and mitochondrial data of seven cave and seven surface populations from Mexico to test whether the evolution of the cave fish represents a case of parallel evolution. Our data revealed that Astyanax invaded northern Mexico across the Trans-Mexican Volcanic Belt at least three times and that populations of all three invasions adapted to subterranean habitats. Significant differentiation was found between the cave and surface populations. We did not observe gene flow between the strongly eye and pigment reduced old cave populations (Sabinos, Tinaja, Pachon) and the surface fish, even when syntopically occurring like in Yerbaniz cave. Little gene flow, if any, was found between cave populations, which are variable in eye and pigmentation (Micos, Chica, Caballo Moro caves), and surface fish. This suggests that the variability is due to their more recent origin rather than to hybridization. Finally, admixture of the young Chica cave fish population with nuclear markers from older cave fish demonstrates that gene flow between populations that independently colonized caves occurs. Thus, all criteria of parallel speciation are fulfilled. Moreover, the microsatellite data provide evidence that two co-occurring groups with small sunken eyes and externally visible eyes, respectively, differentiated within the partly lightened Caballo Moro karst window cave and might represent an example for incipient sympatric speciation.
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