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Sleep is an evolutionarily ancient behavior, yet multiple cave-dwelling populations of the Mexican tetra, Astyanax mexicanus, have converged on sleep loss compared to surface fish. However, most of the 34 cave populations remain unstudied, and sleep in natural habitats is largely unknown. To address this, we measured sleep and activity in 15 representative populations of surface, cave, and hybrid populations. All cavefish and hybrid populations tested exhibited drastically reduced sleep, including hybrid populations with diverse eye and pigmentation phenotypes. Mapping behavior onto the A. mexicanus phylogeny revealed that reduced sleep and elevated locomotor activity evolved independently multiple times. Field experiments confirmed that wild fish also exhibit sleep loss, paralleling laboratory findings. These results demonstrate deep evolutionary convergence on sleep loss across cavefish lineages and suggest that sleep reduction is a primary trait contributing to adaptation in subterranean environments. 

Astyanax mexicanus is an emerging model system used to study development, evolution, and behavior of multiple cavefish populations that have repeatedly evolved from conspecific surface fish. Although surface and cavefish live and breed in the laboratory, there are no rapid methods for distinguishing between different cavefish populations. We present 2 methods for genotyping fish for a total of 16 population-specific markers using methods that are easy and inexpensive to implement in a basic molecular biology laboratory. This resource will help researchers maintain independent stocks within the laboratory and distinguish between fish from different populations.