Secondary evolve and re-sequencing: an experimental confirmation of putative selection targets without phenotyping

Evolve and re-sequencing (E&R) studies investigate the genomic
responses of adaptation during experimental evolution. Because replicate
populations evolve in the same controlled environment, consistent
responses to selection across replicates are frequently used to identify
reliable candidate regions that underlie adaptation to a new environment.
However, recent work demonstrated that selection signatures can be
restricted to one or a few replicate(s) only. These selection signatures
frequently have a weak statistical support, and given the difficulties of
functional validation, additional evidence is needed before considering
them as candidates for functional analysis. Here, we introduce an
experimental procedure to validate candidate loci with weak or
replicate-specific selection signature(s). Crossing an evolved population
from a primary E&R experiment to the ancestral founder population
reduces the frequency of candidate alleles that have reached a high
frequency. We hypothesize that genuine selection targets will experience a
repeatable frequency increase after the mixing with the ancestral founders
if they are exposed to the same environment (secondary E&R
experiment). Using this approach, we successfully validate two overlapping
selection targets, which showed a mutually exclusive selection signature
in a primary E&R experiment of Drosophila simulans adapting to a
novel temperature regime. We conclude that secondary E&R
experiments provide a reliable confirmation of selection signatures that
are either not replicated or show only a low statistical significance in a
primary E&R experiment. Such experiments are particularly helpful
to prioritize candidate loci for time-consuming functional follow-up
investigations.