A repeatedly evolved mutation in Cryptochrome-1 of subterranean animals alters behavioral and molecular circadian rhythms

The repeated evolution of similar phenotypes in independent lineages often occurs in response to similar environmental pressures, through similar or different molecular pathways. Recently, a repeatedly occurring mutation R263Q in a conserved domain of the protein Cryptochrome-1 (CRY1) was reported in multiple species inhabiting subterranean environments. Cryptochromes regulate circadian rhythms, and glucose and lipid metabolism. Subterranean species show changes to their circadian rhythm and metabolic pathways, making it likely that this mutation in CRY1 contributes to adaptive phenotypic changes. To identify the functional consequences of the CRY1 R263Q mutation, we generated a mouse model homozygous for this mutation. Indirect calorimetry experiments revealed delayed locomotor activity, energy expenditure and feeding patterns of mutant mice in the dark phase, but no further metabolic phenotypes – unlike a full loss of function of CRY1. Gene expression analyses showed altered expression of several canonical circadian genes in the livers of the mutant mice, fortifying the notion that CRY1 R263Q impacts metabolism. Our data provide the first characterization of a novel mutation that has repeatedly evolved in subterranean environments, supporting the idea that shared environmental constraints can drive the evolution of similar phenotypes through similar genetic changes. We collected brain and liver tissues at two different zeitgeber times (ZT, ZT12 and ZT22) from wild-type and CRY1 R263Q homozygote mice from both sexes, and then extracted and sequenced the mRNA. Samples from each ZT were collected, sequenced and analyzed separately. Comparisons were made within the same ZT, tissue type and sex, and between genotypes.