Adaptation and convergence in genes of the circadian system in Iberian Squalius freshwater species

Abstract

The circadian clock is a biological timing system that improves the inherent ability of organisms to deal with environmental fluctuations. It is regulated by daily alternations of light but can also be affected by temperature. Fish, as ectothermic, have an increased dependence on the environmental temperature and thus are good models to study the integration of temperature within the circadian system. Here, we studied four species of freshwater fish of Squalius genus, distributed across a latitudinal gradient in Portugal with variable conditions of light and temperature. We identified and characterised the expected sixteen genes belonging to four main gene families (Cryptochromes, Period, CLOCK and BMAL) involved in the circadian system. We conducted phylogenetic analysis and tested these genes for the presence of signatures of selection based on dN/dS that revealed the presence of mutations under positive selection in nine genes. Furthermore, we analysed functional features of the proteins and found that these mutations alter protein isoelectric point and aliphatic index. Additionally, in 50% of the genes we found a pattern of convergence uncovered by the phylogenetic clustering of S. aradensis and S. pyrenaicus from Almargem, which inhabit basins with similar environmental conditions. Although this clustering can be explained by incomplete lineage sorting, evidence of positive selection in one gene in the two species and similar functional features in seven proteins are consistent with convergence at protein level. Our results highlight the importance of integrating sequence-based functional protein characterisation with dN/dS-based methods when studying the molecular adaptation of species.