Organisms are exposed to daily environmental fluctuations in their natural habitats. To overcome them, organisms developed biological timing systems to optimize their physiological and biochemical processes in space and time [1]. These systems work as internal clocks and require a proper synchronization with environmental signals. Thus, understanding the molecular evolution of the genes involved in the circadian system provide important clues to elucidate how species adapt to their environments.
The circadian system is a universal biological timing system found virtually in all organisms [2]. Circadian system is synchronised by light–dark cycle of a day’s period and present oscillations with a period of ~ 24 h called circadian rhythms. Oscillations are generated and regulated at molecular level, but the outcomes have been shown to influence several aspects of physiology, behaviour and ecology of organisms [2, 3]. In fact, circadian rhythms have been shown to improve the inherent ability of several organisms to survive under changing environments, by aiding them to efficiently anticipate periodic events, specifically light changes and climate seasons [2, 3].
The molecular circadian system consists of a network of signalling transduction pathways regulated mainly by interconnected transcription-translation feedback loops (Fig. S1) [4, 5]. The regulatory loops are sustained by the so-called core circadian genes and proteins, requiring about 24 h to complete a cycle [1, 5]. In vertebrates, several genes have been reported to be responsible for the maintenance and regulation of the circadian system [1]. The core circadian-genes belong to four main gene families: Cryptochromes (CRY), Period (PER), CLOCK, and BMAL [5]. These gene families encompass several characterized genes (cry, per, bmal and clock) in vertebrates. In fish, several families possess a larger number of circadian paralogs as compared to other vertebrates [6]. For instance, in zebrafish (Danio rerio), several genes have been identified: six cry (cry1aa, cry1ab, cry1ba, cry1bb, cry2, cry3), four per (per1a, per1b, per2, per3), three bmal (bmal1a, bmal1b, bmal2), and three clock (clocka, clockb, clock2/npas2) [7–10]. Cryptochrome genes encode for a class of flavoproteins that are sensitive to blue light [10], whereas period genes encode for proteins that also display a strong but differential light responsiveness [11, 12]. Both cry and per were found to be key agents in the entrainment of the circadian system, as they constitute the negative elements of the system (i.e. repressors of transcription) [13]. BMAL (Brain and muscle ARNT like) and CLOCK (Circadian locomotor output cycle kaput) families encode for canonical circadian proteins, a highly conserved bHLH (basic-Helix-Loop-Helix)-PAS (Period-Aryl hydrocarbon receptor nuclear translocator- Single minded) transcriptional factors and are the positive elements of the circadian system (i.e. activators of transcription) [1, 5].
Studies in fish allowed to elucidate the different levels of organization of the circadian system. This is because fish are a very diverse group of animals adapted to nearly all aquatic environments and possess a larger number of circadian paralogs as compared to the other vertebrates [6]. Advances in genome sequencing allowed to identify circadian-related genes in several model organisms, including zebrafish.
Homology-based methods allowed the identification of circadian genes in other non-model fish species [7–9, 14]. Some studies cover the evolutionary relationships of the core-clock gene families and the mechanisms driving their molecular evolution [7–10, 15], but several key questions remain open, namely the higher number of paralogs in fish when compared to other vertebrates, and its importance for adaptation of species to different environments [6].
By sequencing the transcriptome of zebrafish exposed to light, two recent studies identified several genes whose expression depends on light, revealing a multi-level regulation of circadian rhythms by light-cycles [16, 17]. Photoreception is particularly interesting in fish as, contrary to most vertebrates that only perceive light through the eyes, fish also possess a photosensitive pineal gland, dermal melanophores, and brain photoreceptors [1]. In addition, fish possess independent peripheral photoreceptors and self-sustaining circadian oscillators in every tissue [1, 18].
Circadian rhythms can also be entrained by temperature [19–22]. In mammals it was demonstrated that peripheral cells in vitro could sense the change of room temperature as a cue for entrainment of circadian system [19]. In zebrafish, temperature has also an important role in circadian clock [20, 22], and it was proposed that temperature could entrain the phase of the system by driving expression levels of per3, and other circadian genes (namely cry2 and cry1ba) via an alternative hypothetical enhancer [20]. In this model, per1b (formerly known as per4) promoter integrates temperature and light regulatory inputs [20]. In agreement with the hypothesis that temperature affects the circadian system, in a study comparing a transcriptome profiling of two freshwater fish species (Squalius carolitertii and S. torgalensis) exposed to different temperatures, Jesus et al. [23, 24] found two differentially expressed genes (cry1aa and per1a) between a control and a thermal stress condition.
In the Western Iberian Peninsula, there are four known species of the genus Squalius Bonaparte, 1837 in Portuguese rivers (S. carolitertii, S. pyrenaicus, S. torgalensis and S. aradensis, Fig. 1). S. carolitertii (Doadrio 1988) inhabits the northern river basins, S. pyrenaicus (Günther 1868) occurs in the Central and Southern basins (e.g. Tagus, Guadiana and Almargem), while sister species S. torgalensis and S. aradensis (Coelho et al. 1998) are confined to small Southwestern basins (e.g. Mira and Arade, respectively). Based on phylogenies of nuclear and mitochondrial markers, the species tree of these species is well known, comprising two main groups: (i) S. carolitertii and S. pyrenaicus; and (ii) S. torgalensis and S. aradensis [25, 26]. In Western Iberian Peninsula there is a transition between two contrasting climate types (Fig. 1): the Atlantic in the Northern region that is characterized by mild temperatures (inhabited by S. carolitertii and S. pyrenaicus), and the Mediterranean in the Southern region (inhabited by S. pyrenaicus, S. torgalensis and S. aradensis) typified by higher temperatures and droughts during summer periods [27–29]. Thus, species inhabiting the southern basins affected by the Mediterranean climate face harsher conditions. Besides differences in spring average water temperature of approximately 5 ºC along the distribution of these species, there are differences in the average spring photoperiod (approximately 15 minutes) between the northern and southern basins. The environmental differences associated with the distribution of Squalius species in Portugal make them a good model to study adaptation to different environmental conditions.
Here, we performed an integrative study on the molecular evolution of circadian system genes in Squalius species. We aimed to identify the genes involved in the core circadian system in these species and assess their evolutionary history. We combined several approaches, conducting phylogenetic analysis of the identified genes within each gene family in the Squalius species and using predicted protein sequences. Finally, we aimed to detect signatures of positive selection and correlate those with predicted functional features of the proteins potentially relevant for the response to environmental differences in light and temperature. These results contribute to a better understanding of the mechanisms of adaptation and response of freshwater fish species to climate change.