Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock

Abstract

Most organisms possess time-keeping devices called circadian clocks. At the molecular level, circadian clocks consist of transcription-translation feedback loops (TTFLs). Although some components of the negative TTFL are conserved across the animals, important differences exist between typical models, such as mouse and the fruit fly. In Drosophila, the key components are PERIOD (PER) and TIMELESS (TIM-d) proteins, whereas the mammalian clock relies on PER and CRYPTOCHROME (CRY-m). Importantly, how the clock has maintained functionality during evolutionary transitions between different states remains elusive. Therefore, we systematically described the circadian clock gene setup in major bilaterian lineages and identified marked lineage-specific differences in their clock constitution. Then we performed a thorough functional analysis of the linden bug Pyrrhocoris apterus, an insect species comprising features characteristic of both the Drosophila and the mammalian clocks. Unexpectedly, the knockout of timeless-d, a gene essential for the clock ticking in Drosophila, did not compromise rhythmicity in P. apterus, it only accelerated its pace. Furthermore, silencing timeless-m, the ancestral timeless type ubiquitously present across animals, resulted in a mild gradual loss of rhythmicity, supporting its possible participation in the linden bug clock, which is consistent with timeless-m role suggested by research on mammalian models. The dispensability of timeless-d in P. apterus allows drawing a scenario in which the clock has remained functional at each step of transition from an ancestral state to the TIM-d-independent PER + CRY-m system operating in extant vertebrates, including humans.

Keywords: timeless; Bilateria; Insecta; circadian clock; gene loss; reverse genetics.

Fig. 1.

Phylogeny of bilaterian clock proteins. (A) A tree illustrating relatedness among mammalian- and Drosophila-type of CRYPTOCHROME (CRY-m, grey; CRY-d, blue), 6-4 PL (deep purple), CPD-PL (orange), and DASH -type CRYPTOCHROME (green). (B) Phylogeny of JETLAG (JET, deep/ligh blue) and FBXL3/21 (purple/red) within FBXL proteins (black); (C) bilaterian PERIOD proteins with single-copy genes are shown in black and three vertebrate paralogs (PER1, PER2, and PER3) in colors. (D) TIM-m, found in all Bilateria, and TIM-d are clearly separated into two clusters. Presented trees were inferred using RAxML maximum likelihood GAMMA-based model. For detailed trees see supplementary figures 1–4, Supplementary Material online.

Fig. 2.

Circadian clock gene losses mapped on the bilaterian phylogeny. Representative insect species are shown at the terminal nodes with indicated gene presence (full circle) or absence (empty circle) where the lineage-specific losses are highlighted with red rectangles (see details in supplementary tables 1 and 2, Supplementary Material online). The phylogenetic tree corresponds to a consensus of recent phylogenomic studies (Misof et al. 2014; Johnson et al. 2018; Kawahara et al. 2019; McKenna et al. 2019; Wipfler et al. 2019). Filled circles indicate the presence of PER (black), TIM-m (brown), TIM-d (bright magenta), JET (dark blue), FBXL3/21 (light purple), CRY-m (grey), CRY-d (blue), 6-4 PL (deep purple), CPD-PL (yellow), and DASH-type cryptochrome (green). For phylogenetic relationship see supplementary figures 1–4, Supplementary Material online. Numbers indicate the presence of multiple paralogs in one taxon. The question mark indicates a suspicious occurrence of DASH in Bemisia. Supplementary figure 16, Supplementary Material online, illustrates clear relatedness of this sequence with DASH from plants and fungi which can either be explained as contamination or as a horizontal gene transfer (HGT) from plant to insect. The latter would be consistent with a recent HGT of a plant detoxification component to Bemisia (Xia et al. 2021).

Fig. 3.

The role of circadian clock genes in P. apterus. (A) Summary of the gene knockdown describing its impact on the behavioral rhythmicity shown as percentage of males demonstrating strong rhythmicity, complex rhythmicity, and arrhythmicity; fr #1 and fr #2 are nonoverlapping dsRNA fragments 1 and 2, respectively. (B) Individual τ values are shown as dots for each male with strong rhythmicity; red bars represent means ± SEM (calculated if >10% individuals demonstrated rhythmicity). Columns depict the mean τ, standard error of the mean (SEM), and statistical difference from the controls (P value) (Kruskal–Wallis test with Dunn’s post hoc; calculated only if >10% individuals were rhythmic).

Fig. 4.

Either cry-m or per depletion completely abolishes circadian rhythmicity in P. apterus, whereas tim-d mutants demonstrate robust rhythmicity with significantly shorter τ. (A) Schematic representation of tim-d gene structure with coding regions, alternative splicing, and engineered mutation (tim⁰³). Corresponding wt and mutant proteins are shown with major functional domains highlighted (for details, see supplementary figs. 13 and 14, Supplementary Material online). Alternative splicing of tim-d was detected in exons 9, 17, and 18. (B) Summary indicating the number and rhythmicity of the tested mutant and heterozygous animals compared with corresponding control siblings. (C) Individual τ values are plotted as a dot for each male; red bars depict means ± SEMs (calculated only if >10% individuals were rhythmic). Statistical difference from the controls is shown as P-value (Kruskal–Wallis test with Dunn’s post hoc). Double-plotted actogram of (D) wt and tim⁰³ P. apterus compared with (E) D. melanogaster wt (Canton S) and tim⁰¹ mutant (arrow indicates the beginning of constant darkness).

Fig. 5.

Proposed scenarios of the circadian clock evolution in Bilateria. In the Drosophila ancestor, loss of cry-m gene resulted in a feedback loop relying on a PER + TIM-d dimer with CRY-d serving for light-mediated resetting of the system (Ceriani et al. 1999). A two-step process, when TIM-d first became a modulator of the τ, and only when it was lost, it allowed for a smooth transition to the PER + CRY-m system, with a functional clock in each step. In P. apterus, TIM-d is still present as a modulator of the clock, whereas CRY-d is absent. However, the timing of cry-d loss might differ between lineages. In one scenario, TIM-d could become a modulator in the presence of CRY-d, which would be lost afterward (situation observed in P. apterus). In the second scenario, the modulatory role of TIM-d led to its loss, excluding CRY-d from the loop and resulting in its subsequent loss. The involvement of TIM-m in the circadian clock is indicated both from mammalian models (Barnes et al. 2003; Kurien et al. 2019) and insect models (Benna et al. 2010; Nose et al. 2017), albeit its role is not established in detail. The third type of path observed in evolution involves the loss of the per gene. This strikingly unusual clock found in two basal deuterostomian phyla, Hemichordata, and Echinodermata, is apparently functional (Peres et al. 2014), although the mechanism remains unknown.