| Summary: | Circadian rhythms are biological rhythms with a period of approximately 24 h. While canonical circadian clock genes and their regulatory mechanisms appear highly conserved, the evolution of clock gene families is still unclear due to several rounds of whole genome duplication in vertebrates. The spotted gar (<i>Lepisosteus oculatus</i>), as a non-teleost ray-finned fish, represents a fish lineage that diverged before the teleost genome duplication (TGD), providing an outgroup for exploring the evolutionary mechanisms of circadian clocks after whole-genome duplication. In this study, we interrogated the spotted gar draft genome sequences and found that spotted gar contains 26 circadian clock genes from 11 families. Phylogenetic analysis showed that 9 of these 11 spotted gar circadian clock gene families have the same number of genes as humans, while the members of the <i>nfil3</i> and <i>cry</i> families are different between spotted gar and humans. Using phylogenetic and syntenic analyses, we found that <i>nfil3-1</i> is conserved in vertebrates, while <i>nfil3-2</i> and <i>nfil3-3</i> are maintained in spotted gar, teleost fish, amphibians, and reptiles, but not in mammals. Following the two-round vertebrate genome duplication (VGD), spotted gar retained <i>cry1a</i>, <i>cry1b</i>, and <i>cry2</i>, and <i>cry3</i> is retained in spotted gar, teleost fish, turtles, and birds, but not in mammals. We hypothesize that duplication of core clock genes, such as (<i>nfil3</i> and <i>cry</i>), likely facilitated diversification of circadian regulatory mechanisms in teleost fish. We also found that the transcription factor binding element (Ahr::Arnt) is retained only in one of the <i>per1</i> or <i>per2</i> duplicated paralogs derived from the TGD in the teleost fish, implicating possible subfuctionalization cases. Together, these findings help decipher the repertoires of the spotted gar’s circadian system and shed light on how the vertebrate circadian clock systems have evolved.
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