Genetic Mechanisms Of Sleep Evolution In Marine Mammals

Marine mammals(e.g.cetaceans,sirenias and pinnipeds),as a special group in the evolutionary history of mammals,have developed unique sleep pattern to adapt to aquatic environments.Cetaceans are fully aquatic marine mammals,and have unique unihemispheric slow-wave sleep(USWS),i.e.one cerebral hemisphere is in a state of SWS while the other is awake.Interestingly,cetaceans lack REM sleep.Sirenias,another fully aquatic mammals,display not only USWS but also typical features of sleep in terrestrial mammals with bihemispheric slow-wave sleep(BSWS)and REM.Similar to sirenias semi-aquatic pinnipeds exhibited a typical USWS,BSWS and REM sleep.However,the genetic bases of USWS in marine mammals remain poorly explored.In the present research,we comprehensively investigated eight circadian clock genes of 9 representative cetaceans and compared with the orthologous genes of other terrestrial mammals to explore the following three scientific questions:1)whether positive selection restrict to lineages with USWS;2)whether the marine mammals with USWS have convergent molecular mechanism;3)if these genes evolution rate associated with the unique USWS.When we used three Maximum Likelihood(ML)methods(site model,FEL,and REL)to test selection in cetaceans dataset and found that a total of 51 codons from six genes(Clock,Cry1,Cry2,Per1,Per2,and Per3)were detected to be under positive selection.These positively selected sites were further revealed that 96.1%(49/51)sites were examined to be radical changes at the protein-level by TreeSAAP.Some positively selected sites were located in or close to the important function of domain of the circadian clock genes.These results suggested the circadian clock genes of cetaceans have undergone adaptive evolution across cetacean phylogeny.To determine if the evidence of positive selection was restricted to some particular lineages,we used branch models,including free-ratio and two-ratio models in all mammals dataset.Free-ratio model showed that evidences for positive selection were mainly found in cetacean lineages at five genes(Clock,Npas2,Per1,Per2,and Per3).Similar result was also found at the two-ratio model that positive selection were only restricted to cetacean lineages at Bmal1,Per2,and Per3.To gain insight into the link between the evolutionary of circadian clock genes and sleep phenotypes,we applied correlation analysis between the evolutionary rate of circadian clock genes(root-to-tip?)and SWS/TST ratio in mammals.The result displayed that the molecular evolution of Cry1 and Per1 genes were significantly and positively correlated with SWS/TST ratio.All these lines of evidence implied that circadian clock genes seemed to be related to the USWS in cetaceans.Finally,to test whether convergent molecular evolution in marine mammals with convergent USWS,we reconstructed ancestral sequences implemented in PAML and identified the parallel/convergent amino acids substitutions in three different groups of marine mammals.A total of 80 parallel/convergent nonsynonymous amino acid substitutions were identified in the six circadian clock genes(Clock,Cry2,Npas2)Per1,Per2,Per3)between cetaceans,sirenias,and walrus,suggesting that convergence molecular evolution for USWS existed in these three lineages.This is the first comprehensive analysis of the molecular evolution of circadian clock genes in marine mammals to explore their unique sleep pattern in aquatic environment.Our result revealed that significant evidence of positive selection at clock genes restricted to cetacean species,which seems to be related to their unique USWS.Our study provides important molecular evidence for aquatic adaptation of marine mammals.