| Cetaceans,including whales,dolphins,and porpoises,have lived in an exclusively aquatic environment for approximately 53-56 Myr after their transition from land to water.Hypoxia was a major challenge faced by cetaceans during the course of secondary aquatic adaptation.Previous studies have revealed anatomical and physiological changes in terms of respiration,hematology,circulation and energetic metabolism in cetaceans compared with their terrestrial relatives.To survive,underwater cetaceans must rely on intrinsic oxygen stores bound mostly in blood to hemoglobin(HB)and in muscle to myoglobin(MB).Therefore,cetaceans have high levels of HB,MB,and a high hematocrit(Hct),resulting in blood and muscle oxygen stores that are higher than those in terrestrial species.In addition,adaptive changes in the cardiovascular system in response to hypoxia tolerance have been found in cetaceans,such as dramatic bradycardia(reduction in heart rate in response to apneic immersion)and selective peripheral vasoconstriction,the latter being responsible for redistributing the blood circulation to maintain blood flow to the central nervous system and heart,and reducing flow to the skin,muscle,and splanchnic organs(e.g.,spleen,kidney).These adjustments are essential to achieve effective oxygen conservation.Moreover,reduced metabolism,hypometabolism,in cetaceans is associated with an increased reliance on anaerobic('without oxygen';nitrate)metabolism;the advantage of which is that energy in the form of adenosine triphosphate(ATP)can be produced in the absence of oxygen.Cetaceans also display an enhanced capacity of glial cells to support anaerobic breakdown of glucose,glycolysis,coupled with an elevated number of astrocytes which store glycogen and provide lactate as an energy source.Higher concentrations or activity of key glycolytic enzymes that enhance the ability to process lactic acid,such as lactate dehydrogenase(LDH),may be associated with greater tolerance to hypoxia.Although physiological traits of hypoxia tolerance in cetaceans have been well characterized,the underlying molecular mechanisms remain unknown.The goal of the present study was to test the evolutionary changes in hypoxia tolerant-related genes in cetaceans,which will allow us to reveal the genetic basis for the adaptation of cetaceans to hypoxia,and improve our understanding of the genetic and evolutionary architecture of cetaceans during the course of secondary aquatic adaptation.To reveal the genetic basis of enhanced oxygen store in cetaceans,we studied molecular evolution of globin genes involved in carrying and transporting oxygen in the blood(hemoglobin-?,HBA and ?,HBB),muscle(myoglobin,MB)and brain(neuroglobin,NGB).The results showed that HBA,HBB and MB appear to have undergone adaptive evolution,evidence for positive selection on their particular sites(HBA:9,HBB:11,MB:15),and radical physiochemical property changes of selected condons.Interestingly,"long-diving" cetaceans had relatively higher ?(dN/dS)values than "short-diving" cetaceans for the HBB,indicating divergent selective pressure presented in cetacean lineages with different diving abilities.It also suggested that deep-diving,pelagic species required more oxygen for metabolism.Strong signals of positive selection in HBA,HBB and NGB were also detected in highland and burrowing species,suggesting the potential functional importance of globin genes in hypoxia tolerant species.In summary,positive selection,divergent selective pressures,and parallel evolution at the molecular level provided some new insights into the genetic adaptation of hypoxia tolerance.To understand the molecular mechnisms for the adaptation of cetaceans to cardiovascular system during hypoxic diving,we investigated the evolutionary pattern of 13 vasoconstriction-related genes,including EDN1,EDN2,EDN3,EDNRA,EDNRB,AGTR1,AGTR2,ADRA1A,ADRA1B,ADRA1D,AVP,AVPRA and AVPRB.Evidences of positive selection were found in A VP gene,indicating A VP is essential for enhanced vasoconstriction abilities for reducing blood flow to the skin in cetaceans.Additionally,the observed positive selection in the ADRA1D gene in cetaceans suggests that it is important for enhanced arterial vasoconstriction ability.Most importantly,strong signals of positive selection were detected in endogenous vasoconstrictor endothelins(EDN1,EDN2,EDN3,EDNRA and EDNRB),which maybe associated with enhanced regulating of basal vascular tone,as well as reduced blood flow in the kidney and ceased renal filtration to save oxygen during cetacean diving.Additionally,parallel positive selection or amino acid changes(ADRA1D:P50A,A53G;AVPR1B:I/V270T)among animals exposed to different hypoxia habitats reflect functional convergence or similar genetic mechanisms of hypoxia tolerance.To provide insights into the mechanisms underlying the evolution of energy metabolism for hypoxia tolerance in cetaceans,we investigated genes in four major energy metabolism pathways,and provide evidence of distinct evolutionary paths to mammalian hypoxia-tolerance.Positive selection of genes in the oxidative phosphorylation pathway mainly occurred in terrestrial hypoxia-tolerant species;possible adaptations to chronically hypoxic environments.The strongest candidate for positive selection along cetacean lineages was the citrate cycle signaling pathway,suggestive of enhanced aerobic metabolism during and after a dive.Six genes with cetacean-specific amino acid changes are rate-limiting enzymes involved in the gluconeogenesis pathway,which would be expected to enhance the lactate removal after diving.Intriguingly,38 parallel amino acid substitutions in 29 genes were observed between hypoxia-tolerant mammals.Of these,76.3%were radical amino acid changes,suggesting that convergent molecular evolution drives the adaptation to hypoxic stress and similar phenotypic changes.This study provides further insights into life under low oxygen conditions and the evolutionary trajectories of hypoxia-tolerant species.Although the natural selection have shaped the evolution of the genes involved in oxidative phosphorylation(OXPHOS).However,how network architecture drives OXPHOS protein sequence evolution remains poorly explored.Here,we investigated the evolutionary patterns of genes in the OXPHOS pathway across six cetacean genomes within the framework of a functional network.Our results show a negative correlation between the strength of purifying selection and pathway position.This result indicates that downstream genes were subjected to stronger evolutionary constraints than upstream genes,which may be due to the dual function of ATP synthase in the OXPHOS pathway.Additionally,there was a positive correlation between codon usage bias and omega(?= dN/ds)and a negative correlation with synonymous substitution rate(ds),indicating that the stronger selective constraint on genes(with less biased codon usage)along the OXPHOS pathway is attributable to an increase in the rate of synonymous substitution.Surprisingly,there was no significant correlation between protein-protein interactions(PPIs)and the evolutionary estimates,implying that highly connected enzymes may not always show greater evolutionary constraints.Compared with that observed for terrestrial mammals,we found that the signature of positive selection detected in five genes(ATP5J,LHPP,PPA1,UQCRC1 and UQCRQ)was cetacean-specific,reflecting the importance of OXPHOS for survival in hypoxic,aquatic environments.Totally,our study investigated the hypoxia tolerant-related genes and pathway in cetaceans,and revealed the genetic basis for the adaptation of cetaceans to hypoxia.Moreover,this study improves our understanding of the genetic and evolutionary architecture of cetaceans during the course of secondary aquatic adaptation. |
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