New Methods To Reveal Adaptive Evolutionary Mechanisms

The major driving force of evolution at the molecular level has been heatedly debated.Positive selection is the major theme of Darwinian evolution theory;namely,the biological evolution is a result of continuous accumulation of the small and advantageous variations.Darwin recognized that the core of evolution is adaption.However,Motoo Kimura,a population geneticist of Japan,proposed neutral selection according to that most of the mutations at the molecular level are neutral or close to neutral,and that these mutations are fixed by genetic drift.Mutations in genes can promote the evolution of speciation.If a mutation in nucleotide sequences does not cause a change of an amino acid,it will be called as a synonymous substitution.Since the functional protein is not changed,it will not be subjected to natural selection.Otherwise,it would be considered as a nonsynonymous substitution.The Ka is calculated by the number of actual nonsynonymous substitutions per nonsynonymous site and the Ks is calculated by the number of actual synonymous substitutions per synonymous site.Thus,Ks can be an indicator of neutral selection.At present,in evolutionary genetics,the Ka/Ks(?)ratio is widely used to estimate the selective constraint.There are three different types of selection:when Ka<Ks(?<1),it is negative(purifying)selection;when Ka=Ks(??1),it is considered as neutral mutation and when Ka>Ks(?>1),it is considered as positive(adaptive)selection.Another method is based on of the fixed differences of nonsynonymous substitutions and synonymous substitutions between species.Since the mutations can be saturated in the first method and the fixed differences require large sampling within each species,these methods limit their use in analysis of homologous genes within and between closely related species but are not proper for homologous genes between species from long evolutionary distance.This study takes the advantage of the merit of these two methods but discards their drawbacks to propose a new method to reveal the evolutionary mechanism.This method based on fixations of nonsynonymous and synonymous sites at each position of all the homologous genes within or between phylogenetic branches rather than within or between species.Since the fixations of a residue is easy to determine and they will never be saturated,this approach can be applied to species with close or far evolutionary distance.This method is first to identify whether all homologous genes within or between phylogenetic branches are the same(fixations)or not(polymorphisms),then to divide these sites into fixed nonsynonymous sites(FNSs)and fixed synonymous sites(FSSs).Next,FN(Fixed nonsynonymous rate)and FS(Fixed synonymous rate)are separately calculated as the ratio of the number of actual nonsynonymous fixed per nonsynonymous site and the number of actual synonymous fixed per synonymous site.Last,since the FS can be used as a marker for neutral selection,the ratio of FN to FS can be used to measure the selective restraint:when FN<FS,it is considered negative selection,when FN=FS,it is considered as neutral mutation and when FN>FS,it is considered as positive selection.The new method was then first applied to the evolutionary study of BR(Brassinosteroids,BRs)receptor family(BRL2,BRL1/3 and BRI1)in this study.The results of the analysis showed that fixed nucleotides were pervasive in the BR receptors and almost all of the fixed sites are nonsynonymous except few synonymous sites.Furthermore,BR receptors include the Island Domains(ID)responsible for BR-binding and the Kinase Domain(KD)accounted for activation and BR-specific targeting,thus they are the most important domains of BR receptors.The IDs have the highest rate of fixed differences of nonsynonymous sites,indicating that IDs play a major role in functional differentiation of BR receptors.This result is consistent with the genetic and biochemical study,fully demonstrating that the fixed nonsynonymous sites play an important role during the functional evolution of BR receptors.To investigate the broad application of this method,it was applied to analyze the whole genomes of Drosophila,Avian and Plants that have been sequenced.The results show that the value of FN/FS tend to gradually increase when the evolution distance increase.This is exactly a process of gradual accumulation of fixed small but beneficial variations defined by Darwin and the positive selection,supporting Darwinian evolutionary theory.In conclusion,this study found that positive selection is the main driving force for gene evolution.This innovated approach is complementary to current approaches used in the study of molecular evolution,can be a powerful tool to identify the driving force of gene functionalization during evolution.