Research On The Prochlorococcus Evolutionary Mechanism Based On The Mutation Accumulation Experiments

Prochlorococcus is the smallest and most abundant photoautotrophic organism on the earth,which is widely distributed among tropical and subtropical oceans.It is a dominant group in oligotrophic environments and one of the most important producers in marine ecosystems,playing an important role in the global cycle of biogenic elements.The evolutionary process of Prochlorococcus is a significant research topic.Prochlorococcus was the first reported free-living bacteria with evolutionary characteristics of endosymbionts,such as genome reduction and low GC content in the genome.These characteristics of endosymbionts are thought to be the results of genetic drift caused by repeated population bottlenecks,while the conventional view is that the effective population size(Ne)of Prochlorococcus is huge,so their evolutionary process is dominated by natural selection and the effect of genetic drift is weak.From the perspective of natural selection,scholars have proposed "oligotrophic adaptation theory" for Prochlorococcus,which was that the genome reduction and the low GC content of Prochlorococcus are in order to adapt to the oligotrophic environment.This view has been widely accepted,but the most crucial evidence,Ne of Prochlorococcus,has not actually been rigorously measured.Effective population size(Ne)is a key parameter to judge the relative strength of natural selection and genetic drift in the evolutionary process of a species.To accurately determine the Ne of a prokaryotic organism,the Mutation Accumulation(MA)experiments are required,which can obtain its unbiased spontaneous mutation rate(μ).MA experiments carry out continuous monoclonal passages through solid media to maintain a low level of Ne during the growth process,minimize the effect of natural selection,and theoretically retain all non-lethal mutations from cells self-replicate,so their results can reflect the true level of spontaneous mutation rate of the species.Due to the difficulties of solid culture and slow growth rate,Prochlorococcus has never carried out MA experiments before this study.Prochlorococcus does not have an accurate mutation rate,so the conjecture of its huge Ne has never been confirmed.In this study,the MA experiments of Prochlorococcus are carried out for the first time.This study set up 150 MA lines of a member in high-light adapted(HL)Ⅱ clade,Prochlorococcus marinus AS9601,and completed single-clonal serial passage for 1065 days.Through whole-genome sequencing and mutation identification analysis,the final results showed that the mutation rate of HL II Prochlorococcus was 3.50 × 10-10 cell division-1 site-1.This mutation rate is slightly lower than the average level of prokaryotes,which denies the hypothesis that Prochlorococcus has a high mutation rate due to the lack of multiple key DNA repair enzymes and does not support the theory that the increase in mutation rate drives the genome reduction of Prochlorococcus.In the results of MA experiments,the substitution rate of GC towards AT is 4.36 times faster than that of AT towards GC,and the corresponding balanced GC content is 18.6%,which is much lower than the actual level of AS9601.This results indicate that reduction of the GC content of Prochlorococcus was mainly driven by mutational direction bias,whereas the natural selection resisted the mutational bias and maintained the GC content of Prochlorococcus genome.Based on the mutation rate,this study delineated the population structure of 418 genomes related to AS9601,and calculated the Ne of HL Ⅱ Prochlorococcus to be 1.68×107.This result is only slightly higher than that of endosymbiotic bacteria and lower than that of most free-living bacteria,breaking the inherent cognition that the evolutionary process of Prochlorococcus is dominated by natural selection,and reveal that genetic drift played an important role in the evolution and ecological adaptation of Prochlorococcus.There are a large number of genetically isolated populations in HL ⅡProchlorococcus,and they each possesses some unique genes and show a low level of recombination.Based on these results,this study propose a new mechanism for Prochlorococcus evolution and ecological adaptation.Different genetically isolated populations of Prochlorococcus occupy different ecological niches,and acquisitions of adaptive mutations in periodic selection lead to the fixation of the entire genome carrying the adaptive mutations,therefore Ne of Prochlorococcus decrease and the genetic drift is strengthened.This mechanism provides a new perspective for the research on the evolution and ecological adaptation of other oceanic dominant taxa.This study add the mutation rate,effective population size and genome size of Prochlorococcus to the data set of prokaryotes to re-test the prokaryotes evolutionary theories and their applicability to Prochlorococcus.The results show that Prochlorococcus accords with the overall trends of prokaryotes evolution,and the effect of natural selection and genetic drift on Prochlorococcus is consistent with other prokaryotes.The new data set supports the "drift barrier hypothesis",but can not fundamentally verify the "drift hypothesis of genome size" and "hypothesis of high-speed mutation" due to the tiny number of prokaryotes in the data set.The evolutionary parameters of some key species need to be further determined.Based on the MA experiment,this study obtains the key evolutionary parameters of Prochlorococcus,proposes a new mechanism of its evolution and ecological adaptation,complements the prokaryotes evolutionary theories and expands the perspective of research on prokaryotes ecological adaptation.