Evolution Of ITS2 Secondary Structiures And Their Phylogenetic Implications In Plants

ITS2 is one of the core DNA barcodes for species identification in plants,and also a most commonly used gene in phylogenetic analysis,which plays an indispensable role in the identification and protection of medicinal plants and the prevention of invasive species.Therefore,it is very important to use its genetic information completely and efficiently.This RNA transcript is involved in the cleavage of spacers in the RNA processing machinery with a secondary structure of "one palm and four fingers",which is very conservative in the entire eukaryotes due to the functional constraints.Although it is widely acknowledged that the function is based on the secondary structure,the ITS2 evolution pattern and its potential phylogenetic significance under this function constraint are still not clear enough at present.Therefore,in this study(1)base-pairs frequency,conservative sequence position,and the conservatism of structure shape of the ITS2 secondary structure were computed by using our split counting method,sequence alignment,base localization and shape quantization under the phylogenetic framework of evolution of phylum,orders,families and genera,in order to explore whether the evolution of ITS2 secondary structure has directivity and corresponding characteristics.(2)In order to examine the mutational dynamics of conservatism and variability of ITS2 secondary structure in evolution,we also used the mcmcphase programe of PHASE 3.0 package to track and count the substitution process and characteristics of secondary structure bases and pairing.In summary,we have drawn the following conclusions:Conservatism of ITS2 secondary structure:(1)Refined conservative characteristics of ITS2 secondary structure in plants were found in addition to their prior knowledge of "four-finger hand" model:the G+C content of ITS2 bases was the highest(61.26%),and G>C>U>A for separate base content;In different regions,the G+C content had some bias:the G+C content in the paired region was much higher than that in the unpaired region(69.88%vs.46.54%).For regions composed dominatly by base pairs,the content of guanine was significantly higher and the content of adenine was significantly lower than that of unpaired regions.In contract,the contents of uracil and cytosine were relatively constant in different regions.Among all components,purine and pyrimidine account for about 50%.The conserved subsequences in ITS2 are related to the position of secondary structure as showed by structural comparisonVariability of ITS2 secondary structure:(2)To our knowledge,it is the first study to explore the evolution mode of ITS2 secondary structure in different taxonomic categories.We found that based on the normal function of the ITS2 secondary structure,its variation was unrelated to the evolution of the taxa,despite we sampled at different phylogenetic ranges.This indicates that some regions of ITS2 are strongly constrained by function during the evolution from green algae to angiosperms,while others showed changes diversity.(3)The genetic distance greatly influenced the common structure of ITS2,and the common secondary structure of ITS2 changes relatively with sampling sequences:the degree of dispersion of each statistical value gradually decreases;the degree of variation of the secondary structure of ITS2 was positively correlated with the variation of sequences.At the higher taxonomic level,this correlation became weaker.This result can be used as a reference for ITS2 phylogenetic reconstruction when secondary structure was taken into account.The evolutionary dynamics of ITS2 secondary structure:(4)Under the functional constrain,both the substitutions of base and base-pairs had some differences between paired region and unpaired region of ITS2.The substitution of unpaired bases fit with the common REV substitution model.In contrast,the base composition and the base-pair substitution of paired regions are mainly toward to the direction of maintaining the thermodynamic stability of the secondary structure.(5)We densely selected Corydalis and comprehensively cloned and sequenced ITS2 to explore the process of compensating base changes(CBCs)in paired regions.Because of the instability of intermediate GU state,the evolution process of CBC is difficult to be observed in specific groups,and its variation and phylogenetic value are not clear yet.This study confirms that the complete CBC was a two-step process that from GC to GU and then AU in actual plants.Based on this finding,we further proved the significance of CBC in species delimitation.Unlike previous studies on phylogenetic relationships in deep phylogeny,we found that CBC did not significantly affect the construction of proper phylogenetic relationships among closely related phylogeny groups.In conclusion,we suggest that when using ITS2 secondary structure through all kinds of methods mentioned above,it should be cautious especially in the taxonomical level higher than family.In addition,this study will deepen our understanding of the secondary structure itself and its evolutionary characteristics,thus laying a foundation for better utilization of the secondary structure.