Study On The Phylogenetic Classification And Molecular Evolution Of Five Medicinal Plants From Botrychium

Botrychium Sw.,which belongs to the family Botrychiaceae,is a genus containing 12 species in China,which are distributed all over the country.The plants in Botrychium have clinical efficacies such as clearing heat,inducing diuresis for removing edema and calming liver wind and are commonly used as folk medicine to treat venomous snake bite and lung heat cough.At present,there are still many controversies about the taxonomic study of Botrychium.The unclear taxonomic status is extremely unfavorable to the resource protection and exploitation of medicinal plants of this genus.In order to clarify the phylogenetic position and the molecular evolution mechanism of important photosynthetic genes in medicinal plants of Botrychium,this study we used designed specific primers to amplify the whole sequence of mat K,rbcL and psaA gene of five species from Botrychium,and combined fifty-four species of ferns which have been reported in GenBank to construct phylogenetic tree via bayesian method based on “relaxed molecular clock” model.The reliability of phylogenetic reconstruction in different data sets was analyzed,and the phylogenetic position and the relationship among five species of medicinal plants from Botrychium were discussed.Phylogenetic results showed that in the phylogenetic studies of ferns,matK was more reliable than rbcL and psaA in the construction of phylogenetic trees,which had great application potential in the molecular systematics studies of pteridophytes.However,the combination of matK with rbcL and psaA could significantly improve the reliability of phylogenetic reconstruction.Among the phylogenetic relationships of 7 different data sets,all the plants of Botrychium were grouped with other members of Ophioglossaceae with a high posterior probability(the posterior probability was more than 99%),indicating that Botrychium belongs to Ophioglossaceae.For the five species of medicinal plants from Botrychium Sw.,Botrychium daucifolium and Botrychium ternatum cluster together while Botrychium japonicum gather with Botrychium virginianum,and Botrychium strictum stands alone.Based on the obtained matK sequence,we carried out bioinformatics analysis of matK gene of 5 species of Botrychium;then we used positive selection model in PAML software and co-evolutionary analysis to analyze the adaptive evolution and co-evolution of matK gene in ferns.The results showed that the full length of mat K gene of five species from Botrychium was 1500~1503bp,with 106 variation sites,most of which were in the third nucleotide of triplet codeword,and the content of C+G ranged from 41.32% to 42.47%.The encoding products contain 498~499 amino acids,the most of which is arginine,and all the MATK protein were unstable hydrophilic proteins without transmembrane structure,and the secondary structure was mainly alpha helix and random coil.There are two types of the tertiary structure: Botrychium daucifolium type and Botrychium strictum type.The evolution analysis results showed that there were some positively selected amino-acid sites in the MATK protein.Multiple groups(pairs)of amino-acid sites in the MATK protein evolved to form a co-evolutionary network.The modification of the MATK protein by site adaptation and the protein intra-network may be important factors that benefited the ability of fern to adapt to the new environment of photosynthesis.The current research will not only provide original data for the furture development of Botrychium,but also imply the reason why these ancient spieces surriving under the uprising of angiosperms.