| In China,the stems of Kadsura interior,known as "Dian Ji Xue Teng",is officially recorded in current Chinese Pharmacopoeia and used to tonify and invigorate blood in the Traditional Chinese Medicine(TCM).However,due to the similar morphological characters,some studies have treated K.interior as the synonym K.heteroclita.In addition,species delimitation and interspecies relationship between K.interior and its related species were not clear,which might led to misuse in folk and hindrance to the development and utilization of "Dian Ji Xue Teng".In this study,a total of 107 individuals of K.interior,K.heteroclita,K.longipedunculata,K.oblongifolia,and K.coccinea were studied via two single nucleotide polymorphisms(SNPs)datasets developed through restriction siteassociated DNA sequencing(RAD-seq).Based on these SNPs,genetic diversity,phylogenetic reconstruction,and population genetic structure were first analyzed.Subsequently,divergence time estimation and differentiation scenario simulation were performed.Meanwhile,according to species distribution records and bioclimatic variables,the Last Glacial Maximum and modern potential distributions of five species were constructed.The main results are as follows:(1)976 G clean data were generated from 107 individuals.We obtained 5,998 RAD loci containing 18,820 SNPs at species-level and 813 RAD loci containing 1,771 SNPs at population-level by Stacks software analysis pipeline.The two SNPs dataset were used to calculate genetic diversity respectively,and species-level SNPs dataset was used for other analyses.The average within-species nucleotide diversity(π)ranged from 0.0121 to 0.0288.The observed heterozygosity(Ho)ranged from 0.0105 to 0.0187,and the expected heterozygosity(He)ranged from 0.0760 to 0.1082.The inbreeding coefficient(FIS)in each species ranged from 0.2124 to 0.3381.K.coccinea exhibited higher genetic diversity and K.interior exhibited the lowest genetic diversity.The fixation index(FST)ranged from 0.0737 to 0.5031.K.heteroclita,K.longipedunculata,and K.oblongifolia with a very low degree of differentiation,and except for K.coccinea,the FST between K.interior and other species were round 0.15,indicating that there was a moderate level of genetic differentiation among them.The highest FST between K.coccinea and other species which indicated there was a great genetic differentiation among them.(2)The results of PCA analysis showed that K.heteroclita and K.longipedunculata clustered closely together.And K.interior,K.oblongifolia,and K.coccinea formed three separate groups.The phylogenetic tree constructed based on SNPs revealed that each species formed a strongly supported monophyletic clade.K.longipedunculata and K.oblongifolia were sister groups,K.heteroclita was the sister group of these two species,and K.interior was the basal clade.However,the results based on chloroplast genomes showed that K.longipedunculata and K.heteroclita were not monophyletic.Results of population structure revealed K=5 as the most likely number of clusters best explaining the ancestral components of five species.The individuals of K.interior and K.coccinea were composed of a consistent genetic cluster respectively.And the individuals of K.heteroclita,K.longipedunculata,and K.oblongifolia populations were an admixture of the three genetic clusters.(3)The historical scenarios of K.interior and its related species differentiation was simulated by DIY-ABC.The best scenario obtained by calculating the posterior probability showed that K.longipedunculata split from K.heteroclita and K.oblongifolia.The results of D-statistic showed that the existence of gene flow or introgression between K.heteroclita,K.longipedunculata and K.oblongifolia.The divergence time estimation based on SNPs indicated that the crown age of Kadsura was estimated to be 20.41 Ma.The crown age of four species was estimated as 6.97 Ma for K.interior,6.01 Ma for K.heteroclita,and 5.77 Ma for K.longipedunculata and K.oblongifolia,respectively.(4)The potential distribution of the five species during LGM,MH and present were simulated by Maxent.The results showed that the potential distribution of different species was consistent with the actual distribution areas.During the LGM to present,the total suitable distribution area of K.interior showed a change process of the contraction.Moderately and highly suitable distribution areas of K.heteroclita,K.longipedunculata,K.oblongifolia and K.coccinea moved to the southeast coast and became wider.In this study,we explained the genetic relationship of K.interior and its related species,and suggested that K.interior should not be considered a synonym for K.heteroclita,and found that there were hybridization or introgression between K.heteroclita,K.longipedunculata and K.oblongifolia.In addition,niche differentiation between K.interior and other species was found.The results of this study revealed the genetic differences and geographical distribution patterns of K.interior and its related species,clarified the classification status of K.heteroclita,and provided a reference for the application and quality control of "Dian Ji Xue Teng". |
