Morphological And Molecular Phylogeny Of Chinese Epimedium

Epimedium is the largest and one of the most important herbaceous genera in Berberidiaceae with about 60 species disjunctly distributed in the Eurasia between Europe and eastern Asia.China is the most important center for the genus with about 50 species,especially well diversified in central China.Plants from the genus are well-known for their beautiful flowers as garden ornaments as well as traditional medicine in China.To understand the systematis and taxonomic of Chinese Epimedium species,an integrative study was applied using morphological,cytological and molecular phylogenetic approaches.Our studies will bring important implications for their better conservation and sustainable utilizations.1.MorphometricsMorphometric studies were carried out in order to elucidate the taxomic relationship of Chinese Epimedium taxa.Based on 29 characters of 52 Epimedium species,the cluster analysis show that Chinese Epimedium has been clustered into two branches related to their flower size.Clade 1 includes all species with small flowers plus four species of Ser.Campanulatae(E.campanulatum,E.platypetalum,E.ecalcaratum,E.shuichengense)with large flowers.Clade 2 comprises of all taxa with large flowers.Our results did not support the division of the four Series of Section Diphyllon by Stearn.In addition,based on the results of numerical taxonomic analyses,we identified 5 controversial species with further treatment,including E.membranaceum,E.coactum,E.obophyllum,E.jingzhouense,E.campanlatum.2.CytologyChromosome numbers and karyomorphology of 24 accessions were examined,representing 18 Epimedium species,five of which were counted here for the first time.A chromosome number of 2n = 2x = 12 was observed for all taxa and all karyotypes were Stebbins' 2A,except for E.acuminatum,E.brevicornu,E.ilicifolium and one accession of E.leptorrhizum as 3A.Their asymmetry index(AI)values vary from 1.71 to 4.52.Intercalary satellites are commonly found on the first or second pair.More than two individuals were sampled for E.franchetii,E.leptorrhizum,E.stellulatum,and E.wushanense and all had consistent and stable cytological traits with respect to their karyotype formula,Stebbins' asymmetry,and AI values,except for E.leptorrhizum.Thesignificance and limits of the cytological results for Epimedium is also briefly discussed.3.Genome sizeGenome sizes of 24 Epimedium Species were detected by flow cytometry.By using Arachis hypogaea as first internal standards and Pisum sativum as second internal standards.The differences in genome size between different Epimedium species were compared,and the correlation between genome size and flower morphology(length of inner sepal and length of inner spur)were analysed,and the correlation between genome size and geographical distribution(Longitude,Latitude,altitude)were analysed.The results showed that genome size varied between 1.34 pg and 5.17 pg,the genome size of E.fangii was smallest,the genome size of E.zhushanense was largest,the latter is nearly 4 times of the former.There were no significant differences in the genome size between length of inner sepal or length of inner spur.Geographically,the genome size tends to decline from north to south.4.Molecular phylogeny based on genomic dataDetermining phylogenetic relationships among very closely related species remains to be a challenge for evolutionary biologists due to interlocus phylogenetic discordance and the difficulty of obtaining variable markers.Here,we used a Genotyping-by-Sequencing(GBS)approach to sample a reduced representation genomic data set and infer the phylogeny of 57 samples representing 41 speces plus 4unidentified speces in the genus Epimedium.We inferred the phylogeny based on using maximum likelihood.The genomic results suggested three clades are recognized in the Chinese group,one including Epimedium yinjiangense and E.simplicifolium distributed in northeastern Guizhou,the second clade containing ca.15 species such as E.membranaceum,E.davidii and E.brevicornu distributed in the north and southwest of China,and the third representing ca.22 speces plus 4 unidentified speces,for example E.ilicifolium,E.sagittatum and E.wushanense,mainly distributed in Central China with a few in Jiangxi and Zhejiang.The results of phylogeny analysis indicated that small flower group mixed in the lineages with large flowers and species with small flowers were evolved at least 5 times independently.In the second clade the E.campanulatum and E.ecalcaratum petal without spur was reversedly evolved.