Phylogeny And Taxonomy Of Damnacanthus(Rubiaceae)

Damnacanthus is a small genus of Rubiaceae,including 13 species,11 of which are distributed in China.They are evergreen shrubs mainly distributed in East Asia.Studies on its phylogeny are rare.Most species of Damnacanthus have fleshy roots.Because its roots in shape look like those of Morinda officinalis,it is often used as a substitute for the Chinese medicine Morinda officinalis.As a taxon with taxonomic confusion in Rubiaceae,Damnacanthus has been placed in different tribes of the family.In this study,chloroplast genomics,multiplex-PCR,and high-throughput sequencing techniques were used to explore the phylogenetic relationships within Damnacanthus,and the application of specimen materials in delimiting species boundaries was also explored.In this study,18 chloroplast genomes were assembled and annotated,including 5individuals from Damnacanthus and 13 from its relatives.The complete chloroplast genome of D.indicus was assembled and annotated.It is 153,997?bp in total length,consisting of a large single-copy region(LSC 85,159?bp),a small single-copy region(SSC17,584??bp)and two inverted repeat regions(25,627 bp for IRA and IRB,respectively).It contains 135 genes,including 89 protein-coding genes,38 tRNA genes,and 8 rRNA genes.The phylogenetic analysis indicated that D.indicus is sister to Mitchella repens,suggesting a close relationship of the two genera.Using chloroplast genome data,we designed specific primers for DNA sequencing of Damnacanthus to facilitate PCR.In this study,we selected the specimen materials of Damnacanthus and carried out experimental verification on a phylogenetic genomic approach,which overcomes the shortcomings of sampling difficulties and insufficient information loci,aiming to better solve the problem of species identification of taxonomically difficult taxa.The small fragments of degraded DNA were repaired by DNA reconstruction technology to obtain high-quality DNA templates.Using primers designed based on complete chloroplast genome sequence,regions with high variation were screened and amplified for phylogenetic analysis.The successful application of the plant specimen materials to the phylogenetic study of Damnacanthus can be extended to other groups of Rubiaceae.In this study,a total of 192 specimens were collected from the Herbarium of Institute of Botany,Chinese Academy of Sciences(PE)and the Herbarium of Guangxi Institute of Botany(IBK),covering 11 of the 13 species of the genus,which can well represent the species and geographical diversity of Damnacanthus.We first selected 5 representative individuals for chloroplast genome comparison,designed 135 molecular markers of high variation regions,and carried out high-throughput sequencing for the DNA of 192 specimen materials of Damnacanthus.Phylogenetic analysis was conducted with the maximum-likelihood method.The results showed that there were four clades with clear relationship:(1)D.major and D.indicus;(2)D.macrophyllus;(3)D.angustifolius;(4)D.tsaii.In this study,the monophyly of four species of D.indicus,D.macrophyllus,D.angustifolius and D.tsaii was well supported,while D.major was incorporated into D.indicus.The taxonomic problems of the genus Damnacanthus have not been completely solved.In order to solve the problems,it is necessary to increase the sampling of non-domestic species.On the basis of molecular systematics,studies of morphological,palynological and anatomical evidence should be carried out to confirm each other,so as to realize the taxonomic revision of Damnacanthus.