The Taxonomic Status And Identification Of Some Species Of The Dendrobium Group In China

The genus Dendrobium belongs to the tribe Dendrobieae(Orchidaceae:Epidendroideae)and is the second largest genera in the family Orchidaceae.There are more than 1400 species in worldwide area.Lindley divided this genus into four sections in 1844,while Schlechter did set 41 sections at 1912 and 14 of those in China.The relationships among Dendrobium sections were discongruent and most sections were confirmed to be paraphyletic in some researches.The phylogenetic relationships within Densiflora section is one of the most complicated in the genus Dendrobium of China.In addition,the relationships of some of these species are not clear until now.Therefore,it is necessary to do some phylogenetic research on Chinese sect.Densiflora.In this study,we reconstructed the phylogenetic tree of 50 Dendrobium species,based on Bayesian inference method using the sequences of the internal transcribed spacers(ITS)of nr DNA and the intergenic region of cpDNA.The results demonstrated that Chinese sect.Densiflora was paraphyletic and the species of sect.Densiflora could be divided into two different clades.In the first clade,the species of D.chrysotoxum,D.harveyanum and D.brymerianum were nested in the Holochrysa section with higher support(PP=0.86);In another clade,D.lindleyi and D.jenkinsii formed a single subclade(PP=1),D.densifloum,D.thyrsiflorum,D.farmer,D.sulcatum formed another subclade(PP=1).We supported that D.lindleyi and D.jenkinsii be involved into a separate section.D.chrysotoxum,D.harveyanum and D.brymerianum should be set to the Holochrysa section using the molecular evidence.Both Dendrobium thyrsiflorum Rchb.and Dendrobium densiflorum Lindl.belong to Chinese sect.Densifora.The stems of D.thyrsiflorum and D.densiforum can be processed to make an important Chinese medicine "Huangcao Shihu".However,their active components and efficacy are different.It is difficult to identify these two species without flower and leaf by classic method and they were defined as confused species in Flora of China(1999).We developed approaches to identify D.thyrsiflorum accurately.The results showed that D.thyrsiflorum and D.densiflorum shared the same haplotype of ITS,and it was difficult to identitied these relative species.The phylogenetic tree with Maximum Likelihood method was constructed using 7 chloroplast sequences(psbA-trnH,rps15-ycf1,psbB-clpP,rpl32-trnL,atpH-atpI,psbK-psbl and accD-psal)to test the relationship between D.thyrsiflorum and D.densiflorum.It showed that D.thyrsiflorum and D.densiflorum could't be differentiated by a single chloroplast sequence.The inter-specific variation was greater than intra-specific of 3 chloroplast sequences(atpH-atpI,psbK-psbI and rps15-ycf1).All D.thyrsiflorum samples were set up a monophyletic group,D.thyrsiflorum and D.densiflorum could be differentiated using two random sequences from atpH-atpI,psbK-psbI and rps15-ycfl.A specific fluorescently-labeled primer pairs(R15/Y1)was designed from rps15-ycf1 region and set up a detection system of D.thyrsiflorum based on SYBR Green ? real-time PCR.We discerned the significant difference between the interval cycle threshold(Ct)value of D.thyrsiflorum[13.83,15.9]and that of the relative species[29.22,37.01]by real-time PCR.It showed that D.thyrsiflorum could be accurately and rapidly identified by the primer pairs.This operation also provided a new insight for the identification of the relative species of Dendrobium.