| Dendrobium officinale Kimura et Migo(Orchidaceae)is a rare and endangered species endemic to China.Its stems have been used as traditional Chinese tonic medicine for hundreds of years.This species has important economic and scientific research values.However,the species relationships of D.officinale and its closely related species(D.tosaense,D.shixingense,D.flexicaule,D.scoriarum,D.aduncum)still remain unclear.Accordingly,the classification and accurate authentication of these species are extremely difficult,which is adverse for effective utilization and conservation of genetic resources.Here,we firstly analyzed the phylogenetic relationships of D.officinale and its closely related species based on both morphological and molecular data,i.e.,the complete plastome sequences and genome-wide SNPs derived from Restriction-site associated DNA sequencing(RAD-seq).Then,we used the complete plastome sequences to authenticate the species in this taxonomically difficult group,and compared the intraspecific plastomic mutational hotspots among these species.Finally,we developed SNP markers and evaluated the population genetic divergence for D.officinale by using RAD-seq.Based on extensive sampling of wild populations,we investigated the plant external morphological and stem microscopic characteristics of 255 individuals from 41 popolation of D.officinale and its closely related species.The results revealed that there were abundant intraspecific variations and little interspecific differences in morphology,which makes it difficult to distinguish these species.Several microscopic characteristics of transverse section of stems could be used to discriminate D.flexicaule,D.scoriarum,D.aduncum,but failed in the other three species.Based on careful comparison,8 morphological characteristics of flowers were selected to distinguish D.officinale and its closely related species.The complete plastome sequences were employed to investigate the phylogenetic relationships of this group.We compared 63 Dendrobium plastomes comprising 40 newly sequenced plastomes of the 6 species and 23 previously published plastomes.The plastomes of D.officinale and its closely related species were shown to have conserved genome structure and gene content.Comparative analyses revealed that small single copy(SSC)region contained higher variation than large single copy(LSC)and inverted repeat(IR)regions,which was mainly attributed to the loss/retention of ndh genes.The Maximum likelihood(ML)and Bayesian inference(BI)analysis were performed for the complete plastome sequences.The individuals of each of the 6 species were resolved as a monophyletic group.The phylogenetic trees have divided the 6 species into three clades.D.aduncum,the earliest diverged species among the six species,formed an isolated clade,while the remaining 5 species can be divided into two clades.One consisted of two species,D.flexicaule and D.scoriarum.In the other clade,D.shixingense was placed as sister to the remaining species of D.officinale and D.tosaense.According to BEAST analysis,we deduced the interspecific divergence of D.aduncum,D.scoriarum and D.flexicaule to be in the Pliocene(5.03-2.82 Ma),while the other three species in the Pleistocene(1.50-1.03 Ma).The rapid radiation of this group was likely driven by the uplift of the Qinghai-Tibetan Plateau(QTP).In view of the maternal inheritance of plastome in Orchidaceae,the genome-wide SNPs derived from RAD-seq were employed to infer the phylogenetic relationships of D.officinale and its closely related species.A total of 269,928 SNPs were identified.The analyses based on these SNPs showed that all of the 6 species were strongly supported as monophyletic.D.aduncum was located at the base of this group and formed a sister group relationship with the other 5 species.Then.D.scoriarum,D.flexicaule and D.shixingense diverged successively.D.tosaense is sister to D.officinale.This tree topology is somewhat different from those derived from the complete plastome sequences.The cytonuclear discordance within this group might be related to introgressive hybridization,which remains to be verified.Since D.officinale and its closely related species are morphologically distinct from each other and are monophyletic in molecular phylogeny,we proposed that they can be treated as 6 separate species.The molecular authentication of D.officinale and its closely related species was carried out.The ML and Neighbor-joining(NJ)trees of 72 samples were reconstructed based on different datasets(complete plastome sequences,8 DNA fragments or their combination,and 5 plastomic regions).By comparison,we found that the complete plastome sequence dataset had the highest discriminatory power for D.officinale and its closely related species,indicating that complete plastome sequences can be used to accurately authenticate Dendrobium species.Furthermore,we estimated the pairwised intraspecific sequence variability(SV)of 90 syntenic non-coding loci of D.officinale and its closely related species.Our results indicated that the mutational hotspots for the intraspecific-level studies of Dendrobium species were diversified.Considering diversified mutational hotspots among different orchid genera and different species,we proposed that the plastome-wide comparison is required for studies of orchid species at different levels.For developing SNP markers and evaluating the population genetic divergence of D.officinale,RAD-seq analysis was conducted on 24 individuals from 8 populations.The phylogenetic analysis and population genetic structure analysis using 653,376 SNPs showed that the eight populations of D.officinale could be divided into three clades,the east(P10,P12,P11,P14),central(P7)and west(P2,P4,P6)clade.By contrast,the genetic divergence between the central population(P7)and other populations was high.Further analyses of gene flow and selective sweep indicated that genetic divergence among natural populations of D.officinale was related to natural selection.Moreover,SNP markers of D.officinale were developed,and a database of SNP markers related to polysaccharide metabolism pathway was established,which could be used for studies of population genetic structure,DNA molecular identification and breeding of D.officinale in the future. |
