DNA Barcoding And Molecular Phylogeny Of Holcoglossum Schltr. (Orchidaceae)

DNA barcode is a diagnostic technique for species-level identification using a short andstandardized DNA region.DNA barcoding aims at identifying all species of life by using the information of one or a few genes .DNA barcode will allow users to efficiently recognize known species and speed the discovery of species yet to be found in nature and contribute to biodiversity research.The universal DNA barcode for plants is still one hotspot disputed.This research aims to evalue utility of eight DNA barcode regions for land plants——atpF-atpH,matK,rbcL,rpoB,rpoC1,trnH-psbA,trnL-F,ITS was evaluated in 12 Holcoglossum species(including closely related species),choose the regions with the highest sequence variation for subsequent steps. In addition, we try to solve the systematic position of Holcoglossum species further. The main results and conclusions are as follows:1. Holcoglossum exhibit low inter-specific sequence divergence, and there is no"barcode gap"between intra- and inter-specific data. However, results from ITS and trnL-F evaluated here show it is possible to group species replicates together, which is a basic requirement for a barcode identification tool.2. The sequences, atpF-atpH,matK,rbcL,rpoB,rpoC1 and psbA-trnH are not suit to be an efficient and universal barcode for Holcoglossum or a recent radiation genus with closely related species.3. The two best regions presenting the highest sequence variation and the best resolution at the species level are ITS and trnL-F. It is seems that the single region ITS or trnL-F will be sufficient as an efficient and universal barcode for Holcoglossum. However, the combination of regions (ITS and trnL-F), is more realistic and effective solution for the identification of Holcoglossum. Our results suggest that the combination of these two sequence provided an efficient and universal barcode for a recent radiation genus with closely related species like Holcoglossum.4. Analysis of ITS matrix with ML and MP methods revealed that Holcoglossum is supported as monophyletic .The phylogenetic analyses revealed three groups which agree well with biodiversity centers of Holcoglossum. One tropical group(TC group) comprising four species was on the base, another forming a polytomy comprising five species restricted to the temperate alpine region of the Hengduan Mountains(AC group), and the third with three species somewhat distributed between the first two regions(HC group), forming sister group(TC(HC, AC)).5. Holcoglossum quasipinifolium was subsumed within HC group.