DNA Barcoding And Phylogeny Of Saussurea Subgen. Eriocoryne (Asteraceae)

Saussurea comprise about 490 species being classified into four subgenera.Among them,S.subg.Eriocoryne consists of about 39 species mainly distributed in the Qinghai-Tibet Plateau(QTP)and adjacent regions.There are a variety of“snowball” plants within this subgenus.To adapt to the cold environment,these plants are usually stemless and covered with dense woolly trichomes.Thus,most species appear highly similar and moreover,different populations varied in different environment and resultantly,it is difficult to discriminate some species.However,the subgenus have many widely used traditional medicinal plants,such as S.gossipiphora,as well as some endangered herb,such as S.medusa.Therefore,it is necessary to develop an object and rapid method of identification and to find alternatives of endangered groups for this subgenus.At the same time,previous studies indicated that these “snowball” plants might derive more than once,but no new classification has been proposed.To explore these problems,we sampled 160 individuals of 74 populations representing 26 species within this subgenus and sequenced ITS,ETS and six chloroplast fragments,i.e.,trn H-psb A,mat K,trn K,rbc L,trn Q-rps16 and rpl32-trn L.Five of them were used to evaluate the possible DNA barcodes while all the fragments were used to reconstruct the phylogeny of the subgenus together with28 species representative of Saussurea.The main results are as follows:1,DNA barcodes.The rate of successful identification was tested with the Best Close Match(BCM),Bayesian Inference(BI)and Neighbor-joining(NJ)using ITS and four chloroplast fragments(trn H-psb A,mat K,trn K and rbc L).The results showed:(1)ITS obtains the highest species discriminatory power,which is 93.4% by BCM or 76.9% by both NJ and BI,while the four chloroplast fragments range from15.8%-61.2% when used separately.(2)The combinations of ITS and anyone of the chloroplast fragments could significantly improve the discrimination rate.Among them,ITS + trn K have the highest identification rates,being 96.2% by BCM(25species could be identified),or 88.5% by NJ or BI(23 species successfully identified);(3)ITS and any combination of two chloroplast fragments could further improve the identification efficiency to 95.4%-99.3%,depending on the fragments used.However,the adding of more fragments fail to improve the delimitation rates,or resulted in slightly lower rates in some cases.(4)the identification rates of the combined four chloroplast fragments was only 71.7%.(5)taking account of both the expenditure and identification efficiency,we recommend ITS+trn K as the DNA barcode of S.subg.Eriocoryne.In addition,alternatives to some medicinal species were discussed based on the genetic affinities.2,Phylogeny and species delimitation.Besides the DNA barcode fragments,we additionally sequenced the nuclear ETS and two chloroplast fragments,i.e.,trn Q-rps16 and rpl32-trn L.Two datasets,one composed of ITS and ETS and the other of all the six chloroplast fragments,were constructed to analyze the phylogenetic relationship of 26 species of S.subg.Eriocoryne and 28 species representatives of Saussurea.The results showed that the species of S.subg.Eriocoryne appeared in multiple clades in phylogenetic trees,indicating the polyphyly of this group.Among them,one clade composed of 12 species including S.gossipiphora,according to both the nuclear gene and the chloroplast sequences.Given the inclusion of the type of this subgenus,we propose to name the group as the newly defined subgenus.Among the remaining 14 species,six species from three sections,i.e.,four from sect.Pseudoeriocoryne,S.uliginosa from sect.Cincta and S.paxiana from sect.Eriocoryne,are distinct with the re-defined S.subg.Eriocoryne in the nuclear and chloroplast phylogenetic trees as well as morphology,which are the solely species without dense woolly trichomes within the subgenus as traditionally defined.Thus,they may be distantly related to the newly defined S.subg.Eriocoryne.However,three species,including S.tridactyla,and five species,including S.glacialis,are nested in S.subg.Eriocoryne as newly defined in the chloroplast tree or the nuclear gene phylogenetic tree,respectively.Thus,the relationship of these species with S.subg.Eriocoryne remains to be studied.In addition,the systematic division of the newly defined subgenus is discussed basing on morphological and phylogenetic results,and the delimitation of some problematic species is discussed or analyzed according to the concept of the integrated species,such as S.medusa,S.gnaphalode,S.pseudosimpsoniana,S.zogangensis and S.inversa.