Phylogenomics And Ultra-barcoding In Panax L.

The Panax genus is an ecomomically and medicinally important group.However,the evolutionary relationships within the genus Panax are controversial and authentication for Panax species is very difficult.In this study,59 complete chloroplast(cp)genomes and 54 rDNA of nine specie were sequenced and successfully assembled.In conjunction with the previously published cp genomes,Genomes feature,SSR,and substitution rate were compared to characterize chloroplast genome of Panax.Phylogenies were reconstructed based on the whole chloroplast genome and rDNA to elucidate the evolutionary relationships within the genus Panax.Furthermore,characteristics of the urtra-barcoding and universal barcoding(matK,rbcL,trnH-psbA),divergences of intra-species and inter-species,and species identification rate were compared to evaluate the urtra-barcoding of Panax.Main results and conclusions are summarized as follows: 1.Comparative analysis of chloroplast genomesThe complete cp genome sequence of Panax included a pair of IRs that divide the genome into LSC and SSC regions.The total length of chloroplast genomes was between 155,992 bp and 156,466 bp,and the total GC content was between 38.00% and 38.10%.The cp genome of Panax possessed 114 unique genes including 80 protein-coding genes,30 tRNAs,and four rRNA genes.The extensions of the IRs into rps19 and the intergenic spacer between rpl2 and trnH occurred respectively at the IRA/ LSC and IRB/LSC boundaries.Although the expansion of the IRs into the ycf1 pseudogene at the IRA/SSC junctions occurred in all species,the overlap between the ycf1 pseudogene and ndhF was only detected in P.notoginseng,P.zingiberensis,P.vietnamensis,P.quinquefolius,and P.wangianus.The total number of SSR was between 34 and 40 in cp genomes.Among them,mostly SSR were distributed in no-coding regions and composed of A and T nucleotides.we did not observe any signatures of positive selection in the protein-coding genes in the ten Panax cp genomes,which may have undergone strong purifying selection during their evolution.In addition,seventeen cpDNA markers—ccsA,matK,ndhF,ndhH,pet L,rbc L,rpl22,rpoA,rps3,ycf1,atpA-atpF,ccs A-ndhD,inf A-rps8,ndhI-ndhA,rpl14-rpl16,rps19-rpl2,trnY-GUA-trnE-UUC— with relatively high levels of variation were identified from the cp genomes.2.Phylogenetic analysisThe phylogeneic analyses based on the whole cp genome and rDNA revealed P.stipuleanutus was placed at the basally branching position.Panax ginseng was sister to P.quiquefolius.The topology of cp tree showed P.notoginseng,P.bipinnatifidus,P.vietnamensis,P.zingiberensis,P.wangianus,P.major,and P.sinensis were clustered into a clade,in which P.notoginseng was placed at the basally branching position,and the clade of P.vietnamensis + P.wangianus was sister to P.zingiberensis.While the ML analyses based on rDNA showed P.notoginseng,P.zingiberensis and P.vietnamensis were clustered into a clade,and the overlap of geographic distribution between P.notoginseng and P.zingiberensis were detected.Therefore,the introgression may be presented between P.notoginseng and P.zingiberensis.P.bipinnatifidus,P.major,and P.sinensis were clustered into a clade,which may be interpreted as the result of inter-specific hybridization.Furthermore,phylogenetic analysis showed P.zingiberensis,P.vietnamensis and P.wangianus were respectively recovered as well-supported monophyletic lineages,which thus could be separated from P.bipinnatifidus complex.3.Evaluation of ultra-barcodingUltra-barcoding and universal barcoding(matK,rbcL,trnH-psb A)harbored more inter-specific divergences than intra-specific divergences,while less variable sites,parsimony informative sites and species identification rate were identified in universal barcoding.Thus universal barcoding was not optimal for species authentication in Panax.Ultra-barcoding harbored the highest sequence variation,species discrimination rate(70%)and reliability,thus it could be suitable for species authentication in Panax.While plastid genomes and rDNA could not authenticate P.bipinnatifidus,P.major,and P.sinensis,the ultra-barcoding did not address the limitation in discrimination where hybridization or repeated introgressions.Those single copy nuclear regions generated by ultra-barcoding sequence can potentially be used to help solve difficult situations involving hybridization or repeated introgressions.Collectively,our results highlighted that the cp genome in the genus Panax is highly conserved and the phylogeny within the Panax genus is reconstructed with well-supported.Besides,our results show the potential of the whole cp genome in authenticating species,which extends the use of barcoding and provides a new method for group with identification difficulties.