Choroplast Genome Evolution And Species Delimitation Of Notopterygium

Speciation and species delimitation have been considered to be the "Mystery of mysteries" in evolutionary biology since Darwin's time,and of which has always been the focal issue in molecular ecology and evolutionary biology.Notopterygium H.de Boissieu species within family Apiaceae are perennial herbs,mainly distributed in the the Qinghai-Tibetan Plateau and adjacent alpine regions in China.Since Notopterygium was established,six species(i.e.N.incisum C.C.Ting ex H.T.Chang,N.franchetii H.de Boissieu,N.forrestii H.Wolff,N.tenuifolium M.L.Sheh & F.T.Pu,N.pinnatiinvolucellum F.T.Pu & Y.P.Wang and N.oviforme R.H.Shan.)were confirmed in this genus.The species have similar morphology,weak species boundary and different distribution ranges,which provide a good opportunity for studying their divergent process and species delimination.In the present study,we determined their phylogenetic relationship via chloroplast genome data,and also integrated morphological and molecular data to eluminate their divergence as well as interspecific delimitation.Our results indicated that the chloroplast genomes of Notopterygium species,similar to other ones of angiosperms,have classical four-phase strctures.Their sizes ranged from 157,462 bp to 162,478 bp in length,which contain 85 protein-coding genes,37 transfer RNA(t RNA)genes and 8 ribosomal RNA(r RNA)genes,respectively.Moreover,the gene contents and orders,the number of introns and the GC contents within chloroplast genome among these species are high conservative.Meanwhile,we identified some high variation regions in the non-coding regions of their chloroplast genomes,such as trn K-rps16,atp I-rps2,pet N-trn E,ycf3-trn S,acc D-ycf4,ycf2-trn L,ndh F-rpl32 and ndh G-ndh I,which could develop potential DNA barcodes for species delimination.In addition,we found three genes(psa I,psb I,rpo A)in this Notopterygium species which might be related with cold-tolerant and subsquently adapt to extreme climatic conditions in alpine regions suffered strong positive selection.Addtionally,a generally accepted convention is numerous nucleotide variations(e.g.,whole chloroplast genomes)have much higher resolution than that of few chloroplast or nuclear gene fragments for species delimination.In order to better classify the interspecific relationships and species delimitation via candidate DNA barcodes in Notopterygium species,we amplified and sequenced four barcoding markers,including mat K,rbc L,trn S-trn G,nr ITS and other five nuclear molecular markers(i.e.,25629,25679,32125,OG29101 and OG29960),then integrated morphological evidences to determine their species boundaries.Our results demonstrated that all these markers each revealed that N.oviforme and N.franchetii have much more closely relationship with a high bootstrap support than other taxon.In addition,we found that four DNA barcodes have higher resolutions than other plastome and nuclear molecular markers for revealing their interspecific relationships.Especially for the nr ITS,which had the highest discriminatory power within the all molecular markers.Furthmore,our phylogenetic analysis indicated the Notopterygium species could be divided into four obvious monophyletic clades with high bootstrap support when using combined nr ITS and rbc L sequences,N.incisum and N.pinnatiinvolucellum is clustered into a single clade and the N.franchetii and N.tenuifolium is clustered into another single branch.Our divergence time estimation uncoverd that the orgination of Notopterygium species dated back to the mid-to late Pleistocene boundary(ca.1.62 Mya),which was agreement to the timescale of the recent large uplifting of the Qinghai-Tibet Plateau.