Phylogenetic Relationships And Evolutionary History Analysis Of Zanthoxylum Based On Chloroplast Genome Sequence

Rutaceae is one of the most numerous families in the order Sapindales,including more than 1,800 species in 160 genera,which are widely distributed in tropical,subtropical and temperate regions.Zanthoxylum L.is the second largest genus of Rutaceae with about225 species,and is the only genus in Rutaceae with a pantropical distribution.Due to the rich fossil record and wide geographic distribution,Zanthoxylum L.is an ideal taxon for studying Cenozoic biogeography worldwide.Meanwhile,with the rapid development of molecular systematics,the worldwide origin and evolutionary history of the Zanthoxylum L.has become a major concern for current research scholars.Therefore,clarifying the molecular phylogenetic relationships among species of the genus Zanthoxylum and the evolutionary history of modern distribution patterns can provide important theoretical support for the molecular breeding of Zanthoxylum and the conservation of wild germplasm resources.In this study,in order to clarify the phylogenetic relationships of Zanthoxylum and its close relatives,selectting suitable outgroups for the next phylogenetic analysis of Zanthoxylum at the species level,three subfamilies of Rutaceae,including 16 genera with a total of 46 chloroplast genome sequences,were first selected,and the affinities among the subordinate genera of Rutaceae were reconstructed using species of Burseraceae Kunth,Simaroubaceae DC.and Surianaceae Arn.as outgroups.The phylogenetic relationships among the genera under the subfamily were analyzed.Secondly,based on the intergeneric affinities of Rutaceae,three species of Phellodendron Rupr.and Tetradium Lour were selected as outgroups,and the phylogenetic relationships of representative species of Zanthoxylum in China were reconstructed based on the whole chloroplast genomic DNA(cp DNA)data of 44 species of Rutaceae,including four species distributed abroad,and the ancestral distribution area was reconstructed to explore the phylogenetic relationships among species of the genus Piper and their evolutionary history.The main findings are as follows.(1)The phylogenetic tree of Rutaceae at the genus level constructed based on cp DNA data showed that Rutaceae species formed a monophyletic lineage and were further divided into two major branches,with all Subfamily Aurantioidae species in branch I clustered into a subbranch and forming a sister relationship with Ruta L.;branch II consisted of species of subfamily Rutoideae and Toddalioideae,with Toddalia species nested within Zanthoxylum.Zanthoxylum forms a sister relationship with Phellodendron and Tetradium,which is the nearest relatives.The phylogenetic tree constructed based on cp DNA data showed high support for each branch.Therefore,this study confirmed that the incorporation of species of the genus Toddalia into species of Zanthoxylum is reasonable.(2)Chloroplast genomes of Zanthoxylum are all typical tetragonal structures,and the sequence length ranges from 157,231 bp-159,072 bp,encoding 132-133 genes,including87-88 protein coding genes,37 r RNA genes and 8 t RNA genes.The genome boundary showed that the IR region of Zanthoxylum species was 26,408 bp-27,639 bp,which showed obvious contraction.Visualization and nucleotide diversity analysis showed that the variation degree of non-coding region was higher than that of coding region,and the variation degree of LSC and SSC region was higher than that of IR region.Six hot spots with obvious variation were screened out,including rps16,psb K-trn G-UCC,trn T-UGU-trn L-UAA,rpl22,ndh F-rpl32 and ycf1.(3)Based on the chloroplast genome,constructed a phylogenetic tree with ML,BI and MP methods at the species level of Zanthoxylum,showing strong support for two Zanthoxylum species from the western Indian Ocean islands forming a branch,i.e.,Clade I at the base,which forms a sister group relationship with other Zanthoxylum.And other Zanthoxylum species further forming two major branches,Clade II mostly found in tropical and subtropical regions,Clade III species are mainly distributed in the northern hemisphere.The species of Z.tragodes from America,is located at the base of Clade III,indicating that the East Asian species is closely related to the North American species.It is noteworthy that according to the traditional Engler classification system species of Zanthoxylum are divided into the subgenus Zanthoxylum and the subgenus Fagara,but in this study,the Z.nitidum in the subgenus Fagara were clustered into one branch with the species of the subgenus Zanthoxylum,while the Z.dimorphophyllum which located in the subgenus Zanthoxylum were clustered together with the subgenus Fagara.Therefore,it may not be necessary to subdivide the genus Zanthoxylum into two subgenera.(4)Rresults of cp DNA time tree and ancestral distribution area reconstruction indicate that the two species from the western Indian Ocean separated from other Zanthoxylum at22.38 Ma in the Tertiary Miocene.Clade II and Clade III,which comprise most of East Asian species,separated at 21.23 Ma and began their separate evolution,at approximately6-5 Ma began their rapid divergence.Significant uplift of the northeastern Tibetan Plateau occurred at 14 Ma,11.3 Ma,8 Ma,1.2 Ma,and 0.15 Ma,and at 8.2-7.4 Ma,the East Asian monsoon began to form in the region,and the uplift of the Tibetan Plateau significantly influenced the climate of the region.During the Quaternary glacial period(since 3 Ma),precipitation was mainly concentrated at low latitudes,and the Tibetan Plateau and adjacent areas may have become a refuge for many plants that may spread to higher elevations during warming and to lower elevations during cooling.Therefore,the uplift of the Tibetan Plateau and Quaternary glaciation may be responsible for the separation of the two major branches of the genus Zanthoxylum in China.(5)According to the comprehensive analysis of fossil records,previous studies and data from the present study,species of Zanthoxylum experienced successive intercontinental or long-range migrations in the Paleocene or Eocene after their origin in Eurasia.It is likely that Zanthoxylum colonized North America through the North Atlantic land bridge in the Eocene,and then migrated back to Eurasia probably through the Bering land bridge in the Oligocene or Miocene.Considering the affinity between East Asian species and North American species,it is likely that the extant Zanthoxylum species in China are the surviving species of North American species that migrated back to Eurasia via the Bering Land Bridge during the Paleocene or Eocene.The diversity of Zanthoxylum may have been influenced by the uplift of the Qinghai-Tibetan Plateau,the fourth ice age and human activities.Our results provide evidence for the origin of Chinese Zanthoxylum on the Tibetan Plateau in southwest China.