Phylogenetic And Biogeographical Research Of Scrophularia Incisa Complex

Scrophularia is a model genus of Scrophulariaceae.It contains about 200–300 species and distributed in temperate regions of Asia,Europe,and North America.Species of Scrophularia are divided into two sections,sect.Scrophularia and sect.Caninae.Scrophularia incisa complex is a monophyly subclade of sect.Caninae.It currently includes four species: S.incisa,S.dentata,S.kiriloviana and S.integrifolia.It is mainly distributed in river beaches,valleys,and wet gravel lands that have been hit by suitable snow or streams.Its distribution range covers the Central AsiaQinghai-Tibet Plateau-Mongolia Plateau area.Due to its special geographical distribution,it is a good model for studying the origin and diversity of vegetation in desert regions of the plateau.Previous reports have conducted pedigree geography studies on the S.incisa complex based on ITS and cpDNA molecular markers.Due to sampling and labeling limitations,the intermediate relationship of the complex has not been clearly resolved,and there are inconsistent research results between the markers.In order to further reveal the evolutionary history of the S.incisa complex,especially the phylogeny and biogeographic process of the S.incisa complex species distributed in China,this study focuses on the S.incisa complex distributed in Central Asia,Tibet,Xinjiang,Qinghai and Gansu,using the complete chloroplast gene data and high-throughput RADTags genomic molecular marker technology for population genetic research at the individual level and population level.Analyzing the process and causes of lineage differentiation,and using niche modeling to simulate the contemporary species suitable areas changes of the Chinese lineage of S.incisa complex,and further speculate on their species suitable areas in the Last Glacial Maximum(LGM)and the future(2070s).The results are as follows:1)Comparative analysis of chloroplast genome Based on the chloroplast genome sequence,the chloroplast genomes of four species of S.incisa complex were compared and analyzed to explore their sequence characteristics and phylogenetic relationship.The sequences consist three S.kiriloviana,two S.incisa,two S.dentata,and one S.integrifolia,a total of 8 individuals.The chloroplast genomes are among 152,088 bp and 152,868 bp in length and contain two identical copies of a large inverted repeat(IR)(25,454–25624 bp)sequence separated by small(SSC)(17,375–17,940 bp)and large(LSC)(83,531–84,454 bp)single-copy regions.Each chloroplast genome contains 115 genes,including 80 protein-coding genes,31 t RNA genes,and 4 r RNA genes.In all chloroplast genomes,the gene content,gene order,AT content,and IR/SC boundary structure are almost the same.However,their length differs due to the shrinking / expanding of the IR/SC boundary.Simple sequence repeat(SSR)analysis further indicates that the most abundant repeats in these chloroplast genomes are A/T.Genome comparative analysis was performed using m VISTA software,and the sequence similarity was 94%.Using DNAsp for nucleotide polymorphism analysis,there are three regions with Pi > 0.02,which are considered to be highly variable regions and can be used to develop molecular markers for future phylogenetic analysis and species identification.Phylogenetic analysis is fully supported on 50% of nodes(PP/BS = 1/100).S.integrifolia is in the basal position,S.kiriloviana and S.incisa were sister branches.The results of the divergence time estimates indicate that the differentiation time of the existing S.incisa complex species is 2.47 Ma(95% HPD: 0.81–4.5 Ma).2)SNPs loci based on RAD-tags In this study,42 populations(278 individuals)of the S.incisa complex were used to simplify the genomic database,and STACKS v2 was used for data processing.A total of 4784 high-quality SNP sites were obtained.Both principal component analysis and genetic structure analysis support that the optimal grouping of the S.incisa complex is 4,which corresponds to the gene pools of four species.S.incisa's gene pool and S.dentata's gene pool have a lot of overlap.S.kiriloviana's gene pool and S.integrifolia's gene pool can be well separated from them.S.incisa and S.dentata have the closest genetic relationship,and S.integrifolia is related to the other three species farthest.Gene exchange analysis showed that the gene exchange between S.integrifolia and S.kiriloviana occurred before the differentiation of the Chinese lineage,and the group that generated the gene exchange was kept in S.kiriloviana,and proved that the direction of gene exchange between the two species was unidirectional,and the genes flow from S.integrifolia to S.kiriloviana.Phylogenetic analysis based on the maximum likelihood method revealed that S.integrifolia was at the base,S.dentata and S.incisa were sister branches,and then S.kiriloviana gathered together.The divergence time of the S.incisa complex calculated by BEAST shows that the divergence time between the Central Asian pedigree and the Chinese pedigree is 6.59 Ma(95% HPD: 4.98 Ma-8.27 Ma).The reconstruction of the ancestral area shows that the S.incisa complex may have originated in Central Asia.3)Ecological niche modeling The ENM analysis based on Maxent showed that the potential distribution area of each species is consistent with the actual distribution area,and the AUC value is significantly greater than 0.9.S.kiriloviana was mainly distributed in the areas near Tianshan and Altai Mountains during the LGM,and then expanded to the north and south sides of the Tianshan Mountains.S.dentata was mainly distributed in the western part of the plateau during the LGM,then gradually migrated and expanded to the east of the plateau.S.incisa was mainly distributed in the northeast of the Qinghai-Tibet Plateau in the LGM,and then the distribution area was compressed to the southeast of the plateau and expanded to the north of the Mongolian plateau.In the future(2070s),the distribution area of S.dentata will expand significantly,and the distribution area of S.kirilovaina will move slightly north.The distribution area of S.incisa in China will be significantly reduced,and the distribution area in Mongolia will spread west.