Hybrid Speciation Of The Endemic Herb Genus Notopterygium In The Qinghai-tibet Plateau And Adjacent Areas

Hybridization is considered as main driving force of species divergence and biodiversity formation in nature.At present,most of studies of hybrid speciation mainly focused on long-lived trees,shrubs,model crops and its related species,while perennial alpine herbs is still relatively rare determined.The Qinghai-Tibetan Plateau（QTP）and its adjacent areas are important biodiversity hotspots in the world.It is of great scientific significance to understand the origin and evolution of global biodiversity to study hybridization in this area.Notopterygium is a rare and endangered medicinal herbaceous genus endemic to QTP and its adjacent areas.This genus presently consists of four species,N.franchetii,N.forrestii,N.incisum and N.oviforme.In this project,we firstly developed the expressed sequence tag-simple sequence repeat（EST-SSRs）and single nucleotide polymorphism（SNPs）data based on transcriptome sequencing（RNA-seq）and simplified genome sequencing（SLAF-seq）methods,respectively.Then,we used the population genetic datasets,combined with niche analysis,to investigate putative the hybridization origin and speciation history of the genus Notopterygium.The main results are as follows:（1）Comparative transcriptome and the development of EST-SSRs of N.incisum and N.franchetiiWe performed high-throughput RNA sequencing（RNA-seq）to characterize the transcriptomes of the roots,stems and leaves of two important medicinal species of N.incisum and N.franchetii based on Illumina Hiseq^TM 2000 platform.After de novo assembly,a total of 81,446 and 63,153 unigenes were generated from the two herbal species,respectively.A total of 6375 single-copy orthologous genes were identified by Ortho MCL between N.incisum and N.franchetii.In addition,nineteen of identified 381positively-selected candidate genes potentially involved in RNA splicing,DNA repair,glutathione metabolism and the plant–pathogen interaction pathway,and 12 transcription factors were detected in both plants.They were further enriched in various functional gene categories possibly responsible for environment adaptation in high mountains.In addition,a total of 1184/1007（N.incisum/N.franchetii）SSR loci primers were identified,of which 60primer pairs between the both Notopterygium species were randomly selected and determined.Finally,17 polymorphic EST-SSRs were successfully characterized for the two endangered species,which provides important insights into population genetics and evolutionary history of Notopterygium.（2）Population genetic based on EST-SSR markersA total of 1088 leaf samples from the four Notopterygium species were collected across 62natural populations.We investigated evolutionary history of four endangered Notopterygium herb in the QTP and adjacent regions based on 17 pairs of EST-SSRs.Genetic diversity analysis showed that N.oviforme had the highest values of variations,followed by N.forrestii,N.franchetii and N.incisum were among before two species.The AMOVA suggested that the greatest degree of genetic variation was within populations.Combined analysis of PCo A,STRUCTURE and phylogeneny indicated that the four species were obviously clustered into four unambiguous groups,which corresponded to their respective species,respectively.Interestingly,N.franchetii and N.oviforme exhibited close genetic relationship.The historical gene flow analysis showed low to medium levels of gene flow between species using MIGRATE,and gene flow between N.forrestii and N.franchetii were more larger than that of between N.incisum and N.forrestii.The BAYESASS analyses showed that low level of recently gene flow among species,which results suggested that there was strong interspecific differentiation among Notopterygium species.（3）Hybrid origin of Notopterygium based on population genomic SNPs analysesThe genome-wide SNPs data obtained by SLAF-seq was used to investigate the hybridization origin and speciation history of Notopterygium.Phylogenetic analysis indicated that four Notopterygium species formed a highly supported monophyletic clade,N.incisum,N.forrestii,and N.oviforme clustered into separate genetic clusters corresponding to their respective species,and N.franchetii was nested in the clade of N.oviforme.The genetic structure analysis based on Bayesian clustering showed that when K=2,individuals of the putative hybrid species,N.forrestii,exhibited mixed ancestry derived from N.incisum and N.franchetii.When K=3,individuals of N.oviforme exhibited mixed ancestry derived from N.forrestii and N.franchetii.When K=4,the genetic clusters mostly corresponded to the four species identified from morphological traits.It was consistent with the results of principal component analysis.Approximate Bayesian Calculation suggest that gave rise to N.forrestii originated from the ancient hybrids from N.incisum and N.franchetii in the early Quaternary about 2.26（CI 2.01–2.35）,and N.oviforme occurred from N.forrestii backcrossed with N.franchetii in the Middle Pleistocene about 1.20（CI 1.20–1.20）Mya.Based on the result of MIGRATE,there was bidirectional gene flow between N.forrestii and N.franchetii and N.oviforme.Treemix and ABBA-BABA test indicated that both N.forrestii and N.franchetii had gene flow to N.oviforme,which further confirmed the recent hybridization origin events of Notopterygium.Regional climate changes,particularly the intensification of East Asian winter monsoon,and tectonic movements（affecting both Qinghai-Tibetan Plateau and Qinling Mountains）may have facilitated the occurrence hybrid speciation of Notopterygium.（4）Niche differences analysisThe effects of ecological differentiation/isolation on hybrid speciation were revealed by quantitative analysis of climate variables and niche differences of Notopterygium.The results indicated that there were significant differences among N.incisum,N.franchetii and N.oviforme in bio 1,bio 3,bio 5,bio 12 and bio 14.These key environmental factors have driven the ecological divergence between hybrids species and its parental species.Based on the niche analysis of G-space and E-space,there were specific or differentiation signals in the natural geographical distribution habitat of Notopterygium.Meanwhile,N.forrestii showed ecological specialization with respect to their parental species,whereas N.oviforme would have completely shifted its niche,which can be attributed to its higher environmental adaptability.