Phylogeography Study Of Akebia Trifoliata Based On Chloroplast Gene And ITS Sequence

Akebia trifoliata（A.trifoliata）is a vine of Akebia Decne.,a family of Lardizabalaceae.It is a new cash crop integrating food,medicine,oil and appreciation,commonly known as‘August melon’and‘August fried’.This species is widely distributed in China,South Korea,and Japan.Continuously distributed in the eastern and southeastern edges of the Qinghai Tibet Plateau（QTP）in China,as well as in subtropical areas,it is a common species found on slopes and dense forests（especially evergreen broad-leaved forests）at altitudes of 200 to2600 meters.This large region has rich vegetation types and ancient origins,and has undergone a complex history of vegetation changes since the late Pliocene.However,the response mode of the region to historical climate change is still unclear,and it is urgent to select species that are continuously distributed in the region and integrate multidisciplinary research methods for in-depth research.In this study,391 materials from 61 populations of A.trifoliata collected in the above areas were selected from the A.trifoliata germplasm resource bank.Through PCR amplification and sequencing of the chloroplast gene rps16 and the ITS sequence of nuclear DNA fragments,the genealogical differentiation The spatial distribution pattern of population dynamic history and genetic variation,as well as the ice age refuge and post ice or inter glacial migration and expansion routes of A.trifoliata.The research results are as follows:（1）Out of 391 samples,75 ITS haplotypes（Ribotype:R1～R75）and 22 rps16haplotypes（Chlorotype:C1～C22）were identified.Comparative studies have found that the genetic diversity（Hd）,nucleotide diversity(π×10^-3),total genetic diversity（h _T）,average genetic diversity among populations（h _S）,and genetic differentiation coefficients(F_ST)of ITS haplotypes are 0.86,3.07,0.88,0.31,and 0.64,respectively;The genetic diversity parameters of rps16 haplotypes are 0.31,0.90,0.31,0.18,and 0.09,respectively.From this,it can be seen that the genetic diversity of the ITS sequence in this study population is significantly higher than that of the rps16 sequence,which may be related to the fact that seeds are commonly used as the transmission pathway of A.trifoliata.（2）The AMOVA analysis results indicate that the ITS sequence variation mainly comes from inter population;On the contrary,the rps16 sequence variation mainly occurs within the population.At the same time,Mantel test found that there was a significant correlation between Genetic distance based on ITS sequence and geographical distance（R²=0.209,p=0.01）,while the correlation coefficient between Genetic distance based on rps16 sequence and geographical distance（R²=0.0005,p=0.17）was small.Finally,genetic differentiation coefficient analysis revealed that the G_ST（0.437）of the ITS sequence was significantly lower than that of the N_ST（0.677）,while the G_ST（0.435）of rps16 was greater than its N_ST（0.305）.This suggests that the ITS sequence may exhibit a distinct lineage geographical structure in the population,while the lineage characteristics of rps16 are unclear.（3）Based on the TCS network diagram and ML tree of ITS sequence haplotypes,we divided the population of A.trifoliata into four lineages,namely the Eastern QTP（ETP）,Central Northern China（NCC）,Central China（CC）,and Eastern China（EC）lineages.According to the distribution and number of haplotypes among different populations,we speculate that R2 and C1 are the ancestral haplotypes of ITS sequence and rps16 gene,respectively.The Qinba Mountains,the Nanling Mountains and the Yunnan-Guizhou Plateau may be the main refuge of A.trifoliata in the ice age.（4）The results of molecular clock showed that the ITS sequence and the haplotype of rps16 gene of A.trifoliata originated from the Late Pleistocene to Early Pleistocene,and there was large-scale differentiation in the middle of Pleistocene.The reconstruction of the ancestral distribution area indicates that in the early stage,A.trifoliata was mainly distributed in the eastern region of the QTP（A）and the central northern region of China（B）;Subsequently,these two regions colonized the central region of China（C）;Finally,it mainly spreads to the eastern region of China（D）through the C region.Although there may be occasional mutual or reverse propagation events in these regions,we have not observed any propagation phenomena from region D to region A.This indicates that the migration route of A.trifoliata is mainly characterized by long-distance one-way migration from west to east,accompanied by short distance migration from north to south.（5）The neutral test showed that the entire population of A.trifoliata,as well as the ETP and EC lineages,had undergone expansion in history.The expansion of the entire population of A.trifoliata occurred during the last glacial ice cycle（150 ka）.The expansion of the ETP lineage mainly occurred in the Sichuan Basin,while the expansion of the EC lineage may include parts of South Korea and Japan.The results of this study propose a new viewpoint that species widely and continuously distributed in the eastern and southeastern edges of QTP and subtropical China have multiple shelters during the ice age and migrate in specific directions.