| Objective:Gaultheria leucocarpa B1.var.yunnanensis(Franch.)TZ Hsu&RC is a variety of G.leucocarpa B1.,which belongs to the Gymnobotrys clade of the tribe Gaultherieae within Ericaceae.It is traditionally an ethnical medicinal plant in China,which is effective in clearing heat-toxicity,expelling wind,removing damness and promoting blood circulation.This variety is widely distributed in the south of the Yangtze River,which with the other two varieties as the same origin of medicine 'Tou Gu Xiang',i.e.G.leucocarpa var.crenulata(Kurz)TZ Hsu and G.leucocarpa var.psilocarpa(Copeland)RC Fang from Taiwan form the G.leucocarpa var.yunnanensis complex.Taxa in the G.leucocarpa var.yunnanensis complex are difficult to be distinguished from one another,especially by the morphological variation.Sometimes variation in the intraspecific level is greater than the interspecific.Previous investigation found that the effective compounds of the G.leucocarpa var.yunnanensis complex were significantly different regionally.Although this plant is widespread,its populations are not the dominant species locally.Moreover,most of the plants reproduce through asexuality by roots.Excavation in long term could lead to extinction of regional resources and subsequent decrease in genetic diversity.In order to understand the infra-specific relationships within this complex and provide accurate taxonomy for the downstream medical research,the G.leucocarpa var.yunnanensis complex was investigated with phylogenetic analysis at first.The phylogenetic inference at population sampling with three individuals for each was applied.The germplasm conservation can act as genetic theory and therefore be evaluated by intraspecific genetic structure and demographic history.An investigation on the correlation among the HPLC chemical composition pattern,geographical distribution,and genetic structure was performed by cluster and PCA analyses,for providing any insights on the quality control of the G.leucocarpa var.yunnanensis complex and germplasm selection for medical usage.Methods:We sampled 241 individuals in 81 populations of the G.leucocarpa var.yunnanensis complex(i.e.,G.leucocarpa var.crenulata,G.leucocarpa var.psilocarpa and G.leucocarpa var.yunnanensis)representing all three Chinese varieties and almost covering recorded distribution ranges in China(Mainland and Taiwan).One population of G.leucocarpa var.psilocarpa from the Philippines and one population of G leucocarpa var.leucocarpa from Malaysia were also included.Based on the data from two plastid DNA loci,i.e.,rpl33-psaJ and trnL-rpl32,we reconstructed phylogenetic trees using both Maximum Likelihood and Bayesian Inference methods,and phylogenetic network using Neighbor-Net method.Meanwhile,the G.leucocarpa var.yunnanensis complex with the south second-step montane areas of east China(as its core distribution range)and adjacent islands was studied.We then applied phylogenetic inference,population differentiation,demographic history and divergence dating based on the two chloroplast DNA loci above across 1060 individuals from 47 populations,representing a comprehensive range-wide sampling covered the core distribution range of this complex.In order to reveal the regional difference in chemical composition of G.leucocarpa var.yunnanensis in context of genetic structure,15 samples from various localities in seven provinces including Hubei,Hunan,Jiangxi,Fujian,Guangdong,and Guangxi were investigated.We performed the analyses of HPLC fingerprints and phylogenetic reconstruction with maximum likelihood,and then employed cluster analysis and PCA analysis to explore the pattern of chemical composition of G.leucocarpa var.yunnanensis at population level.Results:(1)Our results showed that there was a distinct genetic differentiation in the G.leucocarpa var.yunnanensis complex,and the genetic differentiations were more correlated with geographical distribution rather than morphology.This complex is mainly divided into three clades,one clade is composed of populations from Taiwan and the Philippines that are previously categorized as G.leucocarpa var.psilocarpa;one clade includes populations of the G.leucocarpa var.yunnanensis and G.leucocarpa var.crenulata with a distribution in the Hengduan Mountains region;and the remaining clade includes the populations mainly distributed in the southeastern range of China,which is composed of G.leucocarpa var.yunnanensis and G.leucocarpa var.crenulata as well.Molecular evidence therefore supports G.1.var.psilocarpa was treated as a variety but doesn't not support G.leucocarpa var.crenulata as a variety.(2)The G.leucocarpa var.yunnanensis complex forms a strong genetic differentiation in the Hengduan Mountains with other secondary stepped areas,two major lineages,i.e.,MT+islands lineage and PAN lineage were inferred.The former includes populations from southwestern of Sichuan,central of Yunnan and neighboring islands(Taiwan Province,Philippines and Malaysia),and the latter inhabits within the mostly rest areas of the south second-step terrain of China(incl.Yunnan,Guizhou and Guangxi Provinces).The overall genetic diversity is not high:Hd=0.378,and the genetic diversity of the MT+Islands lineage(Hd=0.553)is higher than the PAN lineage(Hd=0.103),which indicated that central Yunnan may be the center of species differentiation.The regional population history of the MT+Islands lineage has developed steadily,and the PAN lineage may have expanded its range during the Pleistocene(14.3kya to 116.3kya).The genetic break between MT lineage(composed of populations from southwestern of Sichuan and central of Yunnan)and PAN lineage could be explained by habitat geographical heterogeneity and historical river diversion with the uplift of the Yunnan Plateau.The island populations were found to be genetically closer to those from the Hengduan mountain in Yunnan,although they have longer geographical distance.This can provide evidence for a plant biogeographic pattern "disjunction between Taiwan and Southwest China" proposed previously.(3)Phylogenetic analysis on the 15 samples of G.leucocarpa var.yunnanensis from various localities in seven provinces indicated these samples clustered into two clades:one group including samples from Yadou Mountains in Guangdong,Longyan and Zhangzhou cities in Fujian,and another group including all the remaining samples.The similarity of HPLC fingerprint in various samples was great difference in general,with overall similarity ranging from 0.538 to 0.902.At provincial level,the similarity was relatively high,ranging from 0.843 to 0.978.Based on the paramters of common chromatographic peaks,the chemical composition in 15 samples can be generally divided into two groups with both the clustering analysis and PCA analysis.However,classification of the two groups is inconsistent with that of two clades based on phylogenetic analysis,indicating no correlation between chemical composition pattern and genetics.In the clustering and PCA analyses on chemical composition,samples from Lichuan in Hubei,Fengjie in Chongqing,Wugang in Chongqing,Jinggang mountain in Jiangxi,Zhangzhou in Fujian,Heyuan in Guangdong,Yadou mountain in Guangdong and Baise in Guangxi form a group,while samples from Nanling in Guangdong,Liannan in Guangdong,Longyan in Fujian,Jingfo mountain in Chongqing,Mangshan mountain in Hunan,Yangming mountain in Hunan,and Xiangxi in Hunan form another group.Conclusion:We first reveal the phylogenetic relationship within the G.leucocarpa var.yunnanensis complex with a sufficient sampling covering its distribution range in China.It was found that there are obvious genetic breaks within the species.Such genetic differentiation pattern may be caused by geographical isolation.This result lays a basic taxonomic framework for the evaluation and protection of germplasm resources of this ethnical medicinal plant.The genetic structure,population differentiation,demographic history of the G.leucocarpa var.yunnanensis complex from the south second-step montane areas of east China and adjacent islands were studied at population level.This result provides theoretical support for subsequent conservative study on its germplasm resources.It also provides the evidence for the geological history,river system changes,and species migration in the the south second-step terrain of China after the Himalayan uplift.No correlation between genetic structure and chemical composition of G.leucocarpa var.yunnanensis is detected.The chemical composition has a regional variation obviously,possibly due to adaptation to environment heterogeneity.In this study,there was no qualitative analysis for identifying the common peaks of chemical composition by HPLC fingerprint,and the pattern of content of economic compounds in various localities was still not clear.Therefore,it is necessary to further investigate the G.leucocarpa var.yunnanensis by increasing sampling both in taxa and methology. 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