Taxonomy And Conservation Of Fritillaria In Tibetan Plateau And Its Adjacent Regions

" Beimu ",a kind of traditional Chinese medicine with a long history,was first recorded in Shennong’s Herbal in which it is listed as a medium-grade medicine.With the continuous development of traditional Chinese medicine,Beimu has gradually been indicated to Fritillaria species(Liliaceae),and the source of the medical material has gradually expanded from the middle and lower reaches of the Yangtze River to the Qinghai-Tibet Plateau,Xinjiang Province and Northeast of China after Ming Dynasty(Fourteenth-Seventeenth Century),and the bulbs of Fritillaria from Qinghai-Tibet Plateau was especially called "Chuan Beimu".The Pharmacopoeia of the People’s Republic of China(Volume I,2020 Edition,as CP below)contains five kinds of Beimu,involving 11 species(or variants)of Fritillaria.Due to the high price of Beimu,in market circulation more Fritillaria species were mixed with those authentic species recorded in CP.Unfortunately,there are more taxonomic problems in Fritillaria of China,such as species delimitation and confusing phylogenetic relationship.There are 20 species and 2 varieties in Flora Reipublicae Popularis Sinicae(Vol.14),but more than 100 names of species and varieties have been published during twenty years since 1980 s.Therefore,the authors of Flora of China(Vol.24)reduced some names into previously published species,and finally recorded 24 species and 2 varieties in China.This paper attempts to study those species of Fritillaria distributed in the Qinghai-Tibet Plateau and its adjacent areas,i.e.socalled Chuan Beimu,through four aspects: plant field population investigation,plant macromorphology,micromorphology and molecular systematics,so as to clarify the taxonomic status and phylogenetic relationship of those species and to provide basic data for the accurate identification of Chuan Beimu.At the same time,considering that the Chuan Beimu mainly sources from the wild populations,we also evaluated the threatened status of 6 species of Fritillaria distributed in the Qinghai-Tibet Plateau according to the IUCN standard,which will provide effective data to support the protection of wild resources of Fritillaria in this area.Through literature and herbarium data along with field survey and interview,this paper conducted a comprehensive studies on Chuan Beimu resource status,involving 9 species(or variants),which include Fritillaria cirrhosa D.Don,F.sichuanica S.C.Chen,F.taipaiensis P.Y.Li,F.przewalskii Maxim.ex Batalin.,F.delavayi Franch.,F.unibracteata Hsiao et K.C.Hsia,F.unibracteata var.longinectarea S.Y.Tang et C.H.Yueh,F.unibracteata var.maculata S.Y.Tang & S.C.Yueh,F.cirrhosa var.ecirrhosa Franch.By consulting the original literature,flora,local county chronicles,local herbarium,digital herbarium and other relevant literature and professional databases,this paper sorted out the medicinal properties,microscopic identification,physical and chemical identification,molecular identification and resource distribution of Chuan Beimu.On this basis,the sample plot and survey route are prepared for further field survey.Through the in-depth investigation of 9 species and 2 varieties,F.delavayi,F.fusca Turrill,F.unibracteata,F.unibracteata var.longinectarea,F.unibracteata var.maculata,F.przewalskii,F.sichuanica,F.cirrhosa var.ecirrhosa Franch.,F.cirrhosa,F.taipaiensis,F.wabuensis S.Y.Tang et S.C.Yueh,of Fritillaria ditributed in Qinghai-Tibet Plateau and its adjacent areas,this paper summarizes their habitat and resource distribution.Wild Fritillaria resources in the Qinghai Tibet Plateau and its adjacent areas is suffering from severe overexploitation because of huge market demand.Although the planting scale of cultivated Fritillaria rapidly developed in past twenty years,the sharp reduction status of wild resources of Chuan Beimu still hasn’t changed due to huge drug market demand and habitat damage.Through the investigation in this area,this study provides a scientific reference for sustainable utilization and development of Fritillaria resources.In the systematic study of Fritillaria species in the Qinghai Tibet Plateau and its adjacent areas,the results of light microscope experiment show that the leaf epidermis of F.delavayi is the most special,and the morphology is very different from that of other Fritillaria species distributed in this area.Besides F.delavayi,the anticlinal walls of leaf epidermis of all of Fritillaria species distributed in this areas is mostly straight or slightly curved,which is different from wavy shape of leaf epidermis of Fritillaria species in the middle and lower reaches of the Yangtze River.The results of structure of pollen grain by Scan electronic microscope investigation showed that there were great differences in grid size between wild Fritillaria species and cultivated Fritillaria species,but no obvious different characters supporting interspecific classification were found.Phylogenetic analysis showed that Fritillaria mellea,F.cirrhosa var.ecirrhosa.,F.wabuensis are clustered into independent branches,which support the taxonomic treatment of these three species as independent species.Wild F.wabuensis S.Y.Tang et S.C.Yueh and cultivated F.wabuensis S.Y.Tang et S.C.Yueh are clustered into two branches in the phylogenetic tree,which proves that there may be gene flow between species under cultivation conditions.Through phylogenetic analysis and morphological comparison,it is confirmed that the Fritillaria species distributed near Zheduo mountain in Sichuan Province is F.cirrhosa var.ecirrhosa Franch.,not Fritillaria mellea.A new species(unpublished)distributed in Seda and its surrounding counties was found by population investigation,herbaria specimens observation and phylogenetic tree construction.Compared with the similar species of F.cirrhosa distributed in Shannan area of Tibet Autonomous Region,this species has great differences in morphology,geographic distribution and other Fritillaria species near the distribution area,and becomes a separate branch in the phylogenetic tree,which needs further research.By the specimens identifying carefully of Fritillaria species in the online herbaria,the coordinate points of the specimens were obtained through the retrospective landmark processing of the specimens,along with those coordinate points got from field survey,the threatened status of F.delavayi,F.unibracteata var.unibracteata,F.unibracteata var.maculata,F.unibracteata var.longinectarea,F.cirrhosa,F.przewalskii,F.sichuanica,F.taipaiensis were evaluated according to IUCN standards.The results show that the above species are under threat.Due to the problem of species classification,the threatened level of F.cirrhosa should be upgraded to Vulnerable(VU),F.unibracteata var.maculata and F.przewalskii should be upgraded to Endangered(EN)and F.sichuanica should be upgraded to Critically Endangered(CR)which was assessed in Evaluating the Threat Status of Higher Plants in China.