Diversity,Conservation And Utilization In Genetic Resources Of Coptis Chinensis And Its Closely Related Species

Huanglian,a Chinese herb,is widely known for its antibacterial and antiinflammatory properties,and has been used in traditional Chinese medicine for centuries.C.chinensis‘Weilian’,C.deltoidea‘Yalian’,and C.teeta‘Yunlian’,are classified as official Huanglians in the Chinese Pharmacopeia,and C.chinensis has the largest cultivation area.However,due to founder effects and disorderly seed exchange,cultivated Huanglian has suffered from a serious genetic bottleneck resulting in degraded germplasm and reduced resistance to diseases.It has had a devastating impact on the industry,making the improvement of germplasm an urgent priority.To achieve this goal,the utilization of wild resources is crucial.However,previous studies have shown that the relationship among various species in the wild resources of Coptis,the genus to which Huanglian belongs,is unclear,and the genetic diversity pattern is poorly understood.This has constrained subsequent conservation and utilization efforts.Therefore,there is a pressing need for further research on the genetic diversity and relationship of wild Coptis populations.Additionally,some wild populations exhibit unusual phenotypic characteristics,which deviate from the Flora of China,suggesting that there may be new species yet to be discovered.Clarifying the diversity and differentiation of genetic resources at the species or population level to elucidate the composition,relationship,and evolutionary patterns have been essential prerequisites for the research on the selection,breeding and conservation of Chinese herbal medicine species.Therefore,in this study,we aimed to investigate at three levels,including intra-genus,closely related species,and intraspecies:1.Evolutionary history of genus Coptis and suitable area dynamic changesThe genus Coptis belongs to the Ranunculaceae family,containing 15 recognized species highly diverse in morphology.It is a conspicuous taxon with special evolutionary position,distribution pattern and medicinal value,which makes it to be of great research and conservation significance.In order to better understand the evolutionary dynamics of Coptis and promote more practical conservation measures,we performed plastome sequencing and used the sequencing data in combination with worldwide occurrence data of Coptis to estimate genetic diversity and divergence times,rebuild biogeographic history and predict its potential suitable distribution area.The average nucleotide diversity of Coptis was 0.0067 and the hotspot regions with the highest hypermutation levels were located in the ycf1 gene.Coptis is most likely to have originated in North America and Japanese archipelago and has a typical Eastern Asian and North American disjunct distribution pattern,while the species diversity center is located in Mid-West China and Japan.The crown age of the genus is estimated at around 8.49 Mya.The most suitable climatic conditions for Coptis were as follows:precipitation of driest quarter>25.5 mm,annual precipitation>844.9 mm and annual mean temperature-3.1 to 19℃.The global and China suitable area shows an upward trend in the future when emission of greenhouse gases is well controlled,but the area,especially in China,decreases significantly without greenhouse gas policy interventions.The results of this study provide a comprehensive insight into the Coptis evolutionary dynamics and will facilitate future conservation efforts.2.Evolutionary dynamics of C.chinensis and its closely related speciesAccording to the Flora of China,there are six species and one variety of the genus Coptis that are distributed in China.These taxa have long been utilized in traditional medicine for their heat-clearing and detoxifying properties.Unfortunately,due to human activities and the biological characteristics of this genus,most domestic species of Coptis are now classified as rare and endangered.In light of the increasing focus on plant resource conservation,there is a critical need for the scientific management and sustainable development of domestic Coptis species.To address this challenge,this study employed a population genomics approach to collect 239 samples from all distributions of Coptis taxa and obtain 1,385,394 population SNP loci.The objective of this research was to clarify the phylogeny and genetic differences among species/populations,as well as to reconstruct the evolutionary dynamics of C.chinensis and closely related taxa.The phylogenetic results showed that,two closely related taxa of C.chinensis,namely C.huanjiangensis and C.linearisepala,were identified as new species,with the latter being rediscovered after being absent for more than thirty years.A clear relationship was established between C.omeiensis and C.deltoidea,which had been inseparable in previous studies,and significant population divergence was found within C.deltoidea.The genetic diversity among the closely related species was assessed by Pi,revealing that C.huanjiangensis(Pi=0.155)had the highest genetic diversity,while C.linearisepala(Pi=0.028)had the lowest.Within the C.chinensis species,C.chinensis var.brevisepala(Pi=0.074)exhibited higher genetic diversity than C.chinensis var.chinensis(Pi=0.069).The pattern of genetic differentiation and genetic distance among species was generally consistent,except for Fst between C.chinensis var.brevisepala and C.huanjiangensis(Fst=0).The results of the ADMIXTURE analysis showed that C.deltoidea,and C.chinensis var.brevisepala had degrees of ancient hybridization,while the genetic background of other species was pure.Interestingly,few gene exchanges occurred between the two closely related variants of C.chinensis.Furthermore,the gene flow results of Treemix demonstrated that three ancient hybridization events had occurred during the evolutionary history of Coptis,with C.huanjiangensis playing a significant role in gene exchange which indicated that it acted as a bridge for gene exchange between evolutionary branches of the genus Coptis and further explained why the genetic differentiation between C.chinensis var.brevisepala and C.huanjiangensis was so low.The effective population size(Ne)of Coptis showed a pattern of expansion followed by contraction over the past million years,with the contraction occurring during the Last glacial period.This study has provided a clear understanding of the evolutionary relationships and population dynamics among the closely related species of Coptis,which can be used to guide their conservation and utilization in China.3.Genetic diversity and population structure within C.chinensisRoot rot has been a major problem for cultivated populations of Coptis chinensis var.chinensis in recent years.C.chinensis var.brevisepala,the closest wild relative of C.chinensis var.chinensis,has a scattered distribution across southwestern China and is an important wild resource.Genetic diversity is associated with greater evolutionary potential and resilience of species or populations and is important for the breeding and conservation of species.Here,we conducted multiplexed massively parallel sequencing of the plastomes of 227 accessions of wild and cultivated C.chinensis using 111 marker pairs to study patterns of genetic diversity,population structure,and phylogeography among wild and cultivated C.chinensis populations.The cultivated resources experienced a severe genetic bottleneck and possess highly mixed germplasm.However,high genetic diversity has been retained in the wild resources,and subpopulations in different locations differed in genotype composition.The significant divergence in the genetic diversity of wild and cultivated resources indicates that they require different conservation strategies.Wild resources require in situ conservation strategies aiming to expand population sizes while maintaining levels of genetic diversity;by contrast,germplasm resource nurseries with genotypes of cultivated resources and planned distribution measures are needed for the conservation of cultivated resources to prevent cultivated populations from undergoing severe genetic bottlenecks.The results of this study provide comprehensive insights into the genetic diversity,population structure,and phylogeography of C.chinensis and will facilitate future breeding and conservation efforts.4.Discovery of C.huanjiangensis and rediscovery of C.linearisepalaThrough comprehensive sampling of populations,morphological and molecular data analysis,this study has identified a new species,Coptis huanjiangensis L.Q.Huang,Q.J.Yuan&Y.H.Wang,sp.nov.,which represents a novel addition to the known species of the genus Coptis.In addition,we have successfully rediscovered a species that was previously lost for thirty years,Coptis linearisepala T.Z.Wang et C.K,Hsieh,sp.nov.Compared to closely related species,C.huanjiangensis have notably longer petiole,scape,bigger leaf blade with lobes obviously remote and robust rhizomes without stolons;C.linearisepala have notably longer linear sepals and linear petals.This discovery further enriches our knowledge of the diversity of the genus Coptis,greatly promoted the taxonomic and evolutionary studies of this genus and highlights the importance of comprehensive sampling and analysis in understanding and conserving biodiversity.Simultaneously,it is noteworthy that these two species constitute the most closely related wild taxa of C.chinensis,and as such,will serve as pivotal genetic resources in future breeding and cultivation.In this study,we developed genome-wide SNPs and chloroplast genome sequences as two types of markers to reflect biparental and maternal genetic characteristics,respectively,based on population genomics through full coverage population sampling of Coptis and closely related species.The two markers were combined with global distribution data and massively parallel amplicon sequencing technology to reconstruct new phylogenetic relationships,clarify intraspecific genetic diversity and population structure,and propose conservation and utilization measures for Coptis species.This study provides a foundation for the sustainable development of Coptis by fully understanding its genetic resource diversity and proposing subsequent conservation and genetic improvement measures.