| Diabelia(Landrein)belongs to the Linnaeoideae of Caprifoliaceae family with a typical discontinuous distribution in China,Japan and Korea.Because of interspecific hybridization,recent interspecific differentiation and convergent evolution,only few samples and molecular fragments have been used in the previous studies.Moreover,the phylogenetic relationship and biogeographical distribution of Diabelia are still unclear,and even the definition of species within the genus is still controversial.Based on 549 individuals from 66 populations of Diabelia,the phylogenetic and biogeographic studies were carried out by using morphological,and statistical methods such as ANOVA and linear regression,chloroplast fragment sequencing,microsatellite site amplification of nuclear genes and shallow whole genome sequencing.Based on the morphological and molecular data,the phylogenetic relationship,biogeographic origin,divergence time and taxonomic revision of Diabelia were discussed.The following main conclusions are drawn from the present study:1.Based on the genetic diversity and pedigree geographic structure of chloroplast fragments(rpl32-trnL,trnH-psbA),37 haplotypes were obtained from the analysis of cpDNA of Diabelia,and haplotype network graph showed typical multi-origin distribution.The phylogenetic tree constructed by the maximum parsimony showed that,except Diabelia serrata,the other species did not form a monophyletic structure,and the phylogeneticposition was very complex.Among the phylogenetic branches of the monophyletic group D.serrata,the D.serrata populations of China was found to be at the base of the phylogenetic branches.In terms of genetic diversity,the higher haplotype diversity and nucleotide diversity(Ht=0.926;? t=0.0072)indicated the long evolutionary history of Diabelia.2.A total of 11 pairs of polymorphic nSSR primers were developed based on transcriptome sequencing data of Diabelia.Population genetic diversity analysis showed that 13%of microsatellite loci possess significant linkage disequilibrium.Because of some invalid alleles,some populations deviated from the Hardy-Weinberg equilibrium.Among the genetic diversity and differentiation,the Diabelia showed higher inter-population genetic differentiation and total genetic diversity as well as lower average population genetic diversity(Het=0.736;mean He=0.385;Fst=0.41 9),and there was a significant correlation between geographical distance and genetic distance(p-value=0.010).In the STRUCTURE analysis of nSSR,K=5 showed the best grouping,the percentages of'molecular variance between gene pools was 22.7%,which is greater than 8.9%of the total molecular variance among four species.The grouping of K=5 also supported the classification system of four species of Diabelia.Due to the limited number of samples,the classification system of K=5 is still not perfect.In addition,the bottleneck effect analysis found that the Japanese population was not affected by severe genetic drift(only 5 of 36 populations displayed bottleneck effect),and has remained relatively stable in recent years.3,Based on the analysis of 28 individual chloroplast genomes,the genome length was found in the range of 156,243 to 157,578 bp,including 89,539-90,801bp in largesingle copy region(LSC),18,818-19,072bp in small single copy region(SSC),23,726-24,034bp in inverted repeat region(IR).There are 81 coding gene regions,130 non-coding gene regions,8 highly variable gene spacers and ycfl,ycf2,ndhB,petG,accD,clpP were pseudogenes.The genomic structures among 28 chloroplast genomes were highly similar.The phylogenetic results of chloroplast whole genome showed that The D.ionostachya var.wenzhouensis populations of China,D.spathulata var.spathulata populations of Korea and D.ionostachya var.ionostachya populations of Yamagata County(Japan)formed an evolutionary branch,at the base of phylogenetic tree(BI/ML/MP:1/80/-);D.spathulata is not a monophyletic tree and is divided into three branches:(1)D.spathulata populations in Korea;(2)D.spathulata populations in Toyama Prefecture(Japan);(3)D.spathulata populations in Japan except Toyama Prefecture.D.serrata formed better monophyletic groups.The species of D.ionostachya var tetrasepala are in the monophyletic group of D.serrata during phylogeny.D.sanguinea and D.spathulata populations in Toyama Prefecture of Japan formed sister groups with high support rate(BI/ML/MP:1/100/100).4,Bayesian divergence time analysis based on chloroplast genome showed that the crown population time point was 28.31 Ma[95%HPD:22.73-32.95 Ma].All four species had completed species differentiation in Tertiary.The early differentiated population in China(20.85 Ma[95%HPD:13.42-27.77 Ma])was a primitive residual population,but the role of the East China Sea in species differentiation was still unknown.At the same time,phylogenetic analysis showed that a large number of lineage differentiation occurred in the population of Diabelia in the middle and late Tertiary,including those of Korea.The analysis of the ancestral state reconstruction showed that there are many important time points in the eastern part of China,Japan and South Korea.It was estimated that the node age of the crown group was 28 Ma in the Middle Oligocene.The northeastern part of Japan is the most likely area of the original ancestral distribution of the Diabelia populations in Japan,and at the same time,it also showed a typical multi-origin distribution characteristic.In conclusion,Diabelia began to differentiate during the Oligocene of the Tertiary.The D.ionostachya populations in China is a primitive inherited population.In the middle and late Tertiary period,due to the influence of climate,Diabelia produced a lot of lineage differentiation,including the differentiation of D.spathulata population in Korea.During the Last Glacial Maximum period,Diabelia was widely distributed in East Asia,including the continental shelf of the East China Sea.In the modern distribution area,only the Zhejiang of China has the distribution of Diabelia,while in Japan,the populations are still widely distributed except Hokkaido.The population in Japan has not experienced a wide range of bottlenecks in recent years.The relatively stable population shows the characteristics of low genetic diversity and high genetic differentiation among populations.Interruption of gene flow and genetic drift are the main factors affecting the current population structure in Japan.And our molecular data supported the taxonomy of four Diabelia species(i.e.D.serrata,D.spathulata,D.sanguinea and D.ionostachya[Landrein]). |
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