Phylogenomic And Biogeographic Study Of An Anticancer Herbaceous Subfamily Podophylloideae

Eastern Asia(EA)and Eastern North America(ENA)are continental regions at low to mid-north-temperate latitudes,which have highly attracted the interests of researchers over the past decades.The contemporary floras of these two regions are considered as Tertiary relicts of a flora formerly more widespread at higher latitudes during warmer times during the Paleogene.According to the previous studies,there are about 65 genera with related species disjunct distributed between these two regions,including the subfamily Podophylloideae.Podophylloideae is the only one in the world containing podophyllotoxin,which is the precursor of the anti-cancer drug,etoposide(VP16).This subfamily comprises four genera(Podophyllum,Sinopodophyllum,Diphylleia,Dysosma),with an intercontinental disjunct distribution between eastern Asia(EA,10 spp.)and eastern North America(ENA,2 spp.).Podophyllum includes only one species,Podophyllum peltatum,endemic to northern America.Sinopodophyllum also comprises only one species,Sinopodophyllum hexandrum,which is restricted to the Himalaya regions.There are three species in Diphylleia,i.e.Diphylleia sinensis(China),Di.grayi(Japan)and Di.cymosa(ENA).Dysosma comprises of seven species,endemic to China.In this study,we employed plastid genome sequencing,transcriptome sequencing and RAD-seq method to elucidate the plastid genome evolution,phylogenetic relationship,speciation,population history and molecular evolution of key genes involved in podophyllotoxin pathway.The main results are as follows:(1)Phylogenetic analysis of Podophylloideae based on plastid genomesThis study is the first to report whole plastid genome sequence data for all 12 species of Podophylloideae(14 individuals)and a close relative,Achlys triphylla.These 15 plastid genomes proved highly similar in overall size(156,240 – 157,370 bp),structure,gene order and content,also when compared to other Ranunculales.Our phylogenomic analysis supported the monophyly of Podophylloideae and its two major genera(EA: Dysosma,EA–ENA: Diphylleia),with P.peltatum(ENA)being more closely related to Diphylleia than to the group's earliest diverging species,S.hexandrum(EA).Furthermore,mat K was identified as the fastest evolving gene,which proved to be under positive selection especially in more recently derived,lowerelevation lineages of Dysosma,possibly reflecting an adaptive response to novel environmental conditions.Finally,several highly variable noncoding regions were identified in the plastid genomes of Podophylloideae and Ranunculales.These highly variable loci should be the best choices for future phylogenetic,phylogeographic,and population-level genetic studies.(2)Phylogenetic analysis of Podophylloideae based on transcriptomes and molecular evolution of key genes involved in podophyllotoxin pathwayHere,transcriptomes of ten species of Podophylloideae and the related A.japonica were obtained.A total of 1,048 co-orthologs were identified from the eleven species.Phylogenetic analyses also strongly supported the monophyly of Podophylloideae,Diphylleia and Dysosma.Sinopodophyllum is the earliest branch of this subfamily,followed by Diphylleia as sister to Podophyllum and Dysosma.The results challenge the prevailing view that Podophyllum and Sinopodophyllum represent a disjunctively distributed sister group between EA and ENA.Besides,a whole genome duplication(WGD)event was detected in 33 transcriptomes of Ranunculales.This WGD event possibly occurred between 102.7 Ma and 142.6 Ma.Phylogenetic analysis of the key genes involved in Podophyllotoxin pathway indicated that CYP71BE54,CYP71CU1 and CYP82D61 duplicated twice in Ranunculales,with three copies in Podophylloideae.Meanwhile,three different gene duplication events were also detected in 2-ODD.For each of these four genes,one copy likely experienced strong positive selection in Podophylloideae.(3)Biogeographic history of PodophylloideaeOur analyses revealed an Oligocene origin for Podophylloideae in EA(based on transcriptomes: c.21.90 Ma;based on plastid genomes: c.19.75 Ma),then spread into Japan and ENA.Podophylloideae diversification was recovered as an early-burst pattern,with a speciation rate(? = 0.20 species/Ma)that was initially very high at 21-22 Ma and had declined ever since.The ancestral floral trait reconstructions suggested that the ancestor of Podophylloideae had upward(p = 1.00)oriented flowers(p = 0.96)with white petals(p = 0.87),which were likely more attractive to pollinators.The Bi SSE analyses showed that species with downward flowers tended to have speciation rates three times higher than those with upward-facing flowers(? = 0.19 species/Ma and 0.06 species/Ma,respectively),suggesting that the flower orientation is primary drivers of species diversification in Podophylloideae.The OMI analysis revealed significant differences of species' niche position between the EA and ENA regions,which was highly associated with temperature seasonality.Altitude and temperature seasonality made the largest contributions to the spatial niche separation among three geographical areas in EA.(4)Comparative population genetics of Dysosma and PodophyllumBased on RAD-seq method,we explored the population structure of the sister group Dysosma and Podophyllum.For the 49 Dysosma samples,a matrix containing 31,883 bp unlinked SNPs was successfully assembled.According to the result of ADMIXTURE analysis,Dysosma populations could be divided into 3 clusters: Western cluster(Dy.tsayuensis and Dy.aurantiocaulis),Central cluster(Dy.delavayi,Dy.difformis,Dy.majoensis,Dy.versipellis)and Eastern cluster(Dy.difformis,Dy.majoensis,Dy.versipellis,Dy.pleiantha).Likewise,phylogenetic analyses also recovered three clusters in Dysosma.Divergent time estimation dated the diversification of Dysosma clusters at c.15.06 Ma.Significant niche separation between these three clusters was highly associated with altitude and temperature seasonality.Within Podophyllum,a matrix containing 12,074 bp unlinked SNPs(46 samples)was successfully assembled.Phylogenetic analyses recovered three clades with high support.Divergent time estimation dated the diversification of Podophyllum clades at c.4.42 Ma.Significant niche separation between these three clades was highly associated with annual precipitation and precipitation seasonality.In summary,in this study we have reconstructed a reliable species tree of Podophylloideae based on genomic data.On this basis,the speciation progress and driving factors of EA–ENA disjunct distribution have been clarified from both species level(Podophylloideae)and population level(Dysosma and Podophyllum).Furthermore,our comparative analysis highlighted that the uplift of QTP increased environmental heterogeneity and landform complexity,which derived the allopatric speciation and breeding system transformation of Dysosma in EA.That could explain the species diversity disparity of Podophylloideae between EA and ENA.This study is meaningful for understanding the origin,evolution and mechanism of species diversity pattern between EA and ENA.