Speciation And Phylogeogrpahy Of Cardicrinum,a Genus Widely Distributed In The Sino-Japanese Floristic Region(SJFR)

The Sino-Japanese Floristic Region(SJFR)harbors the most diverse temperate flora of the world.Major biogeographic events across the SJFR,including the uplifting of the Qinghai-Tibet Plateau,the formation and development of the monsoon since the Late Miocene and Quaternary climatic fluctuations,etc.,are hypothesized to be the main driving forces for generating and maintaining the high species diversity in the SJFR.Revealing phylogeographic structure,speciation and demographic history of SJFR's widely-distributed temperate plant taxa is essential to understand the origin and maintenance of rich species diversity in the SJFR.Cardiocrinum(Endl.)Lindl.,which belongs to the family Liliaceae,comprises three long-lived perennial herbaceous species,C.giganteum(Wall.),C.cathayanum(E.H.Wilson)Steam,and C.cordatum(Thunb.)Makino.All three species are the constituents of montane TDF in the SJFR.The distribution of C.giganteum extends from the Himalaya-Hengduan Mountains to warm lowland in central/eastern China,whereas C.cathayanum is endemic to central and eastern China.Hence,these two species are parapatrically distributed.By contrast,C.cordatum is restricted to the Japanese Islands and adjacent Far East regions Comprising three closely-related species with relatively continuous and widespread geographical distribution,Cardiocrinum thus provides an ideal model system to study on speciation and demography history of temperate plant species in SJFR.In this study,based on an extensive sampling,we employed a multidisciplinary approach including phylogenomics,population genetics and phylogeograpy to investigate the phylogenetic relationships,speciation,population diversity and structure,and demography history of Cardiocrinum species.The major results are listed as follows1)Comparative chloroplast genomic and phylogenetic analyses:The chloroplast genomes of C.giganteum,C.cathayanum,and C.cordatum were found to be 152,653,152,415,and 152,410 bp in length,respectively.All these chloroplast genomes show the typical quadripartite structure and contain 132 genes,with high similarity in gene order,GC content and etc.By comparing complete chloroplast genomes of these three Cardiocrinum species,six rapidly evolving regions(rps19,rpoC2-rpoC1,trnS-psbZ,trnM-atpE,psaC-ndhE,ycf15-ycfl)were identified,which may serve as molecular markers for future studies.Phylogenomic analyses based on the complete chloroplast genomes and 74 protein-coding genes strongly supported the monophyly of Cardiocrinum and a sister relationship between C.cathayanum and C.cordatum2)Speciation and divergence of Cardiocrinum:A total of 45 individuals representing 45 populations were randomly selected for RAD sequencing.Based on 2,852 RAD-SNPs,our phylogenomic analysis strongly supported the monophyly of each Cardiocrinum species,and further identified C.giganteum as sister of C.cathayanum and C.cordatum.Both species tree analyses in SNAPP and f3 estimates indicated no admixture signal among the three Cardiocrinum species.Our DIY-ABC analysis provided strong support for an early diversification(c.9.08 Ma)of C.giganteum from the most recent common ancestor(MRCA)of C.cathayanum and C.cordatum,followed by a more recent divergence between these latter two species(c 5.09 Ma).These time windows coincide with the time periods of abrupt uplift of the eastern part of QTP and adjacent Southwest China(c.9-7 Ma)and increasing aridification since the Late Miocene,and that Japanese islands,for the first time had completely separated from Eurasia?5 Ma.In brief,these results suggested that pre-Quaternary(ancient)climatic changes and tectonic activities played an important role in triggering the speciation of Cardiocrinum.3)Genetic diversity and population structure:19 EST-SSR markers were developed from transcriptome data of C.cathayanum and used for population genetic studies of Cardiocrinum.Our EST-SSR survey revealed that C.giganteum possessed the highest genetic diversity(NA=127,HO?0.589,Rs=6.657)among the three species,while the lower values of C.cathayanum(NA=71,HO=0.367,RS=3.737)and C.cordatum(NA=77,Ho-0.236,Rs=3.956)were of similar magnitude.Our STRUCTURE and DAPC analyses divided all 51 Cardiocrinum populations into three distinct clusters,corresponding to each species4)Population demographic histories:Both our ABC analyses of population size change and paleodistribution prediction of ecological niche models indicated that all three Cardiocrinum species experienced range expansions during the transition from the LIG to the LGM,and contractions since the LGM.Glacial expansions with different directions have a significant role in shaping the current distributions in Cardiocrinum.In conclusion,our phylogenomic and population genetic analyses strongly supported the monophyly of each species,with C.giganteum being identified as sister to a highly supported clade comprised of C.cathayanum and C.cordatum.Coalescent-based analyses demonstrated that Late Neogene tectonic/climatic events triggered speciation of Cardiocrinum,and rapid climate change during the last glacial-interglacial transition had great influence on its current distribution.These results will improve our understanding of the origin,dispersal and evolution of temperate plants in the SJFR,as well as the mechanism of the formation and maintenance of the high species diversity in this area,and also lay the groundwork for the conservation of temperate plant species in the SJFR under ongoing environmental change.In addition,our study also provided abundant genetic resources including plastid genomes,transcriptome sequences,EST-SSR and SNPs for the conservation and utilization of Cardiocrinum.