Studies On The Evolutionary History Of The Widespread Aquatic Plant Species Hippuris Vulgaris

Aquatic plants are an important ecological group in the Arctic flora and play a vital role in maintaining biodiversity,purifying water and maintaining the stability of aquatic ecosystems.As a widespread aquatic plant in the North Temperate Zone,Hippuris vulgaris is an important primary producer of aquatic ecosystems in the Arctic and temperate alpine regions.In this study,we used a variety of molecular markers to investigate the origin and dispersal routes of H.vulgaris,compare the chloroplast genomic characteristics of different genetic lineages,and infer the phylogenetic relationships and divergence times between genetic lineages.The main results are as follows:1.Three genetic lineages of H.vulgaris and H.montana were sequenced using next-generation sequencing technology,and their chloroplast genomes were assembled and analyzed using bioinformatics methods.Their chloroplast genomes were found to be highly conserved with identical number and order of arrangement of genes in each genome.They had similar genome sizes,GC content,contraction and expansion trends of reverse repeat regions,types and numbers of repetitive sequences,and codon usage preferences.Sequence variation analysis showed that variation existed mainly between H.montana and H.vulgaris,while within the three lineages of H.vulgaris,there was a large difference between lineage B and lineages A and C.Nucleotide polymorphism analysis identified 19 spacer regions with Pi values greater than 0.02,which can be used as potential chloroplast DNA molecular markers for subsequent studies of the genus Hippuris.2.Transcriptome of the lineage B of H.vulgaris was sequenced,and 90,291 genes and 158,239 transcripts were obtained after de novo assembly.Through GO functional enrichment,all transcripts of H.vulgaris were annotated into 177 functional gene regions in three categories: molecular functions,cell components,and biological processes,with the largest number of transcripts involved in biological processes,mainly in various metabolic processes.The result can provide a reference for the subsequent exploration of important expression genes in various life processes of H.vulgaris and functional analysis.In addition,3746 single-copy nuclear genes were screened based on the transcriptome of H.vulgaris,which can provide potential molecular markers for genetic and evolutionary studies of Hippuris.3.The molecular phylogeography of H.vulgaris was conducted based on samples collected from 200 sites in Eurasia and North America using chloroplast DNA fragments and nuclear microsatellites.Three genetic lineages A,B,and C with different distribution ranges were identified,corresponding to Northern Eurasia,Southern Eurasia,and North America.Analysis using Approximate Bayesian Computation-Random Forest approach suggested that H.vulgaris originated in China,followed by dispersal into Russia plus Northeast China,then successively westwards into Europe and North America,and finally into the Russian Far East from both North America and Russia plus Northeast China.In addition,no private haplotypes or independent genetic clusters were evident in H.lanceolata or H.tetraphylla,suggesting that these two species should be considered conspecific ecotypes of H.vulgaris.4.Based on the datasets of chloroplast genomes,chloroplast protein-coding sequences and single-copy nuclear genes,the phylogenetic relationships among species and intra-species lineages in Hippuris were explored using maximum likelihood and Bayesian methods.Different datasets and methods obtained the same results: the genus Hippuris was divided into two branches,one for the H.montana and the other for the three lineages of H.vulgaris,where lineage A and lineage C clustered into a subbranch before clustering with lineage B.The support rate for each branch was 100%/1.0.The divergence time between species and intra-species lineages in Hippuris was estimated by using the divergence time of outgroups as the calibration point.The results showed that H.vulgaris and H.montana diverged at8.83 Ma in the Late Miocene,the three lineages of H.vulgaris began to diverge at2.81 Ma in the Late Pliocene,and the more closely related lineages A and C did not begin to diverge until 1.38 Ma in the Pleistocene.In summary,this paper comprehensively reveals the evolutionary history of the widespread aquatic plant species H.vulgaris from the aspects of chloroplast genome characteristics,phylogeographic structure,origin and dispersal routes,phylogenetic relationship and divergence time,providing the first case study for Arctic-alpine aquatic plants,and the results will deepen our understanding of the Arctic-alpine flora.