| The family of Juglandaceae belongs to Fagales and is an important economical tree species in the world,which has important material use,edible,medicinal and artistic values.High-throughput sequencing technology,bioinformatics,and evolutionary biology were used to study the Juglandaceae family.Firstly,we studied Juglans with phylogenetic genomics and population genomics,the results reveal the phylogenetic relationships,the speciation mechanism and the complex dynamic history of the Juglans species from the species level and population level.Secondly,this study uses chloroplast genome data to illuminate the phylogenetic relationship of Juglandaceae,reveal its evolutionary origin center and diversified history.Combined with fossil evidence,we further determined the evolution of Juglandaceae family on time scale.We analysed juglandaceae species differentiation time and climate dynamic history,the results show that they are corresponding to the history,respectively in the early Eocene and middle Miocene.The main results of this study are as follows:(1)A total of five Juglans species,which are including Juglans regia,J,sigillata,J.cathayensis,J.hopeiensis,and J.mandshurica are native to or naturalized in China.We sequenced the complete chloroplast genome of J.regia with the Illumina MiSeq sequencing platform.This is the first complete chloroplast sequence for the Juglandaceae,a family that includes numerous species of economical importance.We determined the complete chloroplast genome sequence of J.regia as 160367 bp in length.This chloroplast genome contained 137 functional genes,including 86 protein-coding genes,three pseudogenes(two ycf15 and one infA),40 tRNA genes and eight rRNA genes.A strongly supported phylogeny of these five species is not available due to lack of informative molecular markers.We compared complete chloroplast genomes and determined the phylogenetic relationships among the five Chinese Juglans using IIumina sequencing.A complete map of the variability across the genomes of the five Juglans species was produced,including single nucleotide variants(SNPs),insertions and deletions(Indels),and large structural variants,as well as differences in simple sequence repeats(SSR)and repeat sequences.Molecular phylogeny strongly supported division of the five walnut species into two previously recognized sections(Juglans/Dioscaryon and Cardiocaryon)with a 100%bootstrap(BS)value using the complete chloroplast genomes,protein coding sequences(CDS),and the introns and spacers(IGS)data.The availability of these genomes will provide genetic information for identifying species and hybrids,taxonomy,phylogeny,and evolution in Juglans,and also provide insight into utilization of Juglans.(2)Comparative transcriptomics and cross-species EST-SSRs molecular marker development in Juglans can provide effective genomic resources for subsequent studies on adaptive differentiation of this species.De novo assembly and characterization of bud,leaf and flowers transcriptome from five closely related Juglans species,Juglans regia,J.cathayensis,J.hopeiensis,and J.mandshurica,using short reads on the Illumina Hiseq 2000 platform.In the present study,16,811,432-49,929,297 clean reads were generated from five different species samples of Juglans by de novo assembly.A total of 9,216-9,3 89 core single copy orthologs were identified from 83,112-103,167 unigene sequences,which were obtained by de novo assembly.Moreover,96 primer pairs were successfully designed,and selected to assess polymorphisms and versatility in five Juglans species.In addition,the phylogenetic analysis of seven Juglans(J.regia,J.sigillata,J.cathayensis,J.hopeiensis,J.mandshurica,J.cinerea,and J.nigra)and three outgroups(Carya cathayensis,Castanea mollissima,and Quercus robur)showed that the gene tree is consistent with the species tree in Juglans.A phylogenetic tree was constructed from the chloroplast genome and 467 single copy orthologs of Juglans.The results showed that there were discrepancy in the systematic positions between J.cinerea and J.hopeiensis.The inconsistent phylogenetic relationships of nuclear DNA and chloroplasts DNA may be result from hybridization and chloroplast capture.(3)Juglans L.as one of the typical phylogeography disjunctions in the Northern Hemisphere,the biogeographic disjunction pattern between closely related plant in Eastern Asian and North American has been received considerable attention.However,because a limited number of DNA regions with insufficient phylogenetically informative sites were selected,multiple markers cannot clearly elucidate the phylogenetic relationships and phylogeographic patterns of Juglans,We resequenced 50 individuals from 10 species representing the entire Juglans genus to explore phylogenetic relationship and biogeographic pattern of Juglans.Our study has clarified previously unresolved phylogenetic relationships among species of Juglans and show a close relationship between J.cinerea and to sect.Rhysocaryon.and whole chloroplast genome data promise to generate adequate variation for phylogenetic reconstruction among species.The phylogenetic analysis showed that J.hopeiensis chloroplasts are most closely related to those of the sect.Cardiocaryon.Juglans were estimated to have originated during the Eocene(43.8 Mya)based on fossils and complete chloroplast genome.According to the ADMIXTURE estimate the genetic structure based on nuclear SNPs,J.hopeiensis is potential hybrid,is made up of 50.0%sect.Cardiocaryon and 50.0%sect.Dioscaryon(J.hopeiensis= Cardiocaryon?× Dioscryon?).However,introgression is not detected in the J.cinerea.According to the phylogenetic tree constructed by Juglans nuclear SNPs,the J.cinerea and sect.Cardiocaryon.were grouped into one clade and were the first divergented from sect.Cardiocaryon.However,the phylogenetic tree also showed that J.hopeiensis formed a mixed clade of sect.Cardiocaryon or sect.Dioscaryon probably hybrid.The results of the combination of the paternal plastid inheritance and the biparental nuclear inheritance showed that the incongruence of the phylogenetic relationships of the J.cinerea was due to the existence of the chloroplast gene capture event in the sect.Rhysocaryon.However,the incongruence of the phylogenetic relationships of J.hopeiensis was due to the hybridization between the closely related species.(4)Phylogenetic relationships in the Juglandaceae are problematic and their historical diversification has not been clarified,because of complex evolutionary history.Based on analysis of seven of the eight genera representing all clades of Juglandoideae and fifteen members of five families as outgroups,we analyzed 26 newly sequenced plastomes together with 16 from GenBank in an attempt to reconstruct deep relationships and reveal temporal diversification of this family.Our results identified and characterized the genetic variation in plastomes of the Juglandoidea,including pseudogenes and divergence hotspots.Six clades representing genera were strongly supported as monophyletic,and their deep relationships were fully resolved at all nodes.Juglandaceae were estimated to have originated during the late Cretaceous(78.58 Mya,95%HPD:76.58-80.50 Mya),while Juglandoideae were estimated to have originated during the Paleocene(61.98 Mya,95%HPD:59.98-63.98 Mya),with evidence for rapid diversification events during several glacial and geological periods.The major lineages rapidly diversified in warm and dry habitats during the middle Paleocene and early Eocene.Rapid species diversification within Juglans occurred in warm and dry environments during the Miocene and later.The robust phylogenetic backbone and time estimates we provide establish a framework for future comparative studies of evolution in Juglandaceae and Fagales. |
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