Phylogenomics And Divergence Time Estimation Of Cannabaceae

Cannabaceae belongs to Rosales of nitrogen-fixing clade,which contains ca.10 genera and 182 species.This family shows a cosmopolitan disjunct distribution in tropical,subtropical and temperate regions of both the New World and the Old World.Many species in Cannabaceae have important economic and ecological values.Previous phylogenetic studies have progressed in resolving some intergeneric relationships of this family except for the phylogenetic relationships between Gironniera Gaud.and Lozanella Greenm.mainly based on morphological traits,chloroplast and ribosomal DNA fragments.However,many deep internodes within Cannabaceae remained unresolved due to limited taxon sampling or fewer available markers.Therefore,a stable and reliable phylogenetic framework of Cannabaceae is still lacking.In this study,we explored with an expanded taxon sampling and performed phylogenomic analyses utilizing 90 targeted single or low-copy orthologous nuclear genes,as well as a number of non-targeted regions(e.g.chloroplast and ribosome DNA regions)from Hyb-seq integrated with published chloroplast and ribosome DNA regions from GenBank and literature for the first time.In addition,we inferred patterns of captured nuclear gene-tree concordance and conflict with species tree(ASTRAL).Meanwhile,we estimated the divergence time of Cannabaceae with tree PL based on ML tree inferred by concatenated captured nuclear genes and 5 fossil records.The main results of this study are as follows:1.Phylogenetic relationships in CannabaceaeThe phylogenetic analysis results of the three types of molecular data(captured nuclear genes,chloroplast and ribosome DNA regions)used were inconsistent.The phylogenetic relationships highly supported in this study include: Cannabaceae and all genera except Trema Lour.and Celtis L.are monophyletic.Aphananthe Planch.was resolved as the first diverged genus,which is sister to all other genera in Cannabaceae.Gironniera,Lozanella,Celtis II were successively diverged.However,the consistency and conflict analysis between the captured nuclear gene-tree indicated that there were obvious gene conflicts in the phylogenetic position of Lozanella and the Celtis II clade.For other intergeneric relationships,Cannabis L.is sister to Humulus L.,Pteroceltis Maxim.and Chaetachme Planch.are sisters,and Parasponia Miq.is nested within Trema.Phylogenetic analysis results of captured nuclear genes highly supported the(Cannabis,Humulus)clade and the(Chaetachme,Pteroceltis)clade form a sister relationship(abbreviated as the CHCP clade),and then the CHCP clade is sister to the Trema(Parasponia)clade with a low support,but it showed obvious gene conflicts and differred from the result of chloroplast phylogenetic analyses. Finally,the Celtis I clade is sister to a clade formed by the CHCP clade and the Trema(Parasponia)clade.2.Revision of the generic classification in CannabaceaeOur phylogenetic analysis of three types of molecular data was conducted on the basis of an expanded sampling,and all results supported the Trema(Parasponia)was monophyletic,and suggested that Parasponia should be merged into Trema.In addition,our results found that Celtis was not monophyletic,we divide it into the previously defined Celtis I clade,which contains tropical evergreen and temperate deciduous Celtis,and the newly defined Celtis II clade,which currently contains only the African species Celtis gomphophylla Baker and the South American species Celtis schippii Standl.Phylogenetic analysis result of captured nuclear genes and chloroplast data supported the division of the Celtis II clade after Aphananthe,Gironniera and Lozanella,and sisterized with the other genera.In morphology,Celtis II species have the following obvious differences compared with Celtis I:(1)C.gomphophylla flowers are usually unisexual,dioecy,pistil style 2,stigma 2,linear without division,and the fruit has persistent calyx.(2)The ripe fruit of C.schippii is green and has persistent calyx,membranous and black endocarp,and the embryonic morphology of C.schippii significantly differed from species within the Celtis I clade.In our research,we recommends that the Celtis II clade should be restored to the genus Sparrea Hunz.& Dottori.3.Origin of Cannabaceae and divergence time of each cladeCannabaceae was referred to originate in the late Cretaceous period(?88.6 Ma,95%HPD: ?87.8–89.8 Ma)based on phylogenetic tree inferred by concatenated captured nuclear genes and 5 fossil records.Divergence times of the existing clades are concentrated in Miocene(?5.3–23.0 Ma).Aphananthe(?76.9 Ma,95% HPD: ?74.2–79.1 Ma),Gironniera(?68.8 Ma,95% HPD: ?66.0–70.8 Ma),Lozanella(?65.9 Ma,95% HPD: ?62.8–67.8),Celtis II and other genera(?63.9 Ma,95% HPD: ?61.1–65.7 Ma)originated successively within ca.25 Ma after the origin of Cannabaceae.The CHCP clade,the Trema(Parasponia)clade and the Celtis I contain over 90% Cannabaceae species,but the divergence time interval among them is ca.4.3 Ma(?53.6 Ma,95% HPD: ?51.6–55.2 Ma to ?57.9 Ma,95% HPD: ?56.0–59.5 Ma).We speculated that the warm and humid climate of this period may facilitate the rapid evolution of the above three clades in Cannabaceae.