Plastome Organization,phylogeny And Divergence Time Among Malvaceae S.l.subfamilies

Malvaceae sensu lato(s.l.),which is widespread throughout tropical and temperate regions,is the largest family of Malvales.Some phylogenetic studies strongly supported monophyletic of Malvaceae s.l..Malvaceae s.l.includes 9 subfamilies: Malvoideae Burnett,Bombacoideae Burnett,Dombeyoideae Beilschm,Tilioideae Arn,Brownlowioideae Burrett,Sterculioideae Burnett,Helicteroideae(Schott & Endl.)Meisn,Byttnerioideae Burnett,Grewioideae Dippel.However,Malvaceae Juss.is wide variety(includes 243 genera,and more than 4225 species),widely distributed and difficult sampling.In addition,most of the studies used fewer samples and molecular fragments.Within Malvaceae s.l.,the phylogenetic relationships among subfamilies remain controversial.To resolve these relationships and explore plastome evolution in Malvaceae s.l.,we generated a dataset of32 species(17 species from Genbank)representing 17 genera and 8 subfamilies of Malvaceae s.l.,lacking only Brownlowioideae.Among these plastomes,4 genera,Burretiodendron Rehder,Colona Cav.,Grewia L.and Urena Dill.ex L.,were reported for the first time.In addition,outgroups from Dipterocarpaceae Blume,Thymelaeaceae Juss.,Brassicales Bromhead,Huerteales Doweld,Sapindales Juss.ex Bercht.& J.Presl,thus our dataset included 45 complete plastomes.The main conclusions are summarized as follows:(1)Malvaceae s.l.species possessed the typical quadripartite structure of angiosperm plastomes.In Malvaceae s.l.,the plastomes of 8 subfamilies investigated differed little in genome size,GC content and other genomic characteristics and structures.Except for the duplicate genes of Inverted Repeat a regions,we found 112 unique genes including 78protein-coding genes,30 t RNA genes and 4 r RNA genes in all species of Malvaceae s.l.We find relative stasis in plastome structural evolution during the evolution of Malvaceae s.l.,although we note that Inverted Repeat boundaries have fluctuated.In Hibiscus tiliaceus L.,Hibiscus hamabo Siebold & Zucc.,Hibiscus taiwanensis S.Y.Hu,Abelmoschus esculentus(L.)Moench and Urena procumbens L.,the Inverted Repeat included ~1000 bp of the 5' end of ycf1,as did Firmiana colorata(Roxb.)R.Br.and Firmiana simplex(L.)W.Wight.In general,the plastomes of Malvaceae s.l showed low sequence divergence,and four divergence hotspots(clp P,ycf1,ndh D-ccs A,rps19-rpl2)with the highest nucleotide diversity were found.These regions may be important for DNA barcoding and investigating phylogenetic relationships in Malvaceae s.l.Our analysis identified nine coding sequences with positive selection evidence(acc D,clp P,ndh C,ndh F,rbc L,rpl22,rps12,ycf1 and ycf2).These positive genes may play a key role in the ability of Malvaceae s.l plants to adapt to different environments.(2)Except for the Inverted Repeat regions,the other six datasets(complete plastomes,complete plastomes minus 9 genes with positive selection regions,78 protein-coding genes regions,introns and intergenic spacer regions,Large single copy regions,Small single copy regions)constructed phylogenetic trees separately,all of the 8 subfamilies are monophyletic.Our results show that Malvaceae s.l.included two clades(Byttneriina,Malvadendrina),this was consistent with previous studies.Byttneriina includs Byttnerioideae and Grewioideae;Malvadendrina includes Malvoideae,Bombacoideae,Dombeyoideae,Tilioideae,Sterculioideae and Helicteroideae.Among Malvadendrina,Malvoideae grouped with Bombacoideae,and Helicteroideae in a basal position in the Malvadendrina.But the phylogenetic relationships among the three subfamilies(Sterculioideae,Dombeyoideae and Tilioideae)were poorly resolved.Among the six datasets,phylogenetic reconstructions based on 78 common protein-coding genes strongly supported nearly all relationships among Malvaceae s.l.subfamilies,which strongly supported monophyletic of Malvaceae s.l.,Dipterocarpaceae and Thymelaeaceae,as well as the monophyletic of main subfamilies of Malvaceae.But lacking only Brownlowioideae.(3)Based on the phylogenetic tree of coding sequence,Bayesian divergence time analysis of Malvaceae s.l.was reconstructed with two fossil constraints and one secondary calibrations.The results showed that the stem age of 8 subfamilies was estimated to have occurred between 44.06–68.45 millions of years ago(Ma);the diversification of the subfamilies of Malvaceae s.l was dated to the late Cretaceous and early Eocene(50.14–75.08 Ma),during a time of global warmth.The stem age of Malvaceae s.l.was estimated as 111.55 Ma,and the crown node age of Malvaceae s.l.most likely appeared in the Late Cretaceous period(74.69 Ma).The crown age of Byttneriina and Malvadendrina was 51.86 Ma and 68.45 Ma,respectively.The crown group age of common ancestor of(Sterculioideae,(Tilioideae,Dombeyoideae))and(Malvoideae,Bombacoideae)was 65.38 Ma,the crown group age of the clade(Malvoideae,Bombacoideae)was 61.41 Ma.The stem group of the temperate subfamily Tilioideae was dated to the mid-Eocene epoch(44.06 Ma),and the divergence of this subfamily may be related to the diffusion of temperate climate caused by climate cooling during early Eocene period.In summary,in our study,the phylogenetic relationships among 8 subfamilies of Malvaceae s.l.were constructed at the family and order level in plastome phylogenomics for the first time.The phylogenetic frame of Malvaceae s.l.was established for further study,and the stem age and crown age of Malvaceae s.l.and subfamilies were estimated.In addition,4 potential DNA barcodes and 9 positive genes which may be related to the adaptive environment of this family were found.However,whether the phylogenetic relationships is consistent with the nuclear genome data remains to be further studied.