Comparative Transcriptome And Chloroplast Genome Analyses And Phylogeography Of Dipteronia Species

Dipteronia Oliver,belongs to family Aceraceae,is an endangered endemic genus found in southwestern and central China with only two extant species,D.sinensis Oliver and D.dyeriana Henry.In particular,D.sinensis is mainly found in central and southwestern China,whereas D.dyeriana is only located in the Yunnan Province in southwestern China.As typical Tertiary relic species,fossil records indicate species of this genus also grew in North America during the Tertiary period.Presently,the two living species only located allopatrically in China.Obviously,complicated evolutionary history must be suffered for this genus since Tertiary.However,up to now,it is unclear about the mechanism of speciation and how both species react to the environmental and geographic change since Tertiary.In the present study,we conducted the comparative transcriptome and chloroplast genome analyses for endangered endemic Dipteronia genus and reveled the differences of transcriptomes and plastomes for both species.Additionally,large amount of genetic markers including SSRs and SCNGs were developed for this genus and the phylogeny of D.sinensis and D.dyeriana was firstly determined using whole chloroplast genomes.Furthermore,we combined the methods and theories of biogeography,population genetics,phylogeography and species distribution model（SDM）to infer the divergent evolution,demographic history and spatiotemporal population genetic structure for Dipteronia species.The results of current study are presented as follows:（1）Comparative transcriptome and the development of genetic markers for DipteroniaThe transcriptomes for D.sinensis and D.dyeriana were sequenced based on Illumina Hiseq 2000 platform.After de novo assembly,52,351 and 53,983 Unigenes were recovered for D.sinensis and D.dyeriana,respectively.More than half of Unigenes（58.90%/51.49%）from both species with at least one significant match to the five public databases.A comparison of the transcriptomes of both species identified a total of 7,814 orthologs.Estimation of selection pressures using Ka/Ks ratios showed that only 30 orthologous pairs had a ratio significantly greater than 1,i.e.,showing positive selection.However,4,041orthologs had a Ka/Ks<0.5（p<0.05）,suggesting that most genes had likely undergone purifying selection.Further,12,377 SSR loci were detected and 4,179 primer pairs were designed for experimental validation.Of these 4,179 primer pairs,435 primer pairs were randomly selected to test polymorphism.Our results show that products from 132 primer pairs were polymorphic.Additionally,based on orthologous Unigenes,314 single copy nuclear genes were identified and some of them were verified in our study.（2）Comparative chloroplast genome analyses of two related Dipteronia speciesThrough a combination of de novo and reference guided assembly,plastid genomes were obtained;that of D.sinensis was 157,080 bp and that of D.dyeriana was 157,071 bp.Both plastid genomes encoded 87 protein coding genes,40 t RNAs,and 8 r RNAs;no significant differences were detected in the size,gene content,and organization of the two plastomes.Furthermore,we detected the chloroplast gene coding regions were more conserved than those of their noncoding counterparts.According to the alignment results,several intergenic regions were found to display high divergence,including trn S（GCU）-trn G,trn T（UGU）-rps4,trn L（UAA）-trn T（UGU）,psb E-pet L,and rpl32-trn L（UAG）.These regions could be selected as candidate gene regions for developing more specific DNA barcodes for the Aceraceae family.Furthermore,we used the whole chloroplast genomes to determine the phylogeny of D.sinensis and D.dyeriana and confirmed that the two species were highly divergent.（3）The phylogeographic analyses for Dipteronia species based on cp DNA and SCNG markersWe identified 17 haplotypes across 45 Dipteronia populations based on three cp DNA fragments（psb A-trn H、psb J-rpl33、trn S-psb Z）.A relatively high genetic diversity(Hd=0.7814,π=0.572×10^-2)and significant phylogeographic structure(N_ST=0.940,G_ST=0.850,P<0.05)were detected.We found the genetic diversity of D.sinensis(Hd=0.73,π=0.112×10^-2)was higher than that in D.dyeriana(Hd=0.54,π=0.049×10^-2).The Network and phylogentic analyses indicated that two unambiguous lineages were formed and no shared haplotypes were detected for two species.AMOVA results indicated86.55%of the total variation was detected between two species and 12.53%was explained among populations within species.The mismatch distribution analysis reveled no expansions were detected for whole genus.The divergence time of two species was estimated approximately at 52.5Ma and then the split time of D.sinensis was dated to Miocene（19.32Ma）.It demonstrated that Dipteronia genus may have a long term evolutionary history.The results based on two SCNGs also reveled relatively high genetic diversity(DS11898:Hd=0.910,π=1.103×10^-2;DS43372:Hd=0.645,π=0.814×10^-2)and significant phylogeographic structure(DS11898:N_ST=0.735,G_ST=0.345,P<0.05;DS11898:N_ST=0.711,G_ST=0.615,P<0.05)for Dipteronia genus.AMOVA results of SCNGs indicated 94.70%/96.69%（DS11898/DS43372）of the total variation was detected between two species and 2.34%/1.65%was explained among populations within species.The MDA analyses based on two SCNGs still showed the no demographic expansion was detected for Dipteronia populations.（4）Genetic structure,gene flow and population history inferred from EST-SSR markersThe Bayesian population STRUCTURE analysis based on 15 EST-SSR loci reveled that two species were clustered into two unambiguous groups,and then the D.sinensis populations were subsequently divided into West and East lineage.We estimated the historical gene flow between two species and two lineages of D.sinensis using MIGRATE.The results showed low to medium levels of gene flow were detected between two species and two lineages,and asymmetrical gene flow was detected between D.dyeriana and East lineage of D.sinensis.The Bayes Ass analyses showed that low level of recent gene flow was detected between two species and two D.sinensis lineages.The population history inferred from ABC analysis indicated that Dipteronia populations were diverged in Miocene.It means that pre-Quaternary climate change had left strong influences on the genetic divergence for Dipteronia species.The SDM results reveled that most suitable habitats of Dipteronia in LGM were consisted with current status.Additionally,the PCA analysis for all environmential variables of 45 populations showed a consistent result with the STRUCTURE analysis.So we inferred the prominent reason for the divergence of two Dipteronia species was geographic isolation and the divergence of D.sinensis popuations should be related to environmental adaptation.