Phylogenomics Of Asian Justicia L.based On Complete Chloroplast Genomes

Justicia s.l.is the largest genus in the family Acanthaceae and belongs to the subtribe Justiciinae.It is a pantropical genus mainly distributed in tropical and subtropical areas whereas Asia is one of its diversity centers.Because of its extensive geographical distribution and complex morphological differences,phylogeneticrelationships within the genus have not yet been resolved until now.Chloroplast genome often exhibits its maternal inheritance and is highly conserved in most angiosperms.Its genome sequence is simple and easy to compare,so it is applicable to plant phylogeny.In present study,we assembled 79 complete chloroplast genome sequences of Justicia s.l.by the technology of genome skimming,and described some basic features of Justicia cp genomes,further compared their differences among various genomes,reconstructed phylogenetic relationships of Justicia in Asia,and finally resolved the problems on generic delimitation with Asian Justicia s.l.The main results are summarized as follows:(1)Complete chloroplast genomes of 79 species of Justicia all performed a closed circular structure from 148,218 to 156,819 bp in genome sizes.All of them showed a typical quadripartite structure,including a large single copy region(LSC),a small single copy region(SSC)and a pair of inverted repeats(IR).Their lengths of LSC varied from 81,434 to 84,088 bp,SSC from 984 to 17,533 bp,and IRs from 24,791 to 36,935 bp,whereas overall GC content ranged from 37.8% to 38.4%.Most species encoded 114 different genes,including 80 protein coding genes(CDS),30 transfer RNA genes(t RNA)and 4 ribosome RNA genes(r RNA).In addition,18 introns and 10 pseudogenes were also found.The gene rps12 was thought to be a trans-splicing gene widely distributed in plastid genomes of Justicia.From the perspective of gene function,43 genes were involved in plant photosynthesis while 61 genes participated in gene self-replication.(2)After comparing genomic differences of 23 representative plants among 11 sections,it is found that IRs structure of most species remained unchanged except IR expansions occurred towards different directions in J.austroguangxiensis and J.multinodis and contraction only occurred in J.grossa.Sequence global visualized alignment depicted that the coding regions are more conservative than non-coding regions and introns,while SSC region has more significant variations than LSC and IR regions.The results of nucleotide polymorphism(Pi)showed that ndh F,trn Nndh F,rps15-ycf1 and ycf1 were highly variable regions in Justicia,and they could be used as specific DNA barcodes for species identification of Justicia in the future.A total of 26-100 dispersed repeats and 36-59 SSR loci were detected in the chloroplast genome,among which the repetitive sequences of J.grossa were significantly different from those of other plants.Selection pressure analysis showed that all the cp coding genes were under negative selection pressure,but ccs A and psb T genes were found to own more positive selection sites.(3)Based on complete chloroplast genome sequences of 79 species,phylogenetic relationship of Justicia s.l.is constructed.The results strongly supported that the genus is not a monophyletic group,and its taxonomic problems need to be redefined.Combined with 79 common coding genes,12 introns and ITS sequences,most realistic species trees were constructed on the basis of genes tandem method and ancestral tracing method.The results are surely similar to chloroplast genome,which shows that the phlogeney is reliable.According to Graham’s(1988)description,typical taxonomic characters of Justicia,i.e.inflorescence,bract morphology,pollen types and chromosome number were selected for encoding.The ancestral state of Justicia was reconstructed by Mesquite,and the ancestral state and evolutionary style of Chinese Justicia were inferred by maximum likelihood.The ancestral states showed that the inflorescence was spike terminal,and bracts were ovate-elliptic or ovate-lanceolate,its pollen type was bilateral symmetry with 2-colporate,and each side had a row of(4-9)insulae.The surface was covered by small holes or punching with finely reticulate exine,as well as the chromosome number was 2n = 30.(4)Based on four cp regions(ndh A,ndh F-trn L,trn S-trn G,trn T-trn L)and the ITS sequence of 359 species representing almost genera of Justiinae and all sections and subsections of Justicia s.l.recognized by Graham,phylogenetic relationships of Asian Justicia are further studied and a new classification of Asian Justicia s.l.is proposed.In present study,we suggested to divide Asian Justicia s.l.into twelve genera,i.e.,Wuacanthus,Seabria,Leptostachya,Gendarussa,Rhaphidospora,Pserdorhaphidospora,Nicoteba,Calophanoides,Adhatoda,Clarkea,Trichocalyx and Rostellularia.Among them,three new genera,Seabria,Pserdorhaphidospora and Clarkea,were described ands 36 new combinations were proposed.