| Nolinoideae,a subfamily of Asparagaceae,has multiple economical uses,especially as pharmaceutical and ornamental.About 80% of the genera within Nolinoideae distributed in China,which have been recorded as source of Chinese medicine.However,in some Chinese herb medicine markets,species of Nolinoideae are often wrongly labeled or mixed up with other herbs.In consideration of the achievements obtained in species delimitation and phylogenetic analyses,54 plastomes of Asparagaceae（37 plastomes of Nolinoideae）were sequenced and annotated in this study.Based on 69 protein coding genes and 54 plastomes,the phylogeneny of Asparagaceae and Nolinoideae were constructed respectively.In result,the phylogenetic relationship within Asparagaceae and some long-standing disputes in Nolinoideae were well resolved.Furthermore,the highly variable regions of Nolinoideae plastomes were evaluated in order to lay a foundation for the selection of barcodes that could be useful for species identification.1.Comparative analysis of plastomesPlastomes of all sequenced individuals contained a large single-copy region,a small single-copy region and two reverse repeat regions.The total length and GC content were153,839 bp–162,227 bp and 37.3%–38.0%,respectively.The number of unique genes was112–114（78–80 coding genes,30 t RNAs and 4 r RNAs）.Pseudogenes or gene losses were detected in all plastomes,especially the pseudogenization of ycf15 and inf A.The pseudogenization of inf A in some plants has been demonstrated to result from plastid-to-nucleus gene transfer.In addition,pseudogenization of rps16 in Cordyline fruticosa and rps19 in Hosta plantaginea,loss of rps16 in Hosta plantaginea and Ruscus aculeatus and clp P in Cordyline fruticosa were also discovered.Furthermore,the loss of rps19 gene in Chlorophytum comosum and Hosta plantaginea may display a distinctive feature of evolution.The IR regions of the Convallaria were obviously longer than those of other genera due to the integration of 3.3 kb mitochondrial region in the plastomes.In Cordyline and Agave plastomes,gene inversions were revealed although the plastomes of Asparagaceae were generally conserved.IR/LSC boundary was located on rps19 or between rpl22 and rps19,which made trn H-rps19 completely or partially enter the repeat region.2.Phylogenetic analyses and divergence timesThe phylogenetic analyses of Asparagaceae showed that the relationship among subfamilies was similar to those of previous studies.Aphyllanthoideae,whose systematic position was controversial,was resolved as sister to Agavoideae in the current study with weak support obtained in BI tree,which implied that Aphyllanthoideae may be suffering from long branch attraction and rapid radiation.Based on 54 plastomes,the phylogenetic relationships among species of Nolinoideae were investigated.The topology of ML and BI tree were inconsistent only in the clade including tribes Ophiopogoneae and Nolineae and genus Maianthemum.Nolinoideae was divided into two major clades,the first clade included Rusceae and Dracaeneae.In Dracaeneae,Sansevieria was nested within Dracaena,which supported the previous proposal of submerging Sanseveria into Dracaena.In the second branch,Convallarieae was sister to the rest tribes.Monophyly of Convallarieae（except Theropogon）was strongly supported,within which Convallaria was determined to be the basal group.The phylogeny did not recover a monophyletic Polygonateae because of the deviation of Maianthemum.However,the systematic positions of the rest 3 genera in Polygonateae were confirmed.The intergeneric relationships within Ophiopogneae have long been disputed but was resolved in the present study.The result supported that Ophiopogon was sister to Liriope.Based on the phylogenetic relationship,the divergence times of Asparagaceae were estimated.The result showed that the split between Nolinoideae and Asparagoideae was estimated in late Eocene ca.35.61 Mya and that the crown group of Nolinoideae was dated to Miocene ca.18.21 Mya.3.Selection pressure analyses of plastome protein-coding genesIn the analysis of nucleotide substitution rate of plastome protein-coding genes in Nolinoideae,it was found that the values of Ka/Ks for six genes atp E,rbc L,ycf4,rps3,rps4,rps19 were all greater than 1（p < 0.05）,indicating that these genes may have undergone positive selection in the process of evolution.4.Evaluation of highly variable regionsA total of four highly variable regions were identified in the studied plastomes,i.e.ycf2-trn L-CAA,rpo B-trn C-GCA,ndh C-trn V-UAC,and ndh F-rpl32.Phylogenetic reconstruction based on rpo B-trn C-GCA,ndh C-trn V-UAC,and ndh F-rpl32 showed that the monophyly of most tribes were obtained with moderate to strong support.In addition,intergeneric relationships within Polygonateae（except Maianthemum）,Ophiopogoneae and Dracaeneae were consistent with the result from plastomes.The region ycf2-trn L-CAA had the lowest resolution compared with other regions probably due to the integration of 3.3 kb in Convallaria.In general,the plastomes were more effective in Nolinoideae phylogenetic analyses than these regions. |
