Taxonomy And Phylogeny Of Pooideae (Poaceae)

Pooideae contains a lot of important food crops and high-quality forages,and has important ecological value.It is the largest subfamily of Poaceae,with 15 tribes,202 genera and more than 3900 species.The number of species accounts for nearly 1/3 of the total species of Poaceae.Pooideae species are widely distributed in warm and cold areas in the northern and southern hemisphere,and a few in tropical or subtropical mountains.Previous scholars have done a lot of research on the classification and phylogeny of Pooideae.At present,the classification and phylogenetic relationships of most tribes have been determined.However,the phylogenetic relationship between Bromeae and Triticeae is still controversial,and there are still some phylogenetic problems in subtribes of Poeae.There are still disputes about the phylogenetic relationship between Leymus and Psathyrostachys in Triticeae,the intergeneric phylogenetic relationship in Aveninea,the phylogenetic relationship between Agrostis and Polypogon,and the phylogenetic relationship of Festuca,Lolium,and Castellia.The phylogenetic problems in Pooideae urgently needed further research by increasing sampling density and increasing phylogenetic information sites.Inflorescence types,spikelet morphology,caryopsis morphology,and glumes,lemma,and palea micromorphology of have important taxonomic value in Pooideae groups.Chloroplast genome is suitable for phylogenetic study of different taxonomic levels because of its many copies and moderate evolution rate.Based on the latest classification system of Poaceae published by soreng et al.in 2017,a total of 218 plastomes in 103 genera in Pooideae were obtained.The caryopsis morphology,inflorescence type,spikelet morphology,and glumes,lemma,and palea micromorphology were combined to explore the morphological or micromorphological classification characteristics that suitable for Pooideae,phylogenetic relationship,and chloroplast genome evolution of Pooideae.The results are as follows:(1)The mature caryopsis of 84 species in 37 genera of Pooideae were analyzed.According to the caryopsis size(length,width and aspect ratio),shape,color,ventral shape,hilum shape,compressed mode,the stylopodium persistent or not,the top hairs present or not,and proportion of embryo to caryopsis,the plants studied are divided into five clade.Clade I includes Phaenospermateae and Diarrheneae.Clade II includes Brachypodieae,Avena,Helictotrichon,Bromeae,and Triticeae.Clade III includes Anthoxanthum,Vulpia,and Stipa.Clade IV includes Alopecurus,Aniselytron,and Phleum.Clade IV includes Melica,Achnatherum,Piptatherum,Koeleria,Trisetum,Phalaris,Hierochloe,Agrostis,Calamagrostis,Polypogon,Deyeuxia,Puccinellia,Sclerochloa,Beckmannia,Poa,Festuca,Lolium,and Dactylis.The stylopodium persistent or not and the top hairs present or not have important taxonomic value in Pooideae groups.According to the caryopsis morphology,only the Pooideae can be roughly divided,but some groups with similar caryopsis morphology under Poaceae can not be well distinguished.(2)Study on inflorescence type,spikelet morphology,and glume,lemma,and palea micromorphologyA total of 99 species belonging to 38 genera of Pooideae were selected as experimental materials.There are three main inflorescence types in Pooideae: spike,raceme,and panicle.Spikelet is the basic unit of inflorescence,which has both 1 floret and many florets.According to the type of inflorescence and the number of florets in spikelets,Pooideae can be divided into four clades: Clade I is spike and spikelets with multiple florets,including Triticeae;Clade II is raceme and spikelets with multiple florets,including Lolium and Brachypodieae;Clade III is panicle and the spikelet has one floret,including Phaenosperma,Piptatherum,Achnatherum,Stipa,Sinochasea,Agrostis,Calamagrostis,Polypogon,Deyeuxia,Aniselytron,Alopecurus,Phleum,and Beckmannia;Clade IV is panicle and spikelets with multiple florets,including Glyceria,Melica,Diarrhena,Bromus,Avena,Helictotrichon,Koeleria,Trisetum,Phalaris,Anthoxanthum,Hierochloe,Puccinellia,Poa,Festuca,Vulpia,and Dactylis.The comparative analysis of glume,lemma,and palea micromorphology of Pooideae shows that long cells are the main cells constituting the epidermis,and the shape of long cells,whether the cell wall is thickened and wavy have certain taxonomic significance.Stomatal complex,papillae,silica bodies,and prickles exist in most species,and macro-hairs is only observed in individual species.The stomatal guard cells of Pooideae species have two forms:parallel(flat top)and dome.The shape of silica bodies mostly oval,crescent,reniform,and irregular dumbbell shaped.The shape of papillae are mostly round,inclined and macular vesicular.According to the shape of long cells,thickening or not,bending degree,the shape of stomatal guard cells,papillae exit or not and its shape,the shape of prickles,the macro-hairs exit or not,and the shape of silica bodies,the studied groups of Pooideae were divided into six clades.Comparative morphology does not support the integration of Bromus into Triticeae,supports the separate of Deyeuxia and Calamagrostis,and it is difficult to distinguish between Agrostis and Polypogon.There are significant morphological differences between Festuca and Lolium,and does not support the establishment of Festuca-Lolium complex.(3)Plastid phylogenomics analysisThe phylogenetic relationship of Pooideae was reconstructed based on 218 plastomes within 103 genera,14 tribes(including 49 newly sequenced)with anthor three Bambusoideae species as outgroups.The phylogenetic tree constructed based on whole plastome sequence,coding sequence,and non-coding sequence has the same topology.The monophyly is strongly supported.All tribes except Triticeae and Stipeae are monophyly with the support value of100%.Bromeae is embedded into Triticeae and the clade composed of Psatherostachys and Leymus differentiate before Bromeae.Ampelodesmeae is embedded into Stipeae with the support value of 100%.Brachyelytreae is the basal group of Pooideae.Poeae can be divided into two large branches,corresponding to chloroplast group1 and group2 respectively.In clade 1,Phalaridinae and Torreyochloinae are sister groups to each other and differentiate first.In turn,Aveninea,Anthoxanthinae,and Brizinae are differentiated,and the branches formed by Agrostidinae and Echinopogoninae are sister groups with Brizinae.In clade 2,the branches of Poinae and Phleinae are sister groups with the branches of Alopecurinae and Ventenatinae.Intergeneric phylogenetic relationship analysis showed that Leymus and Psatherostachys,Trisetum and Koeleria,Deyeuxia and Calamagrostis were nested with each other,and the relationship between the two genera could not be clarified based on plastid phylogenomics.Agrostis gigantea and Polypogon species clustered into one brabch,resulting Agrostis is not monophyletic,and the morphology of the two genera is very similar,which supports the merger of Polypogon into Agrostis.Festuca is not a monophyleticgroup,Lolium and Castellia are embedded in Festuca,but each genus is grouped into one branch,and the morphology of these three genera is significantly different,which does not support merging.(4)Comparative plastomes analysisThe plastomes sequences of 218 Pooideae species belonging to 103 genera,14 tribes were compared and analyzed to explore the evolution of plastomes.The plastomes of Pooideae are typical quadripartite structure,with 126-136 genes and the plastomes size ranged from 133165 bp to 139946 bp.The changes of plastome gene number in Pooideae were mainly caused by genes acc D,ndh B,ndh C,ndh D,rpl2,rpl22,rpl23,rps19,trn H-GUG,trn T-UGU,ycf1,and ycf2.The boundaries of most plastomes are relatively conservative.JLB is mostly located in the rpl22-rps19 spacer,JLA is mostly located in the rps19-psb A spacer,JSB is mostly located in the rps15-ndh F spacer,and JSA is mostly located in the gene ndh H.Forward repeat and palindrome repeat are the main repeat types,with the most repeat sequences with a length of 30-34 bp.SSR analysis showed that the plastomes contained138-184 SSRs,of which single nucleotide repeats were the most,accounting for about 75% of all SSRs,followed by dinucleotide repeats,accounting for 16.8% of all SSRs.The codon usage bias is weak,and codons are more inclined to use base A/T and codons are more inclined to end with A/T.By calculating the percentage of parsimonious informative sites(Pi %)of chloroplast genome CDS region and gene spacer,it was found that the Pi % value of IR region was significantly lower than that of LSC region and SSC region,indicating that the genes in IR region were more conservative.According to the Pi % value,12 molecular markers that could be used for phylogeny research of Pooideae were screened,including six protein-coding genes mat K,ndh F,rpo C2,rpo A,rpl22,and ccs A and six gene spacers ndh G-ndh I,ccs A-ndh D,rps16-2-trnK-UUU-1,trnG-GCC-trnfM-CAU,petN-trnC-GCA,and rpl32-trnL-UAG.