The systematics of grasses has advanced through applications of plastome phylogenomics, although studies have been largely limited to subfamilies or other subgroups of Poaceae. Here we present a plastome phylogenomic analysis of 250 complete plastomes (179 genera) sampled from 44 of the 52 tribes of Poaceae. Plastome sequences were determined from high throughput sequencing libraries and the assemblies represent over 28.7 Mbases of sequence data. Phylogenetic signal was characterized in 14 partitions, including (1) complete plastomes; (2) protein coding regions; (3) noncoding regions; and (4) three loci commonly used in single and multi-gene studies of grasses. Each of the four main partitions was further refined, alternatively including or excluding positively selected codons and also the gaps introduced by the alignment. All 76 protein coding plastome loci were found to be predominantly under purifying selection, but specific codons were found to be under positive selection in 65 loci. The loci that have been widely used in multi-gene phylogenetic studies had among the highest proportions of positively selected codons, suggesting caution in the interpretation of these earlier results. Plastome phylogenomic analyses confirmed the backbone topology for Poaceae with maximum bootstrap support (BP). Among the 14 analyses, 82 clades out of 309 resolved were maximally supported in all trees. Analyses of newly sequenced plastomes were in agreement with current classifications. Five of seven partitions in which alignment gaps were removed retrieved Panicoideae as sister to the remaining PACMAD subfamilies. Alternative topologies were recovered in trees from partitions that included alignment gaps. This suggests that ambiguities in aligning these uncertain regions might introduce a false signal. Resolution of these and other critical branch points in the phylogeny of Poaceae will help to better understand the selective forces that drove the radiation of the BOP and PACMAD clades comprising more than 99.9% of grass diversity. Triticum L.), rice (Oryza L.) and corn (Zea L.), as well as numerous forage, bamboo and biofuel species. Grasses grow on all continents in tropical, temperate and Arctic zones. Grasses are common and often dominant components of open ecosystems , but also occur in association with forests, and they have diversified to inhabit aquatic to desert habitats. The deep phylogenetic framework for Poaceae is well established (Grasses (Poaceae) are the fifth largest family of flowering plants in the world, with some 11,500 species and about 768 genera , and theablished . Three sThe systematics of grasses have been studied throughout the history of botany, with the first dedicated classification by Zea . We also compare and contrast the results of our phylogenomic analyses with current grass classifications and with existing knowledge of phylogenetic relationships in grasses derived from plastid/plastome and nuclear analyses.Microstegium vimineum (Trin.) A. Camus  in Arthraxon prionodes (Steud.) Dandy . The same seed accession was sequenced in A. prionodes, and in M. vimineum. We have corrected this error in GenBank (accession KU291471). For new plastomes, we obtained DNA from either fragments of herbarium mounted leaf tissues or silica-dried seedlings germinated in the greenhouse. For the latter, voucher specimens were prepared from greenhouse material when it reached a flowering stage suitable for identification, and these were deposited in the herbarium (DEK) of the Biological Sciences Department, Northern Illinois University, DeKalb, Illinois. Tissue was homogenized manually in liquid nitrogen before extraction. The DNA extraction protocol was followed using the Qiagen DNeasy Plant Mini Kit .Taxon sampling includes 15 new  and 235 Whiteochloa C.E. Hubb. as part of Panicinae rather than Cenchrinae. Generic classification also follows We follow the subdivisional classification of Poaceae by http://bioinf.spbau.ru/spades) was used for de novo assembly  , under trbcL, ndhF, matK, and the trnK intron) was selected to compare against previously published family wide analyses such as We analysed 14 different data partitions , which vJoinvillea Gaudich. ex Brongn. and Gris) resulted in the loss of substantial data, especially in noncoding regions , given the well-established sister group relationship of Anomochlooideae to the rest of the grassesWe conducted only maximum likelihood (ML) analyses, given the extensive partitioning that was conducted and the large number of comparisons that needed to be made among the many clades in trees from different partitions. Since our study is focused on plastome phylogeny of the entire grass family, it would be ideal to root the tree with non-grass taxa. The closest relatives of grasses are the families Joinvilleaceae, Ecdeiocolaceae and Flagellariaceae. Plastome data based on 77 coding regions and some smaller data sets identify Ecdeiocolaceae and grasses as sister taxa , while o regions . Using JA substitution model was selected using jModelTest v. 2.1.3 . For allTo compare and contrast the phylogenetic trees generated from each of the 14 partitions, we compared branch support for identical  clades across all trees . First, We performed six pairwise comparisons of trees generated from data partitions differing only by the inclusion or exclusion of positively selected codons, to gain insight into the relative effect of these characters on the inferred phylogenies. We conducted similar comparisons of trees differing only by the inclusion or exclusion of gapped sites. To characterize the effect of datasets comprising three-genes and all plastome coding genes on tree topology and support we conducted pairwise comparisons of trees A vs. E, B vs. F, C vs. G and D vs. H.Descriptions of phylogenetic results focus on the plastome trees (not the three-gene trees), and clades with support <50% are not discussed. We use the terms “weak or poor,” “moderate,” and “strong” in reference to clades that received BP values of 50–70%, 71–90% and 91–100%, respectively, and “unsupported” for clades with BP <50%.p < 0.05). However, positively selected codons could be identified in 65 out of the 76 loci , seven photosystem II proteins  and two ribosomal proteins . All 11 were relatively short loci (mean length <150 bases). Of the 65 loci with positively selected codons, the greatest number were found in rpoC2 (184 codons), matK (74), ndhF (67) and rbcL (47).Selection analyses of 76 protein coding loci in the grass plastome indicated that all were under selection. The predominate selective force identified for all loci was purifying selection  in two or more trees and different but overlapping sets of species. In 22 instances one or more taxa are part of two conflicting clades, and in five instances one or more taxa are part of three conflicting clades . NineteeThe following descriptions of phylogenetic relationships refer only to the 10 plastome trees unless otherwise indicated. In all plastome trees, Pharoideae and Puelioideae are successively diverging sisters of a lineage comprising the BOP and PACMAD clades –8. The BIn the plastome trees, Bambusoideae (BP = 100%), Oryzoideae (BP = 87–100%) and Pooideae (BP = 100%) are strongly supported, and Bambusoideae and Pooideae form a maximally supported clade ; Fig. S1Brachyelytrum aristosum (Michx.) P. Beauv. ex Trel. (Brachyelytreae), Lygeum spartum L. (Lygeae) + Nardus stricta L. (Nardeae), Phaenosperma globosum Munro ex Benth. (Phaenospermateae), Melica L. , and Stipeae–Ampelodesmeae diverge successively with respect to the rest of the Pooideae clade in all trees (BP = 88–100%). Within Stipeae–Ampelodesmeae (BP = 100%), Eriocoma hymenoides (Roem. and Schult.) Rydb. + Nassella hyalina (Nees) Barkworth (BP = 100%) and Piptatherum songaricum (Trin. and Rupr.) Roshev. ex Nikitina form a clade (BP = 100%), and Oryzopsis asperifolia Michx. + Ampelodesmos mauritanicus (Poir.) T. Durand & Schinz (Ampelodesmeae) (BP = 88–100%) and Piptochaetium avenaceum (L.) Parodi form a clade (BP = 88–100%).Brachypodium distachyon (L.) P. Beauv. (Brachypodieae), Bromus vulgaris (Hook.) Shear (Bromeae), Poeae and Triticeae, with varying support. Gapped sites in the plastome noncoding alignments contribute strongly to support for the clade. In tree Q, based on noncoding regions including gapped sites, and in the two complete plastome trees including gapped sites , support for the clade is 100%, whereas in tree R, based on noncoding regions excluding gapped sites, support for the clade is less than 50%. Plastome coding regions also identify the clade, but with weaker support, when gapped sites are included  and excluded . When gapped sites are excluded from the complete plastome datasets, however, Diarrhena obovata (Gleason) Brandenburg (Diarrheneae) and Brachypodieae are sister taxa with weak  and moderate  support. In all trees, Bromeae and Triticeae are maximally supported sister taxa, and Bromeae + Triticeae and Poeae form a maximally supported clade.Seven trees identify a clade comprising Hordeum L. (two species sampled), Connorochloa tenuis (Buchanan) Barkworth, S.W.L. Jacobs & H.Q. Zhang, Secale cereale L. and Taeniatherum caput-medusae (L.) Nevski diverge successively with respect to the rest of the clade, with all branches strongly supported in most trees. An Aegilops L. + Triticum clade is variously supported (BP = 60–100%) (Triticum macha Dekapr. & Menabde + Triticum turgidum L. + Triticum aestivum L. (BP = 100%) sister to Aegilops speltoides Tausch + Triticum timopheevii (Zhuk.) Zhuk. (BP = 100%). Relationships among taxa in the former clade conflict among trees (Triticum urartu Thumanjan ex Gandilyan + Triticum monococcum L. (BP = 100%) sister to an eight-species Aegilops clade (BP = 98–100%) in which relationships are mostly strongly supported; however, relationships among Aegilops kotschyi Boiss., Aegilops sharonensis Eig and Aegilops longissima Schweinf. & Muschl. conflict among trees   and diving trees . The othng trees .Phalaris arundinacea L.  and Torreyochloa pallida (Torr.) G.L. Church (Torreyochloinae) are sister taxa (BP =100%); Agrostidinae, comprising Agrostis L. (two species sampled), Calamagrostis breviligulata  Saarela [syn. Ammophila breviligulata Fernald (Gastridium ventricosum (Gouan) Schinz & Thell., is monophyletic; Agrostidinae + Briza maxima L. (Brizinae) form a clade (BP = 80–100%); and Agrostidinae + Brizinae + Anthoxanthum L.  form a clade (BP = 100%). There is conflict among trees for the relative branching order of Phalaridinae + Torreyochloinae and Aveninae (Avena sativa L. and Trisetum cernuum Trin.) with respect to the remainder of the clade  Hitchc. + Zingeria biebersteiniana (Claus) P.A. Smirn. (Coleanthinae) (BP = 100%) and Alopecurus arundinaceus Poir. (Alopecurinae) + Phleum alpinum L. (Phleinae) + Poa palustris L. (Poinae) . Relationships among Phleum, Alopecurus and Poa conflict among trees (Cynosurus cristatus L. (Cynosurinae), Catapodium rigidum (L.) C.E. Hubb. (Parapholiinae), Holcus lanatus L. (Holcinae), Deschampsia antarctica E. Desv. (Aristaveninae), Helictochloa hookeri (Scribn.) Romero-Zarco (Airinae), Dactylis glomerata L. and Lamarckia aurea (L.) Moench (Dactylidinae) and Festuca ovina L., Drymochloa sylvatica (Pollich) Holub and Lolium L. (four species) (Loliinae). Holcinae and Airinae are sister taxa in six trees . In all trees, Cynosurinae and Parapholiinae are sister taxa (BP = 100%) and form a clade with Dactylidinae (BP = 83–100%), and in all trees except R these subtribes form a clade with Loliinae (BP = 92–100%).Poeae chloroplast group 2 is divided into two subclades . One comng trees . The othBuergersiochloa bambusoides Pilg. (Buergersiochloinae) is sister to a clade of Eremitis Döll sp. + Pariana Nakai (three accessions and at least two species)  and Diandrolyra Stapf sp. + Raddia brasiliensis Bertol. + Olyra latifolia L. + Cryptochloa strictiflora (E. Fourn.) Swallen + Lithachne pauciflora (Sw.) P. Beauv. . Relationships within Parianinae and Olyrinae are strongly supported.Within Bambusoideae , ArundinHickelia madagascariensis A. Camus (Hickeliinae), Neololeba atra (Lindl.) Widjaja (Dinochloinae) + Greslania Balansa sp. (Greslaninae) and Bambusa spp. + Dendrocalamus latiflorus Munro (Bambusinae) (BP = 100%). Dinochloinae and Greslaninae form a clade (BP = 98–100%), but relationships among Dinochloinae + Greslaninae, Bambusinae and Hickeliinae conflict among trees , with a maximally supported subclade comprising Bambusa emeiensis L.C. Chia & H.L. Fung and B. multiplex (Lour.) Raeusch. ex Schult. & Schult. f. + B. oldhamii Munro. Relationships among this latter clade, B. bambos (L.) and B. arnhemica Voss vary among trees , Rhipidocladum pittieri (Hack.) McClure (Arthrostylidiinae), and Guadua weberbaueri Pilg., Olmeca reflexa Soderstr. and Otatea acuminata (Munro) C.E. Calderón & Soderstr. . Within Chusqueinae, Chusquea spectabilis L.G. Clark is robustly resolved as sister to the remainder of the genus. Arthrostylidiinae and Guaduinae are sister taxa (BP = 100%). Relationships among the three sampled genera of Guaduinae conflict among trees  is sister to the rest of the subtribe. Gelidocalamus tessellatus T.H. Wen & C.C. Chang and Shibataea kumasaca (Zoll. ex Steud.) Makino are sister taxa in all trees (BP = 52–100%), and these plus Ferrocalamus rimosivaginus T.H. Wen form a clade in all trees . Sasa veitchii (Carrière) Rehder, Arundinaria Michx. , Pseudosasa japonica (Siebold & Zucc. ex Steud.) Makino ex Nakai, Pleioblastus maculatus (McClure) C.D. Chu & C.S. Chao, Acidosasa purpurea (Hsueh & T.P. Yi) Keng f., Indosasa sinica C.D. Chu & C.S. Chao and Oligostachyum shiuyingianum (L.C. Chia & P. But) G.H. Ye & Z.P. Wang form a clade (clade VI) in all trees (BP = 95–100%). Indocalamus wilsonii (Rendle) C.S. Chao & C.D. Chu (clade VIII) forms a clade with clades IV and VI in three trees  and is sister to clade IV in two of those trees . Within clade VI, S. veitchii and Arundinaria form a clade (BP = 63–98%) in all plastome trees except R, in which S. veitchii is sister to the rest of the subtribe (BP = 68%). The remaining taxa form a strongly supported clade, in which Pseudosasa japonica is sister to the rest of the lineage. However, relationships among Pleioblastus maculatus, A. purpurea, I. sinica and O. shiuyingianum are discordant among trees , Thamnocalamus spathiflorus (Trin.) Munro (clade VII), Gaoligongshania megalothyrsa (Hand.-Mazz.) D.Z. Li, Hsueh & N.H. Xia (clade IX), Sarocalamus faberi (Rendle) Stapleton, Drepanostachyum falcatum (Nees) Keng f., Indocalamus longiauritus Hand.-Mazz., Yushania Keng. F. (two species), Bashania fargesii (E.G. Camus) Keng f. & T.P. Yi, Fargesia Franch. (two species) and Phyllostachys Siebold & Zucc. (five species) (clade V). Clades III, IX and V form a clade in all trees except R (BP = 55–85%), and clades III and IX are sisters in all trees except Q and R (BP = 60–93%). Within clade V, I. longiauritus and Yushania form a clade in all trees except G, H and R. Fargesia nitida (Mitford ex Anonymous) Keng f. ex T.P. Yi and Fargesia murielae (Gamble) T. P. Yi form a clade (BP = 55–100%) in all trees except R. In all trees, the five species of Phyllostachys form a clade (BP = 65–100%), Phyllostachys aurea Carrière ex Rivière & C. Rivière and Phyllostachys propinqua McClure form a clade (BP = 68–100%), and Phyllostachys sulphurea (Carrière) Rivière & C. Rivière and Phyllostachys nigra var. henonis (Mitford) Stapf ex Rendle form a clade (BP = 87–100%). In subsets of trees, Phyllostachys edulis (Carrière) J. Houz. and P. aurea + P. propinqua form a clade , and P. aurea, P. propinqua, P. sulphurea and P. nigra var. henonis form a clade . Phyllostachys and Fargesia are sister taxa in two trees , and a broader clade including these genera and Bashania is recovered in all trees (BP = 52–100%).A deep lineage of Arundinarieae is recovered in five trees  comprising Streptogyna americana C.E. Hubb. (Streptogyneae), Ehrharta bulbosa Sm. + Microlaena stipoides (Labill.) R. Br.  and Humbertochloa bambusiuscula A. Camus & Stapf (Phyllorachideae) are successively diverging sisters to Oryzinae + Zizaniinae, with strong support for all branches. Within Oryzinae, Leersia tisserantii (A. Chev.) Launert and Prosphytochloa prehensilis (Nees) Schweick. are sister taxa (BP = 100%), and the multiple species of Oryza form a clade (BP = 100%). Relationships among most species of Oryza are strongly supported in most trees; however, relationships among Oryza sativa L., Oryza nivara Sharma & Shastry and Oryza rufipogon Griff. conflict among trees (Rhynchoryza subulata (Nees) Baill. and Zizania aquatica L. are sister taxa (BP = 100%), and relationships among this clade, Chikusichloa aquatica Koidz. and Potamophila parviflora R. Br. are discordant among trees  . Streptong trees . Within ng trees .Monachather forms a clade with Micrairoideae   .Zeugites pittieri Hack. (Zeugiteae) and Chasmanthium sessiliflorum (Poir.) H.O. Yates (Chasmanthieae) form a clade (BP = 57–100%). Loudetiopsis kerstingii (Pilg.) Conert + Danthoniopsis dinteri (Pilg.) C.E. Hubb.  and Thysanolaena latifolia (Roxb. ex Hornem.) Honda (Thysanolaeneae) + Centotheca lappacea (L.) Desv. (Centotheceae) (BP = 100%) are sister groups. Paniceae, Paspaleae, Andropogoneae and Arundinelleae are each monophyletic, and relationships among these four tribes, which compose the core Panicoideae, are maximally supported in all trees: Paniceae is sister to Paspaleae + (Andropogoneae + Arundinelleae)  . Howevernelleae) .A. prionodes (Arthraxoninae) and Zea mays L. (Tripsacinae) sister to the rest of the tribe. Coix lacryma-jobi L. (Coicinae) and Rottboellia cochinchinensis (Lour.) Clayton (Rottboelliinae) are sister taxa (BP = 93–100%), Coicinae + Rottboelliinae and Ischaemum afrum (J.F. Gmel.) Dandy (Ischaeminae) form a clade (BP = 87–100%), and this three-subtribe clade is sister to the rest of Andropogoneae. The two species of Sorghum Moench are sister taxa (BP = 100%), Sorghum and Saccharum cv. NCo310 are sister taxa in all trees (BP = 87–100%) except R, and these plus Imperata cylindrica (L.) P. Beauv. form a clade (BP = 72–100%). Eulalia aurea (Bory) Ku and Sorghastrum nutans (L.) Nash are sister taxa in all trees (BP = 62–100%) except F, but relationships among these species, the rest of Saccharinae, and Andropogoninae are discordant among trees . Within Andropogoninae, the following lineages diverge successively with strong support: Diheteropogon amplectens var. catangensis (Chiov.) Clayton + Hyparrhenia subplumosa Stapf, Themeda Forssk. sp., Iseilema macratherum Domin and Capillipedium venustum (Thwaites) Bor + Bothriochloa alta (Hitchc.) Henrard.Within Andropogoneae, most aspects of relationships are strongly supported, including the successive branching of ng trees . SaccharDigitaria exilis (Kippist) Stapf (Anthephorinae) is sister to the rest of the tribe (BP = 88–100%) (Alloteropsis J. Presl (three species), Amphicarpum muhlenbergianum (Schult.) Hitchc., Echinochloa P. Beauv. (two species) and Oplismenus hirtellus (L.) P. Beauv. form a clade  in all trees except Y, in which Amphicarpum muhlenbergianum and Thyridolepis xerophila (Domin) S.T. Blake (Neurachninae) form a clade (BP = 97%). Within Boivinellinae, relationships among the four genera are discordant among trees  Hitchc. are sister taxa (BP = 100%). The remaining taxa of Paniceae form a clade in all trees  Gould & C.A. Clark (Dichantheliinae) and T. xerophila (Neurachninae) are sister taxa (BP = 98–100%) in all trees except Y (see above). The two species of Panicum L. and Whiteochloa capillipes (Benth.) Lazarides form a clade , but Panicum is not monophyletic because Panicum capillare L. and W. capillipes are sister taxa (BP = 97–100%). Eriochloa meyeriana (Nees) Pilg. and Urochloa reptans (L.) Stapf are sister taxa (BP = 100%), and these species plus Megathyrsus maximus (Jacq.) B.K. Simon & S.W.L. Jacobs form a clade . Cenchrus americanus (L.) Morrone and the three species of Setaria P. Beauv. form a clade  in all trees, but Setaria is monophyletic only in trees E and F (BP = 98–100%). In all other trees, Setaria geminata (Forskk.) Veldkamp is sister to Cenchrus americanus +  P. Beauv. + Setaria viridis (L.) P. Beauv.). Cenchrinae and Melinidinae are sister taxa (BP = 100%), and Panicinae and Cenchrinae + Melinidinae form a broader clade (BP = 98–100%).Within Paniceae, 88–100%) . Alloterng trees . AlloterAxonopus fissifolius (Raddi) Kuhlm. and Paspalum L. (three species) form a clade . Otachyrium versicolor (Döll) Henrard and Steinchisma laxum (Sw.) Zuloaga are sister taxa (BP = 100%), and these plus Plagiantha tenella Renvoize form a clade . Coleataenia prionitis (Nees) Soreng and Oncorachis ramosa (Zuloaga & Soderstr.) Morrone & Zuloaga form a clade  Cope (Centropodieae), Neyraudia reynaudiana (Kunth) Keng ex Hitchc. (Triraphideae), Eragrostideae, Zoysieae and Cynodonteae diverge successively, with maximum support for all branches  and Uniola paniculata L. (Unioliinae). Zoysieae (BP = 100%) comprises Zoysia macrantha Desv. (Zoysiinae) and three species of Sporobolus R. Br. nom. cons. . Sporobolus michauxianus (Hitchc.) P.M. Peterson & Saarela and Sporobolus maritimus (Curtis) P.M. Peterson & Saarela are sister taxa (BP = 100%). The eight sampled subtribes of Cynodonteae form a clade in all trees (BP = 100%). Bouteloua Lag.  + Distichlis Raf.  and Hilaria Kunth  form a clade corresponding to supersubtribe Boutelouodinae. Melanocenchris abyssinica (R. Br. ex Fresen.) Hochst. and Halopyrum mucronatum (L.) Stapf form a clade , as do the two species of Triodia R. Br. . Trichoneura grandiglumis (Nees) Ekman (Trichoneurinae) and Dactyloctenium aegyptium (L.) Willd. (Dactylocteniinae) are sister taxa (BP = 68–100%). Within Eleusininae (BP = 100%), Oxychloris scariosa (F. Muell.) Lazarides + Eustachys glauca Chapm. (BP = 100%) and Chloris barbata Sw. form a clade (BP = 100%), and relationships among this clade, Astrebla pectinata (Lindl.) F. Muell. ex Benth and Leptochloa pluriflora (E. Fourn.) P.M. Peterson & N. Snow are discordant among trees  there are 10 clades with support ≥70% that in tree Y (excluding positively selected sites) are unsupported. Reciprocally, in tree Y there are 11 clades with support ≥70% that in tree W are unsupported. Furthermore, two cases of strong conflict between trees W and Y can be attributed specifically to inclusion or exclusion of positively selected sites: relationships among Among our analyses, we also compared phylogenies inferred from partitions with all plastome coding loci against exclusively noncoding partitions. This diversity of loci across the functional groups of the plastome would again be expected to reduce any particular selection bias, but possibly at the expense of increasing the noise to signal ratio. Consistent with this observation is that the removal of selected sites in our analyses did not introduce extensive topological incongruities and that a greater range of support values for the included clades was observed.The robust sister group relationship between Pooideae and Bambusoideae is congruent with most previous studies of multiple plastid genes and plastomes  as well incertae sedis, and the temperate Arundinarieae with a single subtribe) and one of herbaceous bamboos   . Bamboo ae clade . In the ae clade . Phylogeae clade .Pariana form a maximally supported clade in our trees, the genus is not monophyletic in plastid and ITS trees in Eremitis, including one now recognized in Parianella Hollowell, F.M. Ferreira & R.P. Oliveira  as sister to the rest the tribe is congruent with other studies (I. wilsonii (clade VIII) is moderately to strongly supported as sister to clade IV in the two complete plastome trees including gapped sites. In the earlier plastome trees, I. wilsonii is also sister to clade IV (I. wilsonii is unresolved within Arundinarieae (Seven of the twelve major lineages (clades I–XII) of Arundinarieae are represented in the plastome trees, which in many cases are better resolved and supported than trees based on a few plastid regions. Within the tribe, only 12 clades of two or more taxa are weakly to strongly supported in all plastome trees, indicating weak or conflicting signal in some of the plastome partitions analyzed. Placement of  studies , but som studies . This saclade IV , but againarieae . In the inarieae , but we inarieae .Gelidocalamus tessellatus and Shibataea kumasaca is congruent with the plastid tree in Ferrocalamus Hsueh & Keng f., Shibataea Makino ex Nakai and Sasa Makino & Shibata form a clade that excludes G. tessellatus  was similarly resolved in earlier plastome trees (Arundinaria spp. and Sasa veitchii) and Sino-Japanese clade   and ML trees based on plastomes and taxon sampling comparable to the current study , is congruent with other plastid trees  and with Pleioblastus maculatus resolved as its sister group, is also identified in the plastome tree in P. maculatus + I. sinica + O. shiuyingianum form a moderately to strongly supported clade . Neither of these conflicting clades is identified in other plastid trees  . Supportnt study , and weant study . The clame trees , in whic a clade  or form  a clade  and in B a clade . The thr a clade . Recoverid trees  but not id trees . In plasubclade” . The Aciid trees . We are T. spathiflorus (clade VII) sister to the rest of the clade in nine plastome trees here is congruent with one earlier plastome tree  and C. longiusculus (clade III) in all plastome trees except the two based on noncoding data, one of which weakly supports clades III and VII as sister taxa, is congruent with other studies  within clade V are poorly supported in the plastome trees, as in previous studies of plastid and GBSSI sequences in which species of Drepanostachyum Keng f. and Himalayacalamus Keng. f., neither of which is monophyletic, form a clade of unresolved affinity within clade V  sister to a maximally supported clade comprising Arthrostylidiinae and Guaduinae is congruent with other studies (Olmeca Soderstr. and Otatea (McClure & E.W. Sm.) C.E. Calderón & Soderstr. are strongly supported sister taxa in most trees, but in tree R, Olmeca and Guadua Kunth form a moderately supported clade. The former topology was previously identified in a plastome study  form a clade sister to a lineage of Oryzopsis asperifolia, Ampelodesmos mauritanicus (Ampelodesmeae), which is a polyploid reticulate species with Duthieeae and Stipeae that obtained its plastome from a stipoid grass  with respect to the rest of the subfamily in the plastome trees are congruent with previous few-gene plastid studies, and the robust support in the plastome trees for the respective branches is in many instances stronger than in few-gene trees . Morpholid grass , and Pipsampling .Psathyrostachys Nevski are included in analysis, Triticeae is paraphyletic because Bromeae is included within it  Link and Brachypodium boissieri Nyman, based on their affinities to the annual species and the core perennial clade in a two-gene plastid tree  and better supported than few-gene plastid trees  Eig, Thinopyrum Á. Löve, Lophopyrum Á. Löve, Crithopsis Jaub. & Spach.) are part of the lineage  MacKey), T. turgidum and T. aestivum “Chinese Spring,” whose relationships conflict strongly among the plastome trees. Close relationships among multiple subspecies of T. turgidum (sometimes recognized at species level) and T. aestivum “Chinese Spring” were found in earlier plastome  Jaub. & Spach, A. sharonensis, A. longissima, A. kotschyi and Aegilops searsii Feldman & Kislev , and Alopecurus arundinaceus (Alopecurinae) + Phleum alpinum (Phleinae) + Poa palustris (Poinae) has been identified in other plastid studies  and all other partitions, which identify Poa palustris and A. arundinaceus as strongly supported sister taxa. Although it is unclear which of the two highly supported topologies is accurate, there is sufficient variation in complete plastomes to robustly resolve relationships among these closely related genera compared to earlier plastid studies in which relationships among clades including these three genera were unresolved and/or poorly supported  and Airinae (Helictochloa hookeri) are strongly supported sister taxa in three plastome trees. This topology conflicts with the combined ITS and plastid tree in Helictochloa bromoides (Gouan) Romero-Zarco (as Avenula bromoides (Gouan) H. Scholz) is sister to a Dacytilidinae + Cynosurinae clade, and Holcus L. + Echinaria Desf. (Sesleriinae) are sister to a clade including taxa of Airinae and Deschampsia P. Beauv. The plastome topology also conflicts with the β-amylase tree in Helictochloa Romero-Zarco is currently classified in subtribe Airinae, few-gene plastid and nuclear analyses indicate the genus is not allied with other taxa of the subtribe  to clarify circumscription of the subtribe.Holcinae  do not form a clade  and Parapholiinae  is congruent with earlier plastid and nuclear ribosomal analyses  spikelets. Our results corroborate previous support for the monophyly of Oryzoideae including Streptogyna, as the subfamily is maximally supported in all plastome trees except the two based on noncoding regions, in which support for the same topology is lower (BP = 87–88%), indicative of some conflict in the noncoding partition relative to the rest of the plastome. Nevertheless, there is robust support in the plastome trees for Streptogyna being sister to the rest of the subfamily.Clarification of the evolutionary affinities of the amphi-Atlantic genus togyneae . It shartogyneae , interpr support . Kelchne grasses , as inceOP clade  and as aOP clade , 2017. SStreptogyna are different, however, in nuclear trees. In a phylogeny based on phytochrome B, Streptogyna is sister to the BOP clade  clade is weakly supported , form a clade, and this clade is robustly placed as sister to the rest of the subfamily except Streptogyna. This topology is congruent with other plastid trees, although not all included Streptogyna  is the most important food crop worldwide, there has been extensive phylogenetic research on the ca. 22 wild and two cultivated species of the genus. Although the 11 plastomes included here have all been published elsewhere, they have not all been combined in a single phylogenomic analysis. The relationships among the species of Oryza in our plastome trees are mostly congruent with similar plastome trees  is present in the gapped sites of the noncoding alignment, which when combined with coding region data seemingly override the conflicting signal in the latter partition. Recent data from the nuclear genome provides further insight into the Zizaniinae evolutionary tree, even though the nuclear trees are discordant, in part, with plastome trees. In trees based on 15 individual nuclear genes, relationships among taxa of Zizaniinae varied considerably and were strongly discordant with each other and with the plastid topologies here and elsewhere, whereas when the same 15 genes were analyzed together, Chikusichloa and Potamophila formed a clade sister to the rest of the subtribe , similar to gap-stripped results in Aristida L., Sartidia De Winter and Stipagrostis Nees, compared to It is surprising that the two trees of complete plastomes excluding gapped sites provide no  or only weak  support for the panicoid-sister topology because (1) this partition includes noncoding regions excluding gapped sites (dataset R), which, when analyzed separately, strongly support the panicoid-sister topology; and (2) this partition includes coding regions that, when analyzed separately, do not provide support greater than 50% for any particular topology. Nevertheless, it is possible there is discordant signal in coding regions that might be contributing to reduced support for the panicoid-sister topology when combined with noncoding regions excluding gapped sites. Indeed, the alternative topology in tree F, even though only weakly supported, supports the idea of discordant signal in coding regions. Furthermore, the lack of support greater than 50% for any topology in tree X differs from the ML tree in Choice of outgroup in a phylogenetic analysis can affect inferences of the location of the root of a clade , especiandhF and phyB data  Verboom, Danthonia californica Bol., Tribolium hispidum (Thunb.) Desv., Tenaxia guillarmodiae (Conert) N.P. Barker & H.P. Linder and Rytidosperma Steud. (two species). This topology is congruent with and better supported than the few-gene phylogenetic tree on which the current classification of the subfamily is based  and three genera incertae sedis (Elytrophorus spicatus (Willd.) A. Camus, and subtribe Molininae includes four genera and is represented here by Hakonechloa macra (Munro) Honda and Phragmites australis (Cav.) Trin. ex Steud., as in Arundinoideae includes Arundineae, with four genera and represented here by ae sedis . CrinipiMicraira F. Muell., Eriachneae (monogeneric) by Eriachne Eck-Boorsb. and Isachneae (six genera) by Isachne R. Br. As in other studies, Micraireae is sister to Eriachneae + Isachneae , represented here by sachneae . Of the sachneae , plastomincertae sedis  . This la a tribe , were hi a tribe .incertae sedis are recognized in tribe Andropogoneae  and C. lacryma-jobi  + R. cochinchinensis  that is sister to Andropogoninae + Saccharinae in the plastome trees has also been found in other plastid studies (Zea) diverge successively as sisters to the rest of the tribe, consistent with the better-sampled nuclear tree in The strongly supported clade comprising  studies , but was studies . At the incertae sedis genus Reynaudia Kunth. Our sampling includes two genera of Paspalinae , three of Otachyriinae  and two of Arthropogoninae (Coleataenia Griseb. and Oncorachis Morrone & Zuloaga). Monophyly of each subtribe is robustly supported in the plastome trees, and despite our limited taxon sampling, the strong support for a sister group relationship between Arthropogoninae and Otachyriinae is an improvement on earlier studies with greater taxon sampling but less sequence data per taxon, in which relationships among the three subtribes were unresolved  to be part of this clade and moderately to strongly supported as sister to Dichantheliinae + Neurachninae. In a combined nuclear (phyB) and plastid (ndhF) tree, however, Sacciolepis is embedded in Panicinae  as was inferred earlier  and the loci sequenced are among those with the greatest number of positively selected sites ; the plastome data were obtained from a different accession. The inflorescence and spikelet morphologies of Whiteochloa C.E. Hubb. are more similar to those of Panicinae than Cenchrinae. Setae, which are synapomorphic for Cenchrinae (hence the common name “Bristle clade”), are notably absent from Whiteochloa  shows characters that are diagnostic for Whiteochloa. However, two duplicates of the voucher J. Risler 1804  are also consistent with the current concept of W. capillipes. (3) W. capillipes is actually a complex of hybrids between species of Panicinae and Cenchrinae. The direction of the cross would determine which of two possible plastome haplotypes (Panicinae or Cenchrinae) was captured, depending on the female parent, whereas the morphological phenotypes of the reciprocal hybrids might be similar. Nuclear sequences of Panicinae and Cenchrinae, which have not been obtained to date, would be needed to test this third point and determine the identities of parent species. (4) There was a labeling mix-up or contamination somewhere in the extraction or sequencing process. This could be clarified by re-extracting and sequencing both specimens.Four possible explanations for this discrepancy are: (1) The use of plastid loci, which have high numbers of positively selected sites, skewed previous phylogenetic analyses. Such phylogenies, especially when based on a single gene, are susceptible to selection artifacts (see above). (2) One of the two plants was misidentified, although it is not clear which other Australian grasses might be mistaken for Cenchrus L. and Setaria, of which we sampled three species, form a clade, as in Cenchrus and Setaria italica + Setaria viridis form a maximally supported clade, whereas in two trees Setaria is strongly supported as monophyletic and sister to Cenchrus. Although the species treated here as Setaria geminata has been recognized in the genus Paspalidium Stapf, many authors have included it in Setaria  from China and South America, Ixophorus unisetus (J. Presl) Schltdl., Zuloagaea bulbosa (Kunth) E. Bess, Stenotaphrum secundatum  Kuntze and Uranthoecium truncatum (Maiden & Betche) Stapf (Subtribe Cenchrinae includes some 24 genera  and most Setaria . In our e) Stapf . SamplinAmphicarpum sister to Oplismenus + Echinochloa), and ML and BI analyses (Oplismenus sister to Amphicarpum + Echinochloa). Relationships among these genera similarly differ among our plastome trees. In nuclear phylogenies, Echinochloa is not part of the Boivinellinae clade  P. Beauv. and Pseudolasiacis leptolomoides (A. Camus) A. Camus  and Oplismenus, and relationships among these taxa were weakly supported in their plastid tree. Our trees include Amphicarpum, Oplismenus, two species of Echinochloa and three species of Alloteropsis. The latter genus is of particular interest to evolutionary biologists  Stapf sister to Alloteropsis paniculata (Benth.) Stapf + Alloteropsis papillosa Clayton; the other comprising Alloteropsis angusta and Alloteropsis semialata. Relationships among the four genera, however, vary among the plastome trees. Similar uncertainty at the base of the Boivinellinae tree is present in other studies based on fewer gene regions but greater taxon sampling  (Triraphis R. Br.), two of the three subtribes of Eragrostideae  and both subtribes of Zoysieae . The relationships we find among the three sampled species of Sporobolus (Sporobolinae), of which two were previously recognized in the genus Spartina Schreb., are congruent with previous studies  and seven genera bal rank . The sucbal rank . However Barker) . Relatio Barker) . Our sam studies , 2014c.Distichlis and Hilaria are sister taxa. Based on a phylogeny using seven plastid and ITS regions, these three subtribes are part of a larger clade recognized as supersubtribe Boutelouodinae, also including subtribes Allolepiinae, Jouveinae, Kaliniinae, Muhlenbergiinae, Scleropogoninae, Sohnsiinae and Traginae (Cynodonteae is a large and variable tribe consisting of 94 genera  for whicTraginae . In PeteTrichoneura grandiglumis), Tripogoninae , Triodiinae (Triodia stipoides (S.W.L. Jacobs) Crisp & Mant and Triodia wiseana C.A. Gardner), Eleusininae  and Dactylocteniinae (Dactyloctenium aegyptium). The sister group relationship between Trichoneurinae and Dactylocteniinae is congruent with and better supported than recent plastid trees in which Neobouteloua Gould + Dactyloctenium (L.) Willd. (Dactylocteniinae) are sister to a large but weakly supported clade including Orcuttiinae and a clade recognized as supersubtribe Gouiniodinae comprising subtribes Cteniinae, Farragininae, Gouiniinae, Hubbardochloinae, Perotidinae, Trichoneurinae and Zaqiqahinae, of which only Trichoneurinae is sampled here (Triodia (Triodiinae), one of which (T. stipoides) was previously recognized in Monodia S.W.L. Jacobs, is congruent with other studies  is congruent with the combined plastid + ITS tree in The branching order of the five sampled taxa of Eleusininae is congruent with the plastid tree that included 28 genera of the subtribe in id trees . HoweverHalopyrum Stapf in a molecular study and found it to be nested within Tripogoninae, where it is now classified. Our results are consistent with this, as we find H. mucronatum and M. abyssinica to be sister taxa, although we lack samples of five additional genera that have been placed in the subtribe  in Poeae group 2; Avenella in Airinae) and genera that have not been included in any phylogenetic study, such as Phyllorachis (Phyllorachideae); (3) sample plastomes representing all lineages of genera known to be para- or polyphyletic and which have not yet undergone taxonomic revision; (4) sample taxa of particular evolutionary interest, such as Aristida longifolia, the C3 sister to all other species of Aristida, which are C4. Attention should also be paid to stems and loop micro inversions and data bias from complementary base pair mutations in stems, in mechanical alignments. Attention to nuclear genomes is needed beyond all this, to clarify reticulation events, as those are not evident in plastid trees.Taxon density is key for resolving contradictory branching patterns. A robust plastome phylogeny of Poaceae with complete genus-level sampling is likely attainable within five years, dependent on the availability of rare species or those restricted to remote areas. Such a phylogenetic tree would contribute to further improvement, refinement and confidence in classification of grasses, and would facilitate broad characterization of the molecular evolutionary histories of plastomes in grasses, allow more precise divergence estimations, and produce complete descriptions of microstructural events and rare genomic changes in the plastome. We suggest that future work on plastome phylogeny of Poaceae should aim to (1) sample representatives from the few tribes  and many subtribes for which plastomes have not yet been published, as well as the few genera that remain unplaced to a tribe or a subtribe, most of which have no sequence data available; (2) complete genus-level sampling of plastomes in all tribes and subtribes, focusing first on genera whose affinities within tribes, subtribes and subfamilies are unclear in existing phylogenies  rbcL, ndhF, matK, and trnK intron, excluding gapped sites and including positively selected sites. (C) rbcL, ndhF, matK, and trnK intron, including gapped sites and excluding positively selected sites. (D) rbcL, ndhF, matK, and trnK intron, excluding gapped sites and positively selected sites. (E) Plastome coding regions, including gapped sites and positively selected sites. (F) Plastome coding regions, excluding gapped sites and including positively selected sites. (G) Plastome coding regions, including gapped sites and excluding positively selected sites. (H) Plastome coding regions, excluding gapped sites and positively selected sites. (Q) Plastome noncoding regions, including gapped sites. (R) Plastome noncoding regions, excluding gapped sites. (W) Complete plastomes, including gapped sites and positively selected sites. (X) Complete plastomes, excluding gapped sites and including positively selected sites. (Y) Complete plastomes, including gapped sites and excluding positively selected sites. (Z) Complete plastomes, excluding gapped sites and positively selected sites. In each subfigure, the bootstrap tree is on the left, with bootstrap proportions indicated along branches, and the best ML tree is on the right.Letters identifying each of the 14 trees correspond to the data partitions as identified in Click here for additional data file.10.7717/peerj.4299/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.4299/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.4299/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.4299/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.4299/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.4299/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.4299/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.4299/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.4299/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.4299/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.4299/supp-13Supplemental Information 13Click here for additional data file.10.7717/peerj.4299/supp-14Supplemental Information 14Click here for additional data file.10.7717/peerj.4299/supp-15Supplemental Information 15Click here for additional data file.10.7717/peerj.4299/supp-16Supplemental Information 16Click here for additional data file.10.7717/peerj.4299/supp-17Supplemental Information 17Click here for additional data file.10.7717/peerj.4299/supp-18Supplemental Information 18Clades were first named based on tree X, a reference tree. Unique clades in non-reference trees were also named and scored across all trees.Click here for additional data file.


Aspergillus terreus isolates from six university hospitals in South Korea. Thirty one isolates of A. terreus, comprising 15 respiratory and 16 ear isolates were assessed. Microsatellite genotyping was performed, and genetic similarity was assessed by calculating the Jaccard index. Virulence was evaluated by Galleria mellonella survival assay. All 31 isolates were susceptible to itraconazole, posaconazole, and voriconazole, while 23 (74.2%) and 6 (19.4%) showed amphotericin B (AMB) minimum inhibitory concentrations (MICs) of ≤ 1 mg/L and > 4 mg/L, respectively. Notably, respiratory isolates showed significantly higher geometric mean MICs than ear isolates to AMB (2.41 vs. 0.48 mg/L), itraconazole (0.40 vs. 0.19 mg/L), posaconazole (0.16 vs. 0.08 mg/L), and voriconazole (0.76 vs. 0.31 mg/L) . Microsatellite genotyping separated the 31 isolates into 27 types, but the dendrogram demonstrated a closer genotypic relatedness among isolates from the same body site (ear or respiratory tract); in particular, the majority of ear isolates clustered together. Individual isolates varied markedly in their ability to kill infected G. mellonella after 72 h, but virulence did not show significant differences according to source (ear or respiratory tract), genotype, or antifungal susceptibility. The current study shows the marked diversity of clinical isolates of A. terreus in terms of antifungal susceptibilities, genotypes and virulence in the G. mellonella model, and ear isolates from Korean hospitals may have lower AMB or triazole MICs than respiratory isolates.We analyzed the antifungal susceptibility profiles, genotypes, and virulence of clinical  Aspergillus species has increased among immunocompromised patients due to their considerably high rates of morbidity and mortality . There was no significant difference in the survival rate of G. mellonella infected with non-WT and WT isolates . Also, the mean survival rate of G. mellonella did not differ according to the source of the infecting larvae   were more frequent among respiratory versus ear isolates .The mean survival rates of to 72.5% , Fig 2.  0.6227) . VirulenG. mellonella model of clinical A. terreus isolates obtained during laboratory-based surveillance of six Korean hospitals. Although A. terreus is known to be intrinsically resistant to AMB [A. terreus are not uncommon, especially in ear specimens. Notably, we report for the first time that isolates from the same body site (ear or respiratory tract) have greater genetic similarity, and respiratory isolates are significantly less susceptible to AMB and triazoles than ear isolates. However, there was no difference in virulence between respiratory and ear isolates in a G. mellonella model.This study characterized the antifungal susceptibilities, genetic relatedness, and virulence in a t to AMB –13, our A. terreus has intrinsic resistance to AMB, with elevated MICs [A. terreus isolates have a wide spectrum of AMB MIC ranging from 0.125 mg/L to 16 mg/L [A. terreus isolates worldwide have low AMB MICs (≤ 1 mg/L) [A. terreus isolates in Korea. The high proportion of AMB susceptible A. terreus isolates in the present study may be due to the high proportion of ear isolates in this Korean collection. Ear isolates showed significantly lower geometric mean AMB MICs (0.48 mg/L) than respiratory isolates (2.41 mg/L). The respiratory isolates exhibited AMB MICs of 0.5 to 8 mg/L, in line with other reports [Many investigations show that ted MICs –13, but  16 mg/L , 28, 29. 1 mg/L) , 11, 21. reports , 31.A. terreus isolates from clinical and environmental sources in India [A. terreus species complex in Austria [A. terreus isolates in our collection are highly diverse and unlikely to originate from a common source. Why A. terreus isolates from ear cultures are more susceptible to AMB and triazoles than those from respiratory cultures is unclear, but the explanation may involve the genetic relatedness of the isolates. Notably, the average genetic similarity value of ear isolates was significantly higher than that of the total 31 isolates. The average genetic similarity value of respiratory isolates was also significantly higher than that of the total 31 isolates. Taken together, these findings suggest that Korean A. terreus isolates exhibit considerable genetic diversity without a predominant genotype, but ear isolates from South Korean hospitals comprise closely related genotypes with lower AMB or triazole MICs than respiratory isolates.Of two previous studies using microsatellite typing, one reported a high genetic diversity in a large collection of geographically diverse in India , while t Austria . In the A. terreus, a multicenter study used this method to conduct genotyping on clinical and environmental isolates from India, North America and Europe [A. terreus from South Korean hospitals.After Rougeron et al.  introducd Europe . In thatd Europe . When weA. terreus has been a focus of interest [in vivo murine model of disseminated aspergillosis, AMB-susceptible strains  were more virulent than AMB-resistant strains  [G. mellonella model, two AMB-resistant strains showed lower virulence than three AMB-susceptible strains [G. mellonella facilitates in vivo screening of A. terreus. In the present study, the virulence of 31 clinical isolates of A. terreus in the G. mellonella model varied markedly, which confirms a previous report that AMB susceptibility in A. terreus is not necessarily associated with loss of in vivo virulence [G. mellonella infected with AMB-susceptible and -resistant isolates   was observed. The reasons for these discrepant results are unclear, but the results might have been influenced by the composition of the isolates. One possible explanation is that the previous three studies tested a few selected isolates, but we assessed 31 clinical A. terreus isolates with a wide range of AMB MICs. Another possible explanation is the AMB-susceptible strains  tested in the previous three studies were from respiratory cultures, while all AMB-susceptible strains in the present study were from ear cultures because all respiratory isolates had an AMB MIC ≥ 1 mg/L.The virulence of AMB susceptible strains of interest –16. In a 4 mg/L) . Similar strains , suggestirulence . Howeverctively) . Rather,A. terreus is found frequently in the environment [A. terreus are less common than those caused by A. fumigatus [A. terreus infections are associated with a lower rate of a response to AMB therapy and are more frequently fatal than A. fumigatus infections [A. terreus isolates vary markedly, and that respiratory isolates comprise diverse genotypes with higher AMB or triazole MICs than ear isolates. Because the main portal of entry and site of infection for Aspergillus in human hosts is the respiratory tract, respiratory isolates of A. terreus may be more likely to cause severe infections than ear isolates. Although we did not find a difference in virulence between respiratory and ear isolates, a significantly higher proportion of respiratory isolates than ear isolates was able to kill infected G. mellonella. These characteristics of A. terreus isolates were also supported by clinical information  or amphotericin B resistance (B).(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Androgen receptor (AR) is a promising therapeutic target for breast cancer. However, its prognostic value remains controversial in triple negative breast cancer (TNBC). Here we present a meta-analysis to investigate the correlation between AR expression and TNBC prognosis.p< 0.001), but more lymph node metastases (p < 0.01). AR positivity was associated with prolonged disease free survival , but had no significant impact on overall survival . No difference in survival existed between subgroups using different AR or estrogen receptor cutoff values.Thirteen relevant studies with 2826 TNBC patients were included. AR positive rate was 24.4%. AR+ patients tended to have lower tumor grade (Literature search was performed in Pubmed, Embase and Cochrane Central Register of Controlled Trials databases to identify relevant articles on AR and TNBC prognosis. Fixed- and random-effect meta-analyses were conducted based on the heterogeneity of included studies. Heterogeneity and impacts of covariates were further evaluated by subgroup analyses and meta-regression.AR positivity is associated with lower risk of disease recurrence in TNBC. Further clinical studies are warranted to clarify its prognostic role on TNBC recurrence and survival. There w= 0.270) .p < 0.01, I-square = 58.9%). By random-effect meta-analysis, AR positivity was not associated with any improvement in OS   , study population , ethnicity (p = 0.054), ER cutoffs  or statistical analysis   nor high cutoff 5 or 10%) subgroup revealed AR-related benefit for OS  Figure . There w= 0.165) . Removal= 0.165) .Meta-regression was performed on outcome data. None of the covariates showed statistically significant effects on DFS or OS .p = 0.537, OS p = 0.945).Funnel plots Figure  for DFS As a novel therapeutic target, AR was widely investigated in many pre-clinical and clinical studies for TNBC , 19, 26.et al. found that AR+ was beneficial to DFS in TNBC subgroup , but not for OS  . St. St27]. 61-1.32) . Kim et ctively) , 23, 24,et al. which demonstrated AR expression incurred better prognosis irrespective of ER co-expression [Several studies revealed the antagonizing effect of AR against ER signaling depended on the relative levels of these two steroids receptors. They competed for co-regulatory molecules, resulting in opposite effects on cancer cell proliferation . Withoutpression . With ERSubgroup analyses showed remarkable decrease in the heterogeneity of OS in subgroups of whole-cohort TNBC patients and high AR/ER cutoffs , suggestOur studies had several limitations. First, it based on population data other than individual patient data, and restrained our ability to conduct analyses for LN metastases and other covariates. Second, all the studies were retrospective. It could potentially increase certain bias, such as selection bias. Third, we were unable to identify correlation between AR and molecular intrinsic subtypes of TNBC, especially for LAR subtype which may be helpful to clarify AR prognostic value.In conclusion, although variability and heterogeneity existed among included studies, AR expression was associated with lower risk of disease recurrence. Since AR had a relatively high prevalence in TNBC, evaluating AR status could provide additional information on prognosis and AR-targeting therapy could be another option for chemotherapy resistant TNBC in both neoadjuvant and metastatic settings. Future clinical trials based on different molecular subtypes of TNBC are warranted to further clarify its prognostic value and efficacy of AR-targeting therapy.We searched the following databases for relevant studies: PubMed , Embase  and Cochrane Central Register of Controlled Trials . The following medical subject headings, Embase Emtree terms and keywords were used: ‘Breast Neoplasms’, ‘Triple Negative Breast Neoplasms’, ‘Breast Cancer’, ‘Triple Negative Breast Cancer’, and ‘Androgen Receptor’. There was no limitation on languages or regions of publications. Reference lists of all the relevant articles were manually screened to ensure the sensitivity of the literature search.et al. [To be eligible, studies had to meet the following inclusion criteria: TNBC patients, or studies that provided data on TNBC as a subgroup; assessment of AR expression in primary breast cancer tissue; available data of OS or DFS on patient subgroups with different AR level. Exclusion criteria included: metastatic disease; review, meta-analysis, editorial, letter or conference abstract. Two independent reviewers (C.J. Wang and B. Pan) evaluated eligibility of studies according to the above criteria. Full text of the potentially relevant studies were obtained and reviewed for inclusion by the same two reviewers. Disagreement was resolved by consensus . Inter-reviewer agreement was assessed by Kappa Statistics according to Higgins et al. The quality of the included studies was assessed according to the STROBE checklist for Cohort Studies . Each itp-value) were extracted from tables or text of included studies, or estimated from Kaplan-Meier curves where applicable [Data were collected using a predesigned data extraction form by two reviewers (C.J Wang and B. Pan). Characteristics of included studies were extracted for stratified analyses and meta-regression. Survival data .Publication bias was assessed by Begg's test and symmetry of funnel plot. Statistical heterogeneity was assessed by Cochrane's Q and I-square statistics. Cochrane's Q test with s et al. . Meta-re


Paradiplozoon homoion adult stage using a combined approach of confocal laser scanning and electron microscopy, with emphasis on the forebody and hindbody. Special attention is given to structures involved in functional adaptation to ectoparasitism, i.e. host searching, attachment and feeding/metabolism. Our observations indicate clear adaptations for blood sucking, with a well-innervated mouth opening surrounded by sensory structures, prominent muscular buccal suckers and a pharynx. The buccal cavity surface is covered with numerous tegumentary digitations that increase the area in contact with host tissue and, subsequently, with its blood. The buccal suckers and the well-innervated haptor (with sclerotised clamps controlled by noticeable musculature) cooperate in attaching to and moving over the host. Putative gland cells accumulate in the region of apical circular structures, pharynx area and in the haptor middle region. Paired club-shaped sacs lying laterally to the pharynx might serve as secretory reservoirs. Furthermore, we were able to visualise the body wall musculature, including peripheral innervation, the distribution of uniciliated sensory structures essential for reception of external environmental information, and flame cells involved in excretion. Our results confirm in detail that P. homoion displays a range of sophisticated adaptations to an ectoparasitic life style, characteristic for diplozoid monogeneans.Diplozoidae (Monogenea) are blood-feeding freshwater fish gill ectoparasites with extraordinary body architecture and a unique sexual behaviour in which two larval worms fuse and transform into one functioning individual. In this study, we describe the body organisation of  Monogenea Bychowsky 1937 are among the most species-rich groups of fish parasites . Blood-fParadiplozoon homoion is a generalist diplozoid species parasitising a number of cyprinid fish and, as such, represents a suitable model parasite for a range of studies. To date, most studies have concentrated on Paradiplozoon spp. genetic characterisation and identification, its life cycle under experimental conditions  octane  or with Hoechst and mounted in VECTASHIELD® . Gomori trichrome staining was used for 3D visualisation of sclerotised structures [Diplozoid worms were flat-fixed between microscopic slides in freshly prepared 4% paraformaldehyde in 0.1 phosphate buffered saline (PBS) for 4 h at 4°C and then transferred into fresh fixative. For the labelling of filamentous actin (F-actin), specimens were washed for 24 h in antibody diluent (AbD) containing 0.1 M PBS, 0.5% Triton X-100, 0.1% bovine serum albumin and 0.1% NaNructures . The hydructures .All slides were examined and documented using an Olympus IX81 microscope equipped with a laser-scanning FluoView 500 confocal unit (Olympus FluoView 4.3 software) or an Olympus BX60 microscope with FluoView 1.26 (Fluoview 2.0 software). Some confocal micrographs were processed using Fiji software .4. The samples were subsequently dehydrated through a graded ethanol series and dried in a Pelco CPD II critical point drying apparatus  using liquid CO2. The dried samples were finally mounted on aluminium stubs with double-sided adhesive tape or disks, coated with gold in a Polaron E5100 sputter coating unit  and examined in a MIRA 3 TESCAN SEM operating at 15 kV.For scanning electron microscopy (SEM), the specimens were first washed several times in tap water to remove any fish mucus, fixed in either hot 4% formaldehyde or 4% glutaraldehyde at 4°C for 24 h and postfixed for 1 h in 1% OsOP. homoion in the adult stage typically resembles a letter X. This X-shaped body is comprised of two forebodies and two hindbodies along with the haptors of the two fused individuals  symmetrically arranged in the middle of the forebody apical end Figs  and 5A, Immunofluorescent labelling of α-tubulin localised the forebody nerve cords, and especially the longitudinal dorsal and ventral cords and transverse connective cords . The α-tThe forebody surface is obviously folded, creating transverse ridges with shallow pits . The tegIn Platyhelminthes, the protonephridial excretory system generally consists of terminal organs, i.e. flame cells consisting of terminal cells and adjacent canal cells, and a system of interconnected collecting ducts opening to the body surface . HoweverThe major part of the hindbody surface is covered with transverse, discontinuous tegumentary folds , while pThis staining revealed the presence of large gland-like cells with prominent nuclei. Majority of these cells are randomly scattered over the body, with increased occurrence in the forebody and haptor regions . Of specEctoparasitic diplozoid monogeneans exhibit a range of unique biological characteristics and sophisticated functional adaptations to their bloodsucking life style. The most significant of these structures are those related to host searching, attachment, feeding/metabolism, pairing and protection against host responses ,17. As pP. homoion resembles that of some other monogenean species, e.g. Allodiscocotyla diacanthi [Empleurosoma pyriforme [Eudiplozoon nipponicum [E. nipponicum [Paranaella luquei [Marcorgyrodactylus congolensis [Diclidophora merlangi [P. homoion.The tegument is the primary surface for host-parasite interaction during the search for a suitable niche on the host’s gills and also plays an essential role during contact with other individuals during pairing and reproduction. With its shallow pits, the tegument of iacanthi , Empleuryriforme  and Eudipponicum . Similarpponicum ,17, but a luquei , Marcorggolensis  or Diclimerlangi , we obseP. homoion are likely related to surface perception of host tissue during attachment and food intake (blood sucking). They could also function as tangoreceptors with a tactile function or rheoreceptors for perception of water current during the parasite’s orientation [P. homoion sensory cilia has been described previously [P. homoion cilium anchoring , facilitating selection of suitable attachment sites on the surface of the host . We showentation . The ulteviously . A compashown in  with tana soleae  indicatea soleae  envelopia soleae . The fixa soleae . We assua soleae ,51,54–56E. nipponicum [E. nipponicum [E. nipponicum, two nerve cords terminated in this area [E. nipponicum termed these as gigantic cells [P. homoion haptor may be also associated with adhesive function as proposed earlier [In addition to the uniciliated sensory structures, the apical part of the forebody has two round projections  similar pponicum . Using Cpponicum . In E. nhis area , while ohis area , 51. Phahis area . Goto in earlier .E. nipponicum [E. nipponicum [The paired bowl-shaped buccal suckers in the mouth cavity appear to be the main attachment organs of the forebody. These suckers also help in sucking the host’s blood. As also observed in pponicum , the U-spponicum , except P. homoion as short, irregular lamellae [E. nipponicum [The buccal cavity surrounding the suckers is covered with numerous foliate and tubular digitations up to the rim of the mouth, thereby expanding the parasite’s surface in contact with the host tissue. These tubular digitations have previously been described for lamellae . Similarpponicum . These dE. nipponicum [E. nipponicum visible under SEM , an adjacent canal cell and a system of associated collecting ducts serving for osmoregulation, as previously described for other flatworms [S. mansoni [P. homoion, we achieved good results by using double fluorescent labelling, which stained the ciliated tuft (i.e. the ‘flame’) and cilia root for α-tubulin and the barrel for F-actin. The nucleus of the flame cell was easily detected using Hoechst counterstaining. While it is likely that the collecting ducts were also visualised using α-tubulin labelling . Var mansoni . Unfortu mansoni ), it was mansoni  correspoP. homoion plays an important role in attachment to host tissue and moving on the host’s gills. Compared to E. nipponicum, where prominent folds and species-typical lobular extensions play an important role in attaching this robust parasite within the gill lamellae [P. homoion has less prominent tegumentary folds and three distinct lobes on the haptor rather than lobular extensions. These lobes are highly mobile and equipped with a conspicuous three-layer musculature innervated with abundant uniciliated sensory structures on the surface. Besides the clamps and putative adhesive glands, these lobes serve presumably for attaching the parasite to the host’s gills. In contrast to E. nipponicum, in which the lateral hindbody is equipped with non-ciliated papillae  [P. homoion had similar non-ciliated papillae clustered above the clamps. Such non-ciliated papillae have also been reported in Entobdella soleae [The hindbody of lamellae ,17, P. hstimuli) , P. homoa soleae ,61. It ia soleae . AlternaParadiplozoon sp. using the Hörens trichrome [E. nipponicum [Diplozoon paradoxum [E. nipponicum [As the primary structures fixing the parasite to the host surface, the clamps are organised in a similar manner to those in other Diplozoidae; i.e. four pairs of clamps in two parallel rows. While the sclerotised parts of the clamps exhibit autofluorescence and also are easily recognised under a light microscope equipped with Nomarski differential interference-contrast NDIC; , the starichrome . Phalloipponicum ,25 and Daradoxum . This muP. homoion. Overall, P. homoion exhibits a number of sophisticated functional adaptations to its ectoparasitic life-style, similar to those previously described for other helminth parasites . The. TheP. hes (e.g. . The par


Physcomitrella patens) to 63 (Glycine max). The phylogenetic analysis shows that the canonical Aux/IAA proteins can be generally divided into five major clades, and the origin of Aux/IAA proteins could be traced back to the common ancestor of land plants and green algae. Many truncated Aux/IAA proteins were found, and some of these truncated Aux/IAA proteins may be generated from the C-terminal truncation of auxin response factor (ARF) proteins. Our results indicate that tandem and segmental duplications play dominant roles for the expansion of the Aux/IAA gene family mainly under purifying selection. The putative nuclear localization signals (NLSs) in Aux/IAA proteins are conservative, and two kinds of new primordial bipartite NLSs in P. patens and Selaginella moellendorffii were discovered. Our findings not only give insights into the origin and expansion of the Aux/IAA gene family, but also provide a basis for understanding their functions during the course of evolution.The plant hormone auxin plays pivotal roles in many aspects of plant growth and development. The auxin/indole-3-acetic acid (Aux/IAA) gene family encodes short-lived nuclear proteins acting on auxin perception and signaling, but the evolutionary history of this gene family remains to be elucidated. In this study, the Aux/IAA gene family in 17 plant species covering all major lineages of plants is identified and analyzed by using multiple bioinformatics methods. A total of 434 Aux/IAA genes was found among these plant species, and the gene copy number ranges from three ( The plant hormone auxin (IAA) regulates various processes in plant growth and development, including apical dominance, gravitropic response, embryogenesis, organogenesis, vascular differentiation, axis and lateral root initiation, and shoot elongation ,2. AuxinThe molecular model of auxin signaling was elucidated in the past two decades, despite this plant hormone having been investigated for nearly a century . Aux/IAAArabidopsis thaliana, the IAA1/AXR5 mutant shows a variety of growth defects related to auxin insensitivity phenotypes [IAA3/SHY2 has effects on auxin homeostasis and lateral root formation [A. thaliana IAA7/ARX2 have negative phototropism and gravitropism defects [AXR3-1/IAA17-1 has enhanced apical dominance and a decreased number of lateral roots, which are associated with an increased auxin response [IAA16 inhibits plant growth and causes decreased response to auxin [Oryza sativa), plants with overexpression of OsIAA4 are characterized by dwarfism, increased tiller angles, reduced gravity response, and less responsiveness to auxin [OsIAA6 shows abnormal tiller outgrowth [OsIAA23 is referenced to the postembryonic maintenance of the quiescent center [Recently, several Aux/IAAs have been characterized in model plants, indicating that Aux/IAAs participate in diverse cellular and developmental processes. In enotypes . Loss-oformation . Inflore defects . The mutresponse . The gaito auxin . In monoto auxin . The losutgrowth . OsIAA23t center . Severalt center ,33.A. thaliana [O. sativa [Populus trichocarpa [Zea mays [Vitis vinifera [Medicago truncatula [Eucalyptus grandis [G. max [Solanum tuberosum [Aux/IAA gene families have been identified in several plant species, such as 29 members in thaliana , 31 in O. sativa , 35 in Pchocarpa , 34 in ZZea mays , 26 in Vvinifera , 17 in Muncatula , 26 in E grandis , 63 in G [G. max , and 26 uberosum . Some Auuberosum . Moreoveuberosum ,44,45,46uberosum . In the uberosum . These guberosum .To gain a better understanding of auxin signaling in the evolution of plants, the evolutionary history of Aux/IAAs needs to be investigated. In this study, we identify 434 Aux/IAA proteins from 17 plant species covering major lineages of plants and we perform bioinformatics analyses to reveal the evolutionary mechanisms of the Aux/IAA gene family. Our study demonstrates that canonical Aux/IAA genes can generally be divided into five groups, and that they have a variety of domain compositions. Additionally, our results indicate that tandem and segmental duplication events play dominant roles in the expansion of the Aux/IAA gene family. Moreover, it is found that both changes in coding sequence and rearrangements in domain organization contribute to the functional diversity of plant Aux/IAAs.A. thaliana and 31 O. sativa Aux/IAAs as the query to perform local BLAST searches against the proteomes of 17 plant species. Then, these candidates were submitted to the NCBI conserved domain database. The presence of AUX_IAA domains was necessary to confirm their identities. As a result, we finally identified 434 Aux/IAA genes . It was found that the copy number of Aux/IAA genes was uncorrelated with genome size. For example, G. max and P. abies contained 56,044 and 58,587 genes, respectively; however, the Aux/IAA gene number in G. max (63) was twice that of P. abies (31)  was more than that of A. thaliana . The regression coefficient analysis was conducted , indicating that the Aux/IAA gene copy number and gene loci number had a weak correlation (t-test with these two variables were found to be remarkably different (p < 0.001). In other words, the genome size was not directly proportional to the number of Aux/IAA genes. Plant species might undergo some forces to prompt diversity in this gene family [P. patens and S. moellendorffii had smaller Aux/IAA gene families compared to higher plants, typically gymnosperms and angiosperms. This phenomenon indicated that the Aux/IAA gene family experienced a dramatic boost from lower plants to higher plants. The extra genes might have functional redundancy and/or generate new functions, leading to adaption to changing environments [In order to identify Aux/IAA genes in plantae, we first used 29 AA genes . The num0 and 40 . There wies (31) . There wrelation . The rese family . In addironments ,51,52,53In order to investigate the motif composition of Aux/IAA proteins, MEME (Multiple Em for Motif Elicitation) analysis was applied to find conservative motifs in Aux/IAA proteins. Consequently, five putative motifs corresponding to Aux/IAAs’ four conservative domains were found: motifs 5, 3, and 2 were related to domains I, II and III, respectively; domain IV contained two independent motifs: motif 1 and motif 4 . Motif 5A. thaliana had both motif 1 and 2. However, in G. max, Glyma.10G000700.1 only had motif 1, and Glyma.13G140500.1 only had motif 2. According to the distribution patterns of conservative motifs, Aux/IAA proteins were generally classified into 11 subgroups. The proteins having all five motifs were regarded as canonical Aux/IAAs and grouped into type I. These canonical proteins had four complete domains, which were confirmed by multiple sequences alignment. In type II, 16 proteins lacked motif 4, which led to incomplete domain IV , Clade B , Clade C , Clade D , and Clade E . Bryophyte Aux/IAA sequences were loosely associated with Clade A, which means that Clade A could probably be traced back to the origin of the plant kingdom. Both P. patens and S. moellendorffii had two canonical Aux/IAA sequences, while higher plants contained more gene copies. This gene family might have undergone a dramatic boost during plant evolution. P. equestris (monocot) and aquatic plant U. gibba (eudicots) sequences were on the edge of other monocots and eudicots in this phylogenetic tree. It was speculated that these two species had a longer evolutionary distance than the other species. The origin of the Aux/IAA gene could be dated back at least to the origin of land plants and the major Aux/IAA and ARF lineages originated before the monocot-eudicot divergence [The evolution analysis of the Aux/IAA family was crucial to understanding auxin signaling in plants. However, it was hard to unveil the origin of this gene family, considering the large gene family size and functional redundancy among gene family members. To solve this question, an unrooted tree was built to reveal the phylogenetic relationship of the Aux/IAA gene family among eudicots, monocots, gymnosperms, and lower eukaryotic plants. The phylogenetic tree containing 434 Aux/IAA sequences from 17 species was weakly supported by the bootstrap test because of the many divergent sequences. In order to improve the quality of phylogenetic analysis, we chose 253 canonical Aux/IAA sequences, accounting for 58.3% of the total proteins, to perform phylogenetic analysis . In addiG. max contained the largest number of sister pairs (17 pairs). There were six and four sister pairs in Z. mays and A. thaliana, respectively. In addition, we found one sister Aux/IAA gene pair each in P. patens, S. moellendorffii, P. abies, and S. tuberosum, while there were no sister pairs in A. trichopoda, O. sativa, B. distachyon, P. persica, or R. communis in the entire tree. The remaining Aux/IAA gene pairs were from cross species, and six sister pairs existed between O. sativa and B. distachyon Aux/IAA genes. We also observed three kinds of sister pairs from cross species  at one time. The non-canonical Aux/IAA genes were excluded from the phylogenetic tree as they have a large sequence diversity that would cause less reliability with low bootstrap values. We assumed that these non-canonical Aux/IAA genes tended to be clustered into additional clades.In the phylogenetic tree, significant sequence similarities were observed among the Aux/IAA gene family, which indicated that these genes probably arose from gene duplication events . AdditioA. thaliana), monocot (O. sativa), gymnosperm (P. abies), and lower eukaryotic plant (P. patens and S. moellendorffii) sequences by ClustalX. It was shown that the structure of the PB1 domain was composed of three α-helices (α1–α3) and five β-sheet strands (β1–β5) (The auxin/indole-3-acetic acid (Aux/IAA) family and the auxin response factor (ARF) family are two transcription factor families that regulate the auxin signaling pathway in the nucleus. In the absence of auxin, the Aux/IAAs carboxyl-terminal PB1 domain specifically binds to the corresponding homologous domain in the ARFs, leading to the repression of ARF activity ,20,54. I (β1–β5) . The remP. patens), indicating that the Aux/IAA and ARF gene families’ differentiation event appeared before the divergence of green algae and land plants.Interestingly, although the sequences conservation in domains III/IV of both ARF and Aux/IAA proteins was noticeable, the difference of this region between two gene families was also observed. The most noticeable difference between ARFs and IAA proteins in this domain was that almost all Aux/IAA proteins had the “GDVP” motif linking β3 and α2, while this motif was “GDDP” in almost all ARF proteins . One morS. tuberosum [G. max [M. truncatula [P. patens and 180 amino acids in 438333 from S. moellendorffii). They were possibly byproducts of the origins of Aux/IAAs and ARFs, because these sequences could not be found in higher plants.A phylogenetic tree was built based on the full-length amino acid sequences of Aux/IAA and ARF proteins. ARFs were highlighted with green; canonical Aux/IAAs were highlighted with blue; and a novel group of N-terminal truncated Aux/IAAs was highlighted with orange . The N-tuberosum , Glyma.0 [G. max , and MtIuncatula . TherefoP. patens and S. moellendorffii contained three and nine Aux/IAA genes, respectively, and each of them had one segmentally-correlated gene pair. P. abies was generally regarded as having undergone numerous duplications [A. thaliana, 22 (75.86%) out of a total of 29 Aux/IAA genes were segmentally correlated, six (20.69%) Aux/IAA genes were tandemly correlated, and three genes  took part in both segmental and tandem duplications. Among 31 Aux/IAA genes in monocot O. sativa, 19 (61.29%) and seven (22.58%) Aux/IAA genes were from segmental and tandem duplication, respectively. Two Aux/IAA genes (LOC_Os03g43400.1 and LOC_Os12g40890.1) were involved in both segmental and tandem duplications. Interestingly, we found segmental duplicated pairs in lower plants (P. patens and S. moellendorffii), which were encoded by canonical Aux/IAAs in these species  and two WGD events (legume WGD and Glycine WGD) [It was found that the expansion patterns observed in the Aux/IAA gene family coincided with whole-genome duplication events (WGD) during evolution. Two WGD events that occurred in progymnosperms might contribute to today’s Aux/IAA gene family size and diversity . During ine WGD) . During ine WGD) . Numerouine WGD) ,47,71. Hine WGD) ,71,72. TP. abies and the Potri.006G161200/Potri.006G161400 gene pair in P. trichocarpa, implying that most of these Aux/IAA paralogous gene pairs had evolved under purifying selection. The Ka/Ks values of MA_10430843g0010/MA_10430843g0020 gene pairs and Potri.006G161200/Potri.006G161400 gene pairs were 1.6720 and 1.1686, respectively  were found to undergo positive selection after being separated by duplication, which might improve the fitness of the organism in a new environment.Estimation of the molecular evolution rates of Aux/IAAs is an essential basis to understand the evolution process of the Aux/IAA gene family in the plant kingdom. The Ka/Ks values were calculated to estimate the molecular evolutionary rates of the paralogous gene pairs among Aux/IAAs. It was found that most Aux/IAA pairs evolved at a Ka/Ks value lower than one, as expected for the MA_10430843g0010/MA_10430843g0020 gene pair in ectively , indicatectively . Aux/IAAectively . The Stuelection . Accordielection . TherefoAux/IAA proteins are cell nucleus-located transcriptional repressors. IAA-GFP (green fluorescent protein) and IAA-YFP (yellow fluorescent protein) fusion proteins could be exclusively localized to the nucleus ,16,49. TS. moellendorffii), the detection of this conservative KR basic doublet failed. In the second bipartite NLS (Part 2), the stretches of K/R residues RxxRK represented this region with substitution within lysine (K) and/or arginine (R). Distinctively, this region in lower plants P. patens and S. moellendorffii was QxxRK (the first R in RxxRK was substituted by Q) and KxxNK , respectively. These amino acid substitutions might alter the protein secondary structures, as well as change their subcellularly-located patterns and functions compared with those in higher plants. To reach a more solid conclusion, we also searched Aux/IAA protein sequences from another two already fully-sequenced species of Bryophyta , and their NLS data are added to M. polymorpha’s Aux/IAA proteins also failed, while S. fallax’s second bipartite NLS (Part 2) was presented by RxxRK with substitution within lysine (K) or/and arginine (R) in the last lysine (K) site. An SV40-like NLS was composed of KxRxxRK in plants, with substitution within lysine (K) or/and arginine (R), especially in the first lysine (K) site  for the first arginine (R) mutated to cysteine (C). Fifty-two percent of Aux/IAA proteins had bipartite NLS Part 1; 43.4% of proteins lacked this region; and the rest of the Aux/IAA proteins had degenerated bipartite NLS Part 1. Meanwhile, 49.2% of Aux/IAA proteins contained the bipartite NLS Part 2; 15.6% of Aux/IAA proteins lack this region; and the remaining Aux/IAA proteins contained degenerated bipartite NLS Part 2 (The first part (Part 1) of the bipartite NLS was relatively conservative in plants, depending on the research. However, in Pteridophyta ((K) site . Many AuS Part 2 .The SV40-like NLS was shown to be more conservative than the bipartite NLS, as 71.7% of Aux/IAAs contained intact SV40-like NLS, and 20.9% of proteins had degenerated SV40-like NLS. Recent research indicated that the Aux/IAA proteins contained degenerated NLS in both bipartite NLS and SV40-like NLS, which were specifically targeted to the nucleus . The absChlamydomonas reinhardtii, Physcomitrella patens, Selaginella moellendorffii, Picea abies, Amborella trichopoda, Phalaenopsis equestris, Zea mays, Oryza sativa, Brachypodium distachyon, Solanum tuberosum, Utricularia gibba, Arabidopsis thaliana, Gossypium raimondii, Prunus persica, Glycine max, Populus trichocarpa, Ricinus communis), multiple searches were conducted. First, the complete proteomes of these species were downloaded from the Phytozome website . Then, A. thaliana and O. sativa Aux/IAA protein sequences were used as queries to perform local BLAST searches with a −3 expect (E) threshold. Second, the conserved domains of all obtained sequences were examined by the NCBI conserved domain database (Available online: http://www.ncbi.nlm.nih.gov/cdd). The presence of the AUX_IAA domain was used to confirm the identity of Aux/IAA genes (In order to identify candidate Aux/IAA genes in 17 species (AA genes . ARF genhttp://memesuite.org/) was used to identify motifs under the following parameters: (1) each motif site distributes zero or one occurrence per sequence; (2) optimum motif widths are between six and 50; (3) the maximum number of motifs is five. To identify the finer domains and motifs, the Aux/IAA protein sequences were aligned by using ClustalX (2.1) with the following parameters: protein weight matrix, Gonnet 250; gap penalty at opening, 10; gap penalty at extension, 0.1.The MEME web server  were converted into MEGA format by using MEGA 7. The neighbor-joining (NJ) tree was built under the p-distance model and G + I distribution. Bootstrap analyses with 1000 replicates were performed for support estimation. Maximum likelihood (ML) trees built under the Jones-Taylor-Thornton (JTT), WAG, and Poisson models were also conducted to prove these results.https://phytozome.jgi.doe.gov/pz/portal.html). Tandem duplication gene pairs were identified by comparing their physical locations on chromosomes and their homology (more than 50%). We defined that paralogous genes that exist in the same chromosome within a 50-kb physical distance were tandem duplicated pairs [We used the annotation information of the Aux/IAA genes on the Plant Genome Duplication Database to determine their chromosomal locations  in the Aux/IAA proteins are conservative except sequence variation in several lower plants. Typically, two kinds of new primordial bipartite NLSs in