Annals of Botany 109: 1341– 1357, 2012 doi:10.1093/aob/mcs054, available online at www.aob.oxfordjournals.org Phylogenetic relationships and generic delimitation of Eurasian Aster (Asteraceae: Astereae) inferred from ITS, ETS and trnL-F sequence data Wei-Ping Li1,*, Fu-Sheng Yang2, Todorka Jivkova3 and Gen-Shen Yin4 1 College of Life Sciences, Hunan Normal University, Changsha 410081, China, 2State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China, 3Department of Structural Biology, Faculty of Natural Sciences, Shumen University, Shumen 9700, Bulgaria and 4Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, The Chinese Academy of Sciences, Kunming 650204, China * For correspondence. E-mail lwp@hunnu.edu.cn Received: 18 September 2011 Returned for revision: 7 November 2011 Accepted: 13 February 2012 Published electronically: 19 April 2012 † Background and Aims The classification and phylogeny of Eurasian (EA) Aster (Asterinae, Astereae, Asteraceae) remain poorly resolved. Some taxonomists adopt a broad definition of EA Aster, whereas others favour a narrow generic concept. The present study aims to delimit EA Aster sensu stricto (s.s.), elucidate the phylogenetic relationships of EA Aster s.s. and segregate genera. † Methods The internal and external transcribed spacers of nuclear ribosomal DNA and the plastid DNA trnL-F region were used to reconstruct the phylogeny of EA Aster through maximum parsimony and Bayesian analyses. † Key Results The analyses strongly support an Aster clade including the genera Sheareria, Rhynchospermum, Kalimeris (excluding Kalimeris longipetiolata), Heteropappus, Miyamayomena, Turczaninowia, Rhinactinidia, eastern Asian Doellingeria, Asterothamnus and Arctogeron. Many well-recognized species of Chinese Aster s.s. lie outside of the Aster clade. † Conclusions The results reveal that EA Aster s.s. is both paraphyletic and polyphyletic. Sheareria, Rhynchospermum, Kalimeris (excluding K. longipetiolata), Heteropappus, Miyamayomena, Turczaninowia, Rhinactinidia, eastern Asian Doellingeria, Asterothamnus and Arctogeron should be included in Aster, whereas many species of Chinese Aster s.s. should be excluded. The recircumscribed Aster should be divided into two subgenera and nine sections. Kalimeris longipetiolata, Aster batangensis, A. ser. Albescentes, A. series Hersileoides, a two-species group composed of A. senecioides and A. fuscescens, and a six-species group including A. asteroides, should be elevated to generic level. With the Aster clade, they belong to the Australasian lineages. The generic status of Callistephus should be maintained. Whether Galatella (including Crinitina) and Tripolium should remain as genera or be merged into a single genus remains to be determined. In addition, the taxonomic status of A. auriculatus and the A. pycnophyllus–A. panduratus clade remains unresolved, and the systematic position of some segregates of EA Aster requires further study. Key words: Asteraceae, Astereae, ETS, Eurasian Aster, generic delimitation, infrageneric classification, ITS, molecular phylogeny, trnL-F. IN T RO DU C T IO N Aster sensu lato (s.l.; Asterinae, Astereae, Asteraceae) has been a taxonomic dumping ground for large numbers of morphologically similar but distantly related taxa (Noyes and Rieseberg, 1999; Dorn, 2003). Aster s.l. occurs mainly in the Northern Hemisphere in both Eurasia (EA) and North America (NA) and is estimated to comprise 250 – 1000 species (Ling et al., 1985; Nesom, 1994b; Ito and Soejima, 1995; Noyes and Rieseberg, 1999). Based primarily on achene morphology and cytology, Nesom (1994b) segregated NA Aster species from Aster s.l. and redistributed them among generic segregates Symphyotrichum, Doellingeria, Eucephalus, etc. At the same time, he kept the remainder, about 180 species, as Aster sensu stricto (s.s.), typified by A. amellus. Consequently, Aster, containing approx. 180 species, is restricted to the Northern Hemisphere of the Old World. Internal transcribed spacer (ITS) sequence phylogenetic data (Noyes and Rieseberg, 1999) support the viewpoint of Nesom (1994b) that a fundamental difference exists between NA and EA Aster. Furthermore, ITS data indicate that EA Aster is nested in the Southern Hemisphere grade and does not form a monophyletic group (Noyes and Rieseberg, 1999, Brouillet et al., 2001, 2009b; Fiz et al., 2002), and African Aster should be separated from Aster s.s. (Brouillet et al., 2009b). The classification and phylogeny of EA Aster have remained poorly resolved, however, because of insufficient sampling in these studies. The circumscription of EA Aster has confused botanists for several decades. Many taxonomists have adopted a broad definition of EA Aster. In Flora Europaea, Merxmüller et al. (1976) maintained Doellingeria, Galatella, Crinitaria (the name Crinitaria is a synonym of Galatella and species that are considered part of Crinitaria should be included in Crinitina) and Tripolium in Aster. Similarly, Grieson (1975) accepted Aster s.l. in Flora of Turkey and the East Aegean Islands because he did not recognize Kemulariella and Tripolium as segregate genera. In Flora of Japan, Ito and # The Author 2012. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com 1342 Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) Soejima (1995) merged Tripolium as section Tripolium into Aster, placed Heteropappus in section Pseudocalimeris, included Kalimeris within section Asteromoea, and associated Doellingeria and Miyamayomena into section Teretiachaenium. Other taxonomists have favoured a narrow generic concept of EA Aster and have recognized small genera endemic to eastern Asia. Tamamschyan (1959) segregated two new genera (Kemulariella and Conyzanthus) from Aster and recognized many small genera such as Doellingeria, Kalimeris, Asterothamnus, Krylovia, Turczaninowia, Galatella, Linosyris (¼ Crinitina) and Tripolium. Czerepanov (1995) followed Tamamschyan (1959) except that he placed Galatella and Linosyris under the genus name Crinitaria (¼ Crinitina). Nesom (1994a, b) made Aster largely equal to EA Aster s.s. and EA Aster s.l. almost equal to sub-tribe Asterinae Dumort. Ling et al. (1985) treated Chinese Asterinae in the narrow sense of Aster, recognizing generic status for Gymnaster (¼ Miyamayomena), Kalimeris, Callistephus, Heteropappus, Doellingeria, Turczaninowia, Krylovia (¼ Rhinactinidia), Asterothamnus, Galatella, Linosyris (¼ Crinitina), Arctogeron and Tripolium. These treatments were followed completely for floras of Chinese provinces (e.g. Zhuang, 2004; Lin, 2007). Despite this, Chinese Aster s.s. remains a large genus with approx. 100 species, of which 75 are endemic to China (Fu, 1983; Ling et al., 1985; Chen, 1988, 1990; Zhu and Min, 1990; Li and Liu, 2002; Li and Zhang, 2004). Therefore, China, especially south-western China (the Qinghai – Tibetan and Yunnan – Guizhou Plateaux and Sichuan Province), is the diversity centre of Aster, as it is for many genera (Huang, 2011). Molecular markers, especially ITS and the external transcribed spacer (ETS) of 35S ribosomal DNA, have frequently been used to investigate phylogenetic relationships in Astereae (e.g. Noyes and Rieseberg, 1999; Lowrey et al., 2001; Markos and Baldwin, 2001; Cross et al., 2002; Fiz et al., 2002; Roberts, 2002; Lowell et al., 2003; Urbatsch and Roberts, 2003; Urbatsch et al., 2003; Roberts and Urbatsch, 2004; Karaman, 2006; Selliah and Brouillet, 2008; Andrus et al., 2009; Brouillet et al., 2009a,b; Karaman-Castro and Urbatsch, 2009; Vaezi and Brouillet, 2009). Molecular evidence implies that neither EA Aster s.l. nor EA Aster s.s. is monophyletic (Gu et al., 1994; Ito et al., 1995, 1998; Xiang and Semple, 1996; Noyes and Rieseberg, 1999; Fiz et al., 2002), but only a few species of EA Aster have been included in previous analyses. Although molecular data support a close relationship among Kalimeris, Heteropappus, Miyamayomena, Sheareria, Rhynchospermum and Aster s.s. (Ito et al., 1995, 1998; Noyes and Rieseberg, 1999; Fiz et al., 2002; Gao et al., 2009), the phylogenetic relationships among these genera are unresolved owing to limited taxon sampling of EA Aster s.s. Recently, 27 species of EA Aster s.l. were included in a phylogenetic analysis of Aster s.l. (Brouillet et al. 2009b), but no statistical support was presented for the clades of the ITS phylogenetic tree. To date, no molecular data have been provided for Turczaninowia, Krylovia, Asterothamnus and Arctogeron, and, in particular, Chinese Aster s.s. has not been phylogenetically studied using DNA sequences even though it represents the overwhelming majority of EA Aster s.s. Thus, a reliable phylogenetic analysis based on extensive taxon sampling is essential to determine the inter- and intrageneric relationships of EA Aster. Principally based on nuclear ribosomal DNA (nrDNA) ITS, ETS and plastid trnL-F sequence data of Sheareria nana, Rhynchospermum verticillatum and 62 species of EA Aster s.l., the present study aims to (1) reconstruct the phylogeny of EA Aster s.l.; (2) redelimit the genus Aster and discuss its infrageneric classification; and (3) discuss the systematic position of EA Aster segregates. M AT E R I A L S A N D M E T H O D S Generic circumscriptions and nomenclature of Astereae follow Nesom and Robinson (2007) except for Turczaninowia, which follows Ling et al. (1985), and Crinitina, which is substituted for Crinitaria (a synonym of Galatella). The name Aster setchuenensis follows the International Plant Names Index. The division of phylogenetic lineages of Astereae refers to Brouillet et al. (2009b). Voucher DBY9206 was deposited in the Wenzhou University Herbarium (WZU) and the others in the Hunan Normal University Herbarium (HNNU; see the Appendix). Taxon sampling Seventy-six species of Astereae and three outgroup species were collected from China and Bulgaria and examined for sequence variations in nrDNA ITS, ETS and plastid DNA trnL-F (GenBank accession numbers are given in the Appendix). The vouchers of all accessions were identified using published keys and compared with herbarium specimens in the Institute of Botany, Chinese Academy of Sciences Herbarium (PE), Northwest Agriculture and Forestry University Herbarium (WUK), Sichuan University Herbarium (SZ), Chengdu Institute of Biology Herbarium (CDBI), HNNU, Herbarium of Kunming Institute of Botany, the Chinese Academy of Sciences (KUN), the Herbarium of the South China Botanical Garden, Chinese Academy of Sciences (IBSC), Guangxi Institute of Botany Herbarium (IBK), Institute of Botany, Jiangsu Province and Chinese Academy of Sciences Herbarium (NAS), Guizhou Academy of Sciences Herbarium (HGAS), Central China Normal University Herbarium (CCNU), Wuhan Botanical Garden, Chinese Academy of Sciences Herbarium (HIB), Inner Mongolia University Herbarium (HIMC) and Fudan University Herbarium (FUS). Of the 76 species included in this study (see the Appendix), 41 represent three sections and 20 series of EA Aster s.s. (Ling et al., 1985; Chen, 1988; Li and Liu, 2002), 21 represent 12 segregate genera of EA Aster s.l., four generic groups of Nesom’s (1994b) Asterinae, and two recently recognized close relatives of EA Aster s.s. (S. nana and R. verticillatum; Fiz et al., 2002; Brouillet et al. 2009b; Gao et al., 2009). The data matrix for ITS comprises 110 accessions from 48 genera and 110 species of tribe Astereae (see the Appendix). Seventy-six accessions were newly sequenced, and the remaining 34 were obtained from GenBank (Appendix). Of the 110 accessions, 41 species belong to EA Aster s.s., 21 species are 12 separate genera of EA Aster s.l., one is Astereae incertae sedis, three are members of Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) Bellidinae or Grangeinae, and 44 represent the six phylogenetic lineages of Astereae (Brouillet et al. 2009b). These phylogenetic lineages of Astereae are the early diverging lineages (e.g. Madagaster madagascariensis, Felicia filifolia and Printzia polifolia), the palaeo-South American clade (e.g. Chiliotrichum diffusum), the New Zealand clade (e.g. Olearia covenyi), the Australasian lineages, the South American lineages (e.g. Baccharis neglecta) and the NA lineage (e.g. Conyza sumatrensis and Symphyotrichum subulatum). Brouillet et al. (2009b) divided the Australasian lineages into seven genus or species groups, whereas ten genus or species groups are, in fact, included in the depiction of the grouping (fig. 37.1 C in Brouillet et al. 2009b). In the current analysis, 19 species (Appendix) were sampled to represent these ten groups. Because Brouillet et al. (2009b) consider Olearia s.s. to be a sister to EA Aster, five species were sampled to represent sub-clades of the Olearia s.s. clade. In the combined matrix of ITS, ETS and trnL-F, 78 accessions from 25 genera and 78 species of tribe Astereae were included (Appendix). Seventy-six accessions were newly sequenced, and the remaining two were obtained from GenBank (Appendix). Of the 78 accessions (Appendix), 41 belong to EA Aster s.s., 21 belong to 12 segregate genera of EA Aster s.l., one is Astereae incertae sedis, and three are members of Bellidinae or Grangeinae. The remaining 12 accessions represent three phylogenetic lineages of tribe Astereae (Brouillet et al. 2009b), the palaeo-South American clade (e.g. C. diffusum), the Australasian lineages (e.g. two Myriactis spp.) and the NA lineage (seven species such as C. sumatrensis and S. subulatum). In all analyses, Chrysanthemum coronarium and Dendranthema indicum of tribe Anthemideae and Calendula officinalis of tribe Calenduleae were selected as outgroups for the rooting of the phylogenetic trees (Appendix) because in molecular phylogenetic analyses Anthemideae and Astereae are sisters, and Calenduleae is a sister to tribes Gnaphalieae, Anthemideae and Astereae (Panero and Funk, 2008; Garcia et al., 2010). DNA extraction, polymerase chain reaction (PCR) and sequencing Total genomic DNA was isolated from fresh leaf material or silica gel-dried leaves using a modified cetyltrimethylammonium bromide procedure (Doyle and Doyle, 1987). Amplification and sequencing were performed using the primers ITS1 and ITS4 (White et al., 1990) for the ITS region, Ast-8 (Markos and Baldwin, 2001) and 18S-ETS (Baldwin and Markos, 1998) for the ETS region, and c and f (Taberlet et al., 1991) for the plastid DNA trnL-F region (trnL UAA-trnL UAA-trnF GAA). The PCR mixture contained 1 mL (50 –100 ng) of sample DNA, 2 × 2 mL of primer (10 pmol), 5 mL of 10 × PCR buffer, 3 mL of Mg2+ (25 mM), 0.8 mL of deoxyribonucleotide triphosphate (each 25 mM), 0.5 mL of Taq DNA polymerase (5 U mL21) and sterile water for a final volume of 50 mL. The PCR parameters were as follows: initial denaturation for 4 min at 95 8C followed by 30 cycles of denaturation (95 8C, 1 min), annealing (56 8C, 40 s) and extension (72 8C, 1 min), and a final extension of 10 min at 72 8C. 1343 PCR products were purified using a UNIQ-10 Spin Column PCR Product Purification Kit (Sangon Biotech Co., Ltd, Shanghai, China) following the manufacturer’s instructions. Sequencing reactions were performed in both directions by Sangon Biotech Co., Ltd. Sequence alignment and phylogenetic analysis Boundaries of the ITS, ETS and trnL-F regions were determined through comparison with previously published sequences of tribe Astereae (Noyes and Rieseberg, 1999; Liu et al., 2002; Urbatsch et al., 2003). All DNA sequences were aligned initially using Clustal X1.83 (Jeanmougin et al., 1998) and then adjusted manually in BioEdit (Hall, 1999). The ITS region was analysed separately and in a combined data set with the ETS and trnL-F regions. The incongruence length difference test (Farris et al. 1994) was carried out to test the homogeneity between data sets using PAUP* version 4.0b10 with 1000 replicates. Maximum parsimony (MP) and Bayesian inference (BI) methods were performed for the data sets using PAUP* version 4.0b10 (Swofford, 2001) and MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003), respectively. In the MP analysis, characters were equally weighted and treated as unordered, gaps were treated as missing data, and a heuristic search was implemented with 1000 random additional sequence replicates and sub-tree pruning– regrafting branch swapping. Bootstrap analyses based on 1000 replicates with ten random additions per replicate were used to estimate the confidence of the clades. The MaxTrees setting in PAUP* was set to 5000 for the searches and bootstrap tests. For BI analysis of the ITS region and combined data set, the best-fitting model of each sequence partition (ITS1, ITS2, 5.8S, ETS, trnL-F intron, exon, the internal guide sequence) was determined using MrModeltest 2.2 (Nylander, 2004). The SYM + G model was chosen for the 5.8S region, and the GTR + I + G model for the ITS1, ITS2 and ETS regions. The GTR + G model was chosen for the intron and the internal guide sequence partitions of the trnL-F region and the K80 model for the exon partition. The Markov chain Monte Carlo algorithm was run for 1 000 000 generations, resulting in an overall sampling of 10 000 trees. The first 3000 trees were discarded as a conservation burn-in, and the remaining trees were used to construct the 50 % majority rule consensus tree. R E S ULT S Characterization of nucleotide data The aligned ITS sequence matrix of 110 taxa contained 689 base pairs, of which 394 were variable and 315 were potentially parsimony informative. Pair-wise distance within ingroup varied from 0 to 18.7 % (average ¼ 6.7 %). The incongruence length difference test indicated that the data sets were not significantly heterogeneous (P ¼ 0.01). Therefore, a combined analysis of the three regions was performed using PAUP* and MrBayes. The combined data set of 78 taxa consisted of 2313 positions, with 641 potentially parsimony-informative characters and 283 phylogenetically uninformative variable 1344 Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) 0·99/– 8 0·99/98 9 A –/63 0·99/– 10 1·00/98 –/54 1 ·00/97 11 1 ·00/98 12 13 14 1·00/93 15 16 1·00/91 –/0·89 –/67 0·98/100 0·98/94 0·96/62 0·96/56 0·94/– 0·95/94 0·95/– 0·95/– 20 1·00/100 0·98/54 0·97/86 –/75 0·99/78 –/54 0·89/– 23 1·00/92 22 0·89/58 0·99/62 0·90/57 0·99/93 0·99/97 1·00/– 0·99/– 0·99/100 The Aster clade (B, below) A. hersileoides A. nitidus A. asteroides A. flaccidus A. brachytrichus A. diplostephioides A. yunnanensis A. setchuenensis Myriactis nepalensis Myriactis wightii A. auriculatus A. pycnophyllus A. panduratus A. argyropholis A. lavanduliifolius A. albescens albescens Callistephus chinensis Kalimeris longipetiolata A. fuscescens A. senecioides Minuria macrorhiza A. batangensis Olearia tomentosa Olearia astroloba Olearia ballii Olearia cordata Olearia rudis Olearia calcarea Olearia ciliata Remya kauaiensis Camptacra gracilis Kippistia suaedifolia Minuria integerrima T. humile humile Calotis hispidula Brachyscome rigidula S. subulatum S. novi-belgii Eurybia sibirica Ma. tanacetifolia Boltonia asteroides Solidago decurrens Pentachaeta aurea Erigeron breviscapus Erigeron annus Conyza sumatrensis Chrysopsis mariana Astranthium integrifolium Doellingeria umbellata Baccharis neglecta Crinitina villosa Crinitina linosyris Galatella dahurica Tripolium vulgare Bellis perennis Dichrocephala auriculata Grangea maderaspatana Conyza japonica Madagaster madagascariensis P. camphorata camphorata Oritrophium hieracioides Felicia filifolia Mairia hirsuta Celmisia mackaui Olearia covenyi Pleurophyllum hookeri Chiliotrichum diffusum Nannoglottis delavayi Printzia polifolia C. coronarium Dendranthema indicum Calendula officinalis AL SEA AL AL NA SA SEA BE GR AIS BL PSA BL NZ PSA BL OG F I G . 1. The 50 % majority rule consensus tree from the Bayesian analysis of nuclear ribosomal DNA internal transcribed spacer sequences. (A) Bayesian posterior probabilities (≥0.89) and bootstrap values (≥50 %) are indicated above the branches; ‘– ’ indicates that Bayesian posterior probabilities are ,0.89 or bootstrap percentages are ,50 %. Some clades are indicated by numbers below the branch. Abbreviations: A., Aster; C., Chrysanthemum; S., Symphyotrichum. Triangles, ‘Kalimeris group’; squares, Doellingeria; circles, ‘Galatella group’. (B) The Aster clade (continued part of A). Bayesian posterior probabilities (≥0.89) and bootstrap values (≥50 %) are indicated above the branches; ‘–’ indicates that Bayesian posterior probabilities are ,0.89 or bootstrap percentages are ,50 %. Some clades are indicated by numbers below the branch. Abbreviations: A., Aster; As., Asterothamnus; H., Heteropappus; M., Miyamayomena; R., Rhynchospermum. See key for symbols. Some clades are indicated by numbers below the branch. Abbreviations of the lineages are identical to those given in the Appendix and are shown on the right side of the taxa. The labelled species are discussed in groups in the text. Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) 1·00/79 B 1·00/82 0·99/88 1·00/81 ‘Rhynchospermum group’ ‘Kalimeris group’ Turczaninowia Doellingeria ‘Asterothamnus group’ 0·89/– 1·00/76 –/60 1·00/99 1 0·93/62 1·00/99 1·00/100 2 0·94/– 1·00/97 5 0·99/– 1·00/100 1·00/93 6 0·96/– 3 From (A) 0·99/– 8 4 0·99/96 0·99/79 7 1·00/100 –/65 1345 H. altaicus millefolius SEA H. crenatifolius A. smithianus A. heterolepis A. vestitus A. souliei A. ageratoides lasiocladus A. dolichopodus A. mangshanesis A. oreophilus A. tongolensis Kalimeris integrifolia Kalimeris indica SEA Kalimeris indisa A. homochlamydeus A. handelii A. fanjingshanicus AL Sheareria nana A. baccharoides A. jishouensis AL R. verticillatum A. turbinatus M. piccolii M. simplex Rhinactinidia limonifolia SEA Rhinactinidia eremophila A. tataricus Turczaninowia fastigiata SEA A. taliangshanensis A. sampsonii A. alpinus A. maackii A. amellus M. angustifolius Arctogeron gramineum SEA As. fruiticosus As. centrali-asiaticus A. sikuensis A. poliothamnus A. falcifolius SEA Doellingeria scaber Fig. 1. Continued characters. Pairwise distance between sequences varied from 0.1 to 11.7 % (average ¼ 4.6 %). Phylogenetic analyses For convenience, some clades were numbered (Figs 1A, B and 2). Phylogenetic analyses using ITS and combined data sets yielded generally consistent phylogenetic trees (Bayesian trees; see Figs 1A, B and 2), although the BI and MP analyses based on the combined data generated trees with higher bootstrap support (BS) and Bayesian posterior probability (PP), and some clades (e.g. 17 and 18; Fig. 2) of the combined tree were unresolved in the ITS trees (Fig. 1A). The Aster clade (clade 8; Figs 1 and 2) with A. amellus (the type species of Aster) was strongly supported (PP ¼ 0.99 in Fig. 1; PP ¼ 1.00 and BS ¼ 98 in Fig. 2) by the ITS and combined data set analyses. Sheareria, Rhynchospermum and some EA Aster segregates such as Heteropappus, Kalimeris (excluding section Cordifolium), Miyamayomena, Turczaninowia, Rhinactinidia, Arctogeron, Asterothamnus and eastern Asian Doellingeria were deeply nested within the Aster clade (clade 8), whereas other segregates (e.g. Callistephus, Galatella, Crinitina, Tripolium and K. longipetiolata) and 17 species of Aster s.s. (e.g. A. nitidus, A. asteroides and A. panduratus) occurred in other clades and showed close (clade 7; Figs 1A and 2), remote (e.g. clade 18 in Fig. 1A; clade 22 in Fig. 2) or unresolved (e.g. clade 9 – 16; Fig. 1A) relationships with the Aster clade. Callistephus, K. longipetiolata, two Myriactis spp. and 15 Aster spp. formed a moderately supported clade (clade 18; PP ¼ 100; Fig. 2) that was unresolved in the ITS tree (Fig. 1A). Tripolium, Galatella and Crinitina constituted a well-supported clade (clade 22: PP ¼ 1.00, BS ¼ 92 in Fig. 1A; PP ¼ 1.00, BS ¼ 95 in Fig. 2) sister to Bellis perennis, but this relationship was weakly supported (clade 23: PP ¼ 0.89 in Fig. 1A; PP ¼ 0.91, BS ¼ 57 in Fig. 2). The monophyly of the NA clade (clade 20) was moderately to strongly supported in both phylogenetic analyses (PP ¼ 0.95 in Fig. 1; PP ¼ 1.00, BS ¼ 100 in Fig. 2). DISCUSSION Relationship between EA Aster and NA asters In this study, the ITS and combined data set analyses (Figs 1 and 2) clearly indicate that the Aster clade (clade 8 in Figs 1A and 2) is strongly supported (PP ¼ 0.99 in Fig. 1; PP ¼ 1.00, BS ¼ 98 in Fig. 2) in an unresolved Astereae polytomy (Fig. 1A) or is embedded within clade 19 which includes Myriactis (sub-tribe Lagenophorinae) of the Australian lineages (see Fig. 2), whereas NA Astereae forms a moderately to strongly supported clade (clade 20: PP ¼ 0.95 in Fig. 1; PP ¼ 1.00, BS ¼ 100 in Fig. 2). Therefore, EA Aster has no close relationship to NA asters. These results support the viewpoint of Nesom (1994b) that a fundamental difference exists between NA and EA Aster; they do not support the opinion of Xiang and Semple (1996) that Aster s.s. comprises not only EA taxa but also the segregate genus Eurybia and that 1346 Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) 1·00/100 H. altaicus millefolius H. crenatifolius A. smithianus A. heterolepis A. vestitus 100/73 A. souliei A. ageratoides lasicladus –/75 A. dolichopodus 1·00/74 A. mangshanensis A. oreophilus A. tongolensis 1·00/100 Kalimeris integrifolia 0·93/– Kalimeris indica Kalimeris indisa 1·00/100 A. homochlamydeus A. handelii A. fanjingshanicus Sheareria nana A. baccharoides A. jishouensis 1·00/– R. verticillatum A. turbinatus 1·00/84 M. piccolii M. simplex 1·00/100 Rhinactinidia limoniifolia Rhinactinidia eremophila A. tataricus Turczaninowia fastigiata A. taliangshanensis A. maackii A. sampsonii 0·97/– A. alpinus A. amellus M. angustifolius Arctogeron gramineum 1·00/62 1·00/100 As. fruticosus As. centrali-asiaticus 1·00/100 1·00/71 A. sikuensis A. poliothamnus A. falcifolius Doellingeria scaber A. hersileoides A. nitidus 0·93/61 A. asteroides A. flaccidus A. brachytrichus 1·00/98 10 1·00/72 1·00/100 A. diplostephioides A. yunnanensis A.setchuenensis 1·00/87 Myriactis nepalensis Myriactis wightii 1·00/100 A. auriculatus 11 1·00/– A. pycnophyllus A. panduratus 1·00/99 A. argyropholis 1·00/100 A. lavanduliifolius 12 A. albescens albescens Callistephus chinesis 13 Kalimeris longipetiolata 141·00/100 A. fuscescens A. senecioides 15 A. batangensis 16 1·00/100 S. subulatum 1·00/100 S. novi-belgii 1·00/62 Eurybia sibirica Solidago decurrens Erigeron breviscapus 1·00/100 0·99/– Erigeron annus Conyza sumatrensis Crinitina villosa –/81 1·00/97 Crinitina linosyris 1·00/95 Galatella dahurica 21 22 Tripolium vulgare Bellis perennis Dichrocephala auriculata Grangea maderaspatana Conyza japonica Chiliotrichum diffsum 1·00/100 C. coronarium Dendranthema indicum Calendula officinalis 1·00/66 1·00/100 1 2 1·00/68 5 1·00/100 1·00/– 3 6 1·00/98 8 0·99/54 17 4 1·00/92 7 1·00/100 9 0·95/– 19 1·00/– 18 1·00/99 –/100 1·00/– 1·00/100 20 0·91/57 23 1·00/100 SEA –/74 SEA AL AL SEA SEA SEA SEA AL SEA NA SEA BE GR AIS PSA OG F I G . 2. The 50 % majority rule consensus tree from the Bayesian analysis of the combined data set (nuclear ribosomal DNA internal and external transcribed spacer sequences and plastid genome DNA trnL-F sequences). Bayesian posterior probabilities (≥0.89) and bootstrap values (≥50 %) are indicated above the branches; ‘–’ indicates that Bayesian posterior probabilities are ,0.89 or bootstrap percentages are ,50 %. Some clades are indicated by numbers below the branch. Abbreviations: A., Aster; As., Asterothamnus; C., Chrysanthemum; H., Heteropappus; M., Miyamayomena; R., Rhynchospermum; S., Symphyotrichum. Abbreviations of the lineages are identical to those given in the Appendix and are shown on the right side of the taxa. Some species are labelled with the symbols shown in Fig. 1. Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) EA Aster is derived from NA Aster. Aster alpinus, distributed in both EA and NA, is deeply nested within the EA Aster clade (clade 8; see Figs 1B and 2), which implies that this species originated in EA and dispersed to NA. Relationship between EA Aster and Australasian lineages According to Brouillet et al. (2009b), Australasian lineages are part of a large polytomy at the crown of Astereae. Although our data sets did not include a large sample of Australasian taxa, the ITS tree (Fig. 1) included 19 sampled species that represented the ten genus or species groups of Australasian lineages of Brouillet et al. (2009b). The ITS tree (Fig. 1A) showed that the Aster clade (clade 8) is a clade of the large polytomy of the crown of Astereae, but it does not group with any of the Australian (e.g. Olearia astroloba and Remya kauaiensis), Hawaiian (Tetramolopium humile) or Asian (Myriactis) species of the Australasian lineages. Brouillet et al. (2009b) have proposed that EA Aster s.s. was a sister to the Australasian Olearia s.s. and had Australasian ancestors. Three species (including the generic type Olearia tomentosa) of the Australasian Olearia s.s. (Brouillet et al. 2009b) constitute a clade (Fig. 1A) but not a sister to the Aster clade. The phylogenetic tree from the combined data set, which is more resolved, includes only a few species (Myriactis and Callistephus) of the Australasian lineages. In the combined tree (Fig. 2), clade 17 (PP ¼ 0.99, BS ¼ 54) with the Aster clade (clade 8) is a sister to clade 18 (PP ¼ 1.00) that includes Myriactis and Callistephus (representatives of the Australasian lineages) and clades 17 and 18 group further into clade 19 (PP ¼ 0.95) which might correspond to the Australasian lineages. Therefore, EA Aster (clade 8; Fig. 2) and some of its segregates (clades 9, 10, 12– 16, and Aster spp. of clade 11; Fig. 2) belong to the Australasian lineages. A more extensive taxon sampling of Australasian Astereae for an analysis of combined DNA sequences is needed to study the origin of both EA Aster and its segregates. Status of the ‘Rhynchospermum group’ According to Nesom (1994a) and Nesom and Robinson two monotypic genera, Sheareria and (2007), Rhynchospermum, belong to the Rhynchospermum group of sub-tribe Lagenophorinae. The present study shows that these genera are well nested within the Aster clade, however, and not closely related to each other (Figs 1B and 2). Sheareria. Endemic to China, this was first placed in tribe Astereae and later transferred to tribe Heliantheae (Hoffmann, 1890). Chen (1979) recognized it as belonging to sub-tribe Milleriinae of Heliantheae. Robinson (1981) redelimited Heliantheae and considered Sheareria to be a member of Astereae. Li et al. (2008) provided micromorphological, anatomical and cytological evidence for moving the genus from Heliantheae to Astereae but did not determine its systematic position within tribe Astereae. Nesom (1994a) and Nesom and Robinson (2007) placed Sheareria in sub-tribe Lagenophorinae, but Nesom (1994a) doubted a natural alignment with Lagenophorinae. Gao et al. (2009) used an ITS data set to show that Sheareria formed a strongly supported clade with 1347 Kalimeris integrifolia and A. amellus rather than with Myriactis humilis, a species of Lagenophorinae, which implies that Sheareria should be transferred from Lagenophorinae to Asterinae. Both the ITS (Fig. 1B) and the combined (Fig. 2) trees show that Sheareria is well nested within the Aster clade. Sheareria nana differs noticeably from other species of the Aster clade owing to its somewhat reduced leaves (bract-like, linear) and assimilating branches, solitary head with only 5 – 8 florets, functionally staminate disc flowers and epappose and glabrous achenes. Sheareria nana forms a single-species subclade (clade 2) of clade 8 in all analyses (Figs 1B and 2), and it could be designated as a section of Aster. Rhynchospermum. The monotypic genus Rhynchospermum is distributed in eastern and southeastern Asia (Nesom and Robinson, 2007). Ling et al. (1985) included the genus in subtribe Bellidinae, and Zhang and Bremer (1993) placed it into their ‘Bellis group’ with Bellis and Bellium, whereas Nesom (1994a) and Nesom and Robinson (2007) assigned it to the Rhynchospermum group of sub-tribe Lagenophorinae. A previous phylogenetic analysis of ITS (Fiz et al., 2002) suggested that Rhynchospermum is related to neither Bellis nor Myriactis (sub-tribe Lagenophorinae) but to A. amellus and K. integrifolia. Brouillet et al. (2009b) also showed Rhynchospermum nested within Aster s.s., which is supported by our ITS and combined data sets (see Figs 1B and 2). In the phylogenetic trees (Figs 1B and 2) R. verticillatum is nested within the Aster clade (clade 8) and belongs to a clade (PP ¼ 0.94 in Fig. 1B; PP ¼ 1.00 in Fig. 2) with three species of series Turbinati of section Aster (see the Appendix). Although Rhynchospermum has some unique characters, such as a caducous pappus and biseriate pistillate ray florets with a short ligule, our results (Figs 1B and 2) suggest that it should be merged in Aster s.s. and placed in series Turbinati. Status of the ‘Kalimeris group’ Nesom (1994a, b, 2000) has suggested that the Kalimeris group is composed of five small genera: Boltonia, Callistephus, Heteropappus, Kalimeris and Miyamayomena. This arrangement is unsupported by previous reports and the present study. Kalimeris. This is native to eastern Asia, and one of its diagnostic characters is short pappi. Its complex taxonomic history has been reviewed in detail by Gu and Hoch (1997). Kalimeris shares several floral and achene characters with the small NA genus Boltonia, which led Bentham (1861, 1873) to place Kalimeris in Boltonia as one of three sections. Tamamschyan (1959), Ling et al. (1985) and Nesom (1994b, 2000) retained Kalimeris as a segregate genus, however. Gu and Hoch (1997) made a detailed comparison of the achenes and pappi of Boltonia and Kalimeris and concluded that their similarity was rather superficial. Based on ITS data, Fiz et al. (2002) and Brouillet et al. (2009b) demonstrated that Kalimeris and Boltonia are in divergent clades. Our phylogenetic analyses show that Kalimeris and Boltonia belong to different, strongly supported clades (clades 8 and 20, respectively; Fig. 1), supporting the view that no close relationship exists between Kalimeris and Boltonia. 1348 Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) Kalimeris was sub-divided into two sections by Kitamura (1937): Kalimeris and Cordifolium. Section Cordifolium has cordiform leaves with long petioles, two or three series of subequal phyllaries and cylindrical achenes with 4 – 7 ribs. The section includes two species, K. miqueliana, endemic to Japan, and K. longipetiolata, endemic to China (Kitamura, 1937; Ling et al., 1985). Gu and Hoch (1997) excluded section Cordifolium from Kalimeris and left it as part of Aster, and Ito and Soejima (1995) merged the section within Aster section Aster, although restriction fragment length polymorphisms (RFLPs) of plastid DNA supported a close relationship between K. miqueliana and Doellingeria scaber (Ito et al., 1995, 1998). Nesom (1993) transferred K. longipetiolata to Doellingeria, as D. longipetiolata, but the present results show that it is related to neither NA Doellingeria species nor Asian Doellingeria species. In the ITS tree, K. longipetiolata occupies an unresolved position (clade 14 in Fig. 1B) within the big polytomy, and in the combined tree it belongs to a polytomy (clade 18 in Fig. 2) with two Myriactis spp. and many other species of the Australasian lineages. Kalimeris longipetiolata should be treated as a new monotypic genus and be placed with the Australasian lineages. Kalimeris (excluding section Cordifolium) has been recognized as having a close relationship with EA Aster s.s. and Heteropappus according to morphological comparisons (Gu and Hoch, 1997), cytological studies (Huziwara, 1950; Tara, 1972, 1973), RFLPs of plastid DNA (Ito et al., 1995, 1998) and ITS data (Noyes and Rieseberg, 1999; Fiz et al., 2002; Brouillet et al. 2009b). The taxonomic status of Kalimeris remains to be determined, however (Gu and Hoch, 1997). In the two trees in our study, three Kalimeris spp. (excluding section Cordifolium) are well nested in the Aster clade and form a highly supported clade (PP ¼ 1.00 and BS ¼ 99 in Fig. 1B; PP ¼ 1.00 and BS ¼ 100 in Fig. 2). Kalimeris (excluding section Cordifolium) is characterized by laterally compressed achenes with short pappus bristles no longer than the length of the corolla tube (Gu and Hoch, 1997) and S-type chromosomes (Li, 2006). Thus, Kalimeris (excluding section Cordifolium) is monophyletic, and treating Kalimeris as series Kalimeris of Aster is reasonable. In the phylogenetic trees (Figs 1 and 2) Kalimeris is nested in clade 1 with A. ageratoides and Heteropappus, whereas Miyamayomena belongs to clade 3 with A. amellus. Natural hybridizations between Kalimeris and A. ovatus (formerly A. ageratoides subsp. ovatus; Tara, 1972, 1989), between Kalimeris and A. ageratoides (Li, 2006), and between Kalimeris and Heteropappus (Tara, 1973) support a close relationship with the A. ageratoides complex and Heteropappus, as do morphological studies (Gu and Hoch, 1997). Hu (1967) transferred a few species of Aster, including A. smithianus, to Kalimeris based on their short pappi, whereas our analyses showed that A. smithianus is not closely related to Kalimeris (Figs 1 and 2). Miyamayomena. This was separated from Kalimeris and initially named Gymnaster (Kitamura, 1937, 1982; Chen, 1986). It is characterized by a lack of pappi (Kitamura, 1937, 1982; Ling et al., 1985; Chen, 1986). Although there are only five species (Chen, 1986), Miyamayomena is as variable morphologically as the large genus Aster and may in fact be an artificial assemblage (Gu and Hoch, 1997). Ito and Soejima (1995) treated M. savatieri, the generic type, as a species of Aster section Teretiachaenium which also includes A. scaber (¼ D. scaber). In the phylogenetic trees based on RFLPs of plastid DNA, two species of Miyamayomena did not form a clade: M. koraiensis was nested in the Aster clade, and M. savatieri was a sister to the Aster clade. Therefore, Miyamayomena could be polyphyletic (Ito et al., 1994, 1998). Our analyses (Figs 1B and 2) show that three Chinese Miyamayomena spp., M. piccolii, M. simplex and M. angustifolius, are nested within the Aster clade (clade 8) and should be merged into Aster. These species belong to different clades, implying that a lack of pappi is not a homologous synapomorphy and that Miyamayomena is not monophyletic. Miyamayomena angustifolius (clade 4) is sister to clade 5 (Figs 1B and 2) and might be designed as a section of Aster. Miyamayomena piccolii and M. simplex form a strongly to weakly supported clade (PP ¼ 0.99 in Fig. 1B; PP ¼ 1.00 and BS ¼ 84 in Fig. 2) embedded within the A. amellus clade (clade 3 in Figs 1B and 2) and might be treated as a series of section Aster, whereas the taxonomic positions of Miyamayomena koraiensis and M. savatieri, endemic to Japan and North Korea, respectively, remain to be determined. Heteropappus. In 1832 the genus Heteropappus was established and the type species, H. hispidus, was transferred from Aster (Lessing, 1832). Heteropappus includes approx. 30 species distributed in eastern and central Asia and the Himalayan region (Ling et al., 1985). The genus is characterized by its two series of sub-equal herbaceous phyllaries and dimorphic pappi (shorter on the ray achenes and longer on the disc achenes; Ling et al., 1985; Gu and Hoch, 1997). Some species such as H. altaicus have a monomorphic pappus, so Grierson (1964) redefined Heteropappus by the unequal corolla lobes of the disc florets. Zygomorphic disc florets are also found in some species of Aster and Kalimeris, however. RFLPs of plastid DNA show that H. hispidus is embedded in Aster (Ito et al., 1998), implying that Heteropappus should be included in Aster. Our analyses (Figs 1 and 2) also strongly support the placement of Heteropappus in Aster. The two sampled species, representing two sections, form a highly to weakly supported (PP ¼ 1.00 and BS ¼ 79 in Fig. 1B; PP ¼ 1.00 and BS ¼ 100 in Fig. 2) sub-clade of the A. ageratoides clade (clade 1), which might indicate that Heteropappus should be treated as a series of section Ageratoides (corresponding to clade 1). Callistephus. This is a monotypic genus native to China. Based on its double pappus and unique involucre (outer bracts foliaceous and innermost white scarious), it was distinguished from Aster in 1817 by Cassini (Ling et al., 1985; Nesom, 2000). Heteropappus hispidus was placed in Callistephus by de Candolle as Callistephus biennis (Nesom, 2000), implying that Callistephus and Heteropappus might be related to each other. Zhang and Bremer (1993) suggested that Callistephus, Gymnaster, Heteropappus and Kalimeris are closely related to each other and to Aster. Nesom (1994b) thought that Callistephus is similar to some species of Myriactis (sub-tribe Lagenophorinae) in habit and tendency toward pappus Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) reduction, but he placed Callistephus within the Kalimeris group given the similar morphology of leaves, receptacles, disc corollas, and papillate collecting appendages of the style branches, the arrangement of the capitulum and the tendency toward pappus reduction (Nesom, 1994a, b, 2000). Our analyses (Figs 1 and 2) reveal that Callistephus has no close relationships with the other four genera of the Kalimeris group or with Myriactis. In the combined tree (Fig. 2), Callistephus and Myriactis occur in the same polytomy (clade 18) that is part of the Australasian lineages, which is concordant with the result of Brouillet et al. (2009b) that placed Callistephus in the large Australasian polytomy. We suggest that Callistephus maintain its generic status. Status of Turczaninowia Turczaninowia fastigiata is native to north-eastern Asia (Tamamschyan, 1959; Ling et al., 1985; Ito and Soejima, 1995) and is characterized by its dense vestiture and small heads (with flowers and fruits reduced correspondingly) in a compact capitulescence. Turczaninowia fastigiata was originally published as Aster fastigiatus in 1812 (Ling et al., 1985) and was segregated as the monotypic genus Turczaninowia by de Candolle in 1836 (Nesom, 1994b). Tamamschyan (1959), Ling et al. (1985) and Bremer (1994) followed de Candolle’s treatment, whereas Nesom (1994b) and Nesom and Robinson (2007) supported the inclusion of the species in Aster, and Ito and Soejima (1995) placed this species in Aster section Aster. The ovarian sterility of some of the inner disc flowers of this species and the triangular collecting appendages of its style branches are considered hallmarks of a possible close relationship with Galatella (Ling et al., 1985; Nesom, 1994b). Our phylogenetic trees (Figs 1 and 2) suggest that T. fastigiata does not merit generic rank or have a close relationship to Galatella; rather it should be transferred to Aster section Aster. Status of Doellingeria Nees established Doellingeria in 1832, typified by D. umbellata. Bentham (1873) advocated a conglomerated Aster and included Doellingeria within a larger Aster. Some botanists continued to recognize Doellingeria as a distinct genus, however. Its phylogenetic position is equivocal. Zhang and Bremer (1993) placed Doellingeria in the Aster group. Nesom classified it first in sub-tribe Solidagininae (Nesom, 1993), then in sub-tribe Symphyotrichinae (Nesom, 1994a) or in sub-tribe Asterinae (Nesom, 1994b), and recently as an unplaced genus of Astereae (Nesom and Robinson, 2007). Doellingeria includes 11 species, of which three are NA and eight are eastern Asian species (Nesom, 1993, 1994b). RFLPs of plastid DNA show that eastern Asian Doellingeria is embedded in Aster s.s. (lto et al., 1994), and hybridization between eastern Asian Doellingeria and Aster has been reported (Saito et al., 2007), whereas ITS data support an earlybranching position of NA Doellingeria (represented by D. umbellatus) in the NA Astereae clade (Noyes and Rieseberg, 1999; Brouillet et al., 2001). In our trees (Figs 1 and 2) NA Doellingeria belongs to the NA clade (clade 20; Fig. 1A), and eastern Asian Doellingeria (represented by 1349 Doellingeria scaber) is embedded in clade 8 (the Aster clade; Figs 1B and 2), which implies that Doellingeria is biphyletic and that eastern Asian Doellingeria should be moved from Doellingeria (which is typified by NA D. umbellatus) to Aster. Ito and Soejima (1995) placed eastern Asian Doellingeria and Miyamayomena together in Aster section Teretiachaenium. Our analyses (Figs 1B and 2) show that eastern Asian Doellingeria and Miyamayomena belong to different sub-clades (clades 6 and 7, respectively) of the Aster clade (clade 8), however. In clade 7 (Figs 1B and 2) eastern Asian Doellingeria is a sister to a clade with Arctogeron, Asterothamnus and three species of Aster s.s., showing that it diverged early in Aster evolution and suggesting that eastern Asian Doellingeria should be treated as an independent section of Aster. Status of Aster segregates of the ‘Asterothamnus group’ Nesom (1994a, b) set up an Asterothamnus group consisting of five small genera, Asterothamnus, Krylovia (¼ Rhinactinidia), Arctogeron, Kemulariella and Psychrogeton, of which the first four are segregates of Aster. The Asterothamnus group occurs primarily in central Asia and is characterized by a woody stem base, caespitose habit, sessile – glandular and tomentose stems and leaves, few or solitary heads and strongly coiling rays (Nesom, 1994b). Most of these features may be convergent characters resulting from adaptive modification under harsh environmental conditions (drought or cold), however. Our samples were limited to Asterothamnus, Rhinactinidia and Arctogeron (Appendix) because Kemulariella and Psychrogeton materials were unavailable. Asterothamnus. This was segregated from Aster in 1950 by Novopokrovskiy, and its generic status has been accepted by several authors (Tamamschyan, 1959; Ling et al., 1985; Zhang and Bremer; 1993; Bremer, 1994; Nesom, 1994b; Czerepanov, 1995; Nesom and Robinson, 2007). The genus comprises seven species endemic to deserts and desert steppes in central Asia (Ling et al., 1985; Zhao, 1996). Asterothamnus has distinctive characters: it is a strongly branching sub-shrub with a woody rhizome, linear or narrowly elliptic leaves, densely or thinly tomentose stems and leaves and solitary or few heads in a loose corymb, reflecting adaptation to drought. In our phylogenetic trees (Figs 1B and 2) Asterothamnus belongs to the Aster clade (clade 8) and should be treated as a member of Aster. The two species sampled form a well-supported sub-clade (PP ¼ 1.00 and BS ¼ 100) that is nested in clade 7 with A. sikuensis, A. poliothamnus, A. falcifolius, Arctogeron and eastern Asian Doellingeria in both phylogenetic analyses (Figs 1B and 2). Asterothamnus is obviously different in morphology from the other members of clade 7 and should be regarded as a section of Aster. Arctogeron gramineum. This is the only species of Arctogeron and is distributed in north-eastern China, Mongolia and eastern Russia. It occurs on dry mountain slopes or stony slopes and displays characters linked to drought adaptation such as low-growing and mat-forming habit and linearsubulate leaves. The species was originally described in 1350 Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) 1753 by Linnaeus as a member of Erigeron and then established as a separate genus in 1836 by de Candolle and transferred to Aster in 1907 by Komarov (reviewed by Ling et al., 1985). Like Asterothamnus, Arctogeron belongs to the Aster clade (clade 8; Figs 1B and 2) and should be treated as a member of Aster. It is well nested in clade 7 (Figs 1B and 2) and should be treated as a monotypic section of Aster. Rhinactinidia. This is a genus of four species native to central Asia and Siberia (Ling et al., 1985; Czerepanov, 1995). It was established as a genus in 1831 by Lessing and was later included in Aster s.l. (Ling et al., 1985). Its generic status is currently generally accepted (Tamamschyan, 1959; Ling et al., 1985; Zhang and Bremer, 1993; Bremer, 1994; Nesom, 1994b; Czerepanov, 1995; Nesom and Robinson, 2007). Nesom (1994b) suggested that Asterothamnus and Krylovia (¼ Rhinactinidia) are closely related in terms of similarities such as keeled phyllaries, a coiling – reflexing disc corolla, and two-veined achenes with glandular surfaces. Rhinactinidia is considered different from Aster in its diagnostic characters and zygomorphic disc corollas (Ling et al., 1985), but these features can also be found in Aster s.s. Our study shows (Figs 1B and 2) that Rhinactinidia is well nested within the Aster clade, belongs to the A. amellus clade (clade 3) and has no close relationship with Asterothamnus. Two samples of Rhinactinidia form a wellsupported clade (PP ¼ 1.00 and BS ¼ 100 in Figs 1B and 2), and Rhinactinidia should be treated as a series of section Aster. Status of the ‘Galatella group’ According to Nesom (1994a, b), the Galatella group of Asterinae s.s. includes three genera, Galatella (approx. 30 species), Crinitina (13 species) and Tripolium (a monotypic genus). These genera have been treated as three sections of Aster (Galatella, Linosyris and Tripolium, respectively) by some botanists but as segregate genera in other studies (reviewed by Ling et al., 1985; Nesom, 1994b). Furthermore, Nesom was indecisive about whether Galatella and Crinitina might belong in Solidagininae (Nesom, 1991) or whether they are more closely related to typical Aster (Nesom, 1994b). Based on ITS data, Fiz et al. (2002) and Brouillet et al. (2009b) found that Galatella and Crinitina form a well-supported clade, and a few studies have shown that Galatella or Crinitina are weakly related to Bellidinae rather than to Aster (Noyes and Rieseberg, 1999; Fiz et al., 2002; Karaman, 2006). Our phylogenetic analyses (Figs 1 and 2) show that Galatella, Crinitina and Tripolium constitute a well-supported clade (clade 22: PP ¼ 1.00 and BS ¼ 92 in Fig. 1A; PP ¼ 1.00 and BS ¼ 95 in Fig. 2). Furthermore, in the combined analysis (Fig. 2), Crinitina linosyris, Crinitina villosa and Galatella dahurica form a well-supported clade (clade 21: PP ¼ 1.00, BS ¼ 97), which would support the merger of Crinitina into Galatella. Whether Tripolium deserves generic status or whether the three genera should be merged into a single genus remains to be determined. If the latter is reasonable, the oldest name would have to be used for the genus, i.e. Galatella. In our analyses the Galatella – Crinitina– Tripolium clade (clade 22; Figs 1A and 2) is closely related to neither the Aster clade nor Solidago decurrens (a representative of sub-tribe Solidagininae). Similarities between the Galatella group and Aster in leaves, disc style branches, achenes and heads (Nesom, 1994b) are superficial and have developed in parallel, and the Galatella group should be separated from Aster. The trees show a moderate to weak relationship (clade 23: PP ¼ 0.89 in Fig. 1A; PP ¼ 0.91 and BS ¼ 57 in Fig. 2) between the three genera of the Galatella group and Bellis, which is consistent with the conclusions of Fiz et al. (2002). The systematic position of the Galatella group remains unresolved. Redelimitation of Aster According to our data, all existing generic delimitations of Aster are problematic. The EA Aster as delimited by some botanists (e.g. Ling et al., 1985; Nesom, 1994b; Nesom and Robinson, 2007) is paraphyletic because it excludes some of the descendants of the most recent common ancestor. Therefore, monophyletic Aster should include such genera as Sheareria, Rhynchospermum, Kalimeris (excluding K. longipetiolata), Heteropappus, Miyamayomena, Rhinactinidia, Turczaninowia, Asterothamnus, Arctogeron and eastern Asian Doellingeria. Conversely, EA Aster as delimited by other botanists (e.g. Merxmüller et al., 1976; Ito and Soejima, 1995) is polyphyletic because it includes morphologically similar but distantly related taxa. Callistephus, Galatella, Crinitina and Tripolium should be excluded from Aster. The Aster clade (clade 8) is strongly supported in both the ITS tree (PP ¼ 0.99; Fig. 1B) and the combined tree (PP ¼ 1.00, BS ¼ 98; Fig. 2), so the Aster clade is the recircumscribed genus Aster. Molecular data (Figs 1 and 2) revealed, however, that many Chinese Aster spp. should be excluded from Aster, although their status as Aster species, except for series Albescentes, has not been doubted. Of 41 sampled species of Aster s.s. (Ling et al., 1985; Chen, 1988; Ito and Soejima, 1995; Li and Liu, 2002), 17 should be removed from the genus. Series Hersileoides (Aster section Orthomeris, sensu Ling et al., 1985) is endemic to western China and consists of two restricted species, A. hersileoides and A. nitidus (Ling et al., 1985; Yin et al., 2010). They are characterized by a shrubby habit, solitary capitula at the apex of branches, membranous receptacular bracts and a short outer pappus. A karyotypic study of these species (Yin et al., 2010) showed that they are diploid and have shorter chromosomes and higher asymmetry of karyotype than that with A. ageratoides. Our study demonstrates that the series is a well-supported monophyletic group (clade 9: PP ¼ 0.99 and BS ¼ 98 in Fig. 1A; PP ¼ 1.00 and BS ¼ 100 in Fig. 2). Although the systematic position of the series has never been questioned, the ITS phylogenetic tree (Fig. 1A) shows that clade 9, series Hersileoides, is not closely related to clade 8, the Aster clade, and in the combined tree (Fig. 2) the sister relationship between clades 8 and 9 is only weakly supported (BS ¼ 54), even though the Bayesian PP is high (0.99). Therefore, the series should be removed from Aster, and it might be reasonable to elevate the series to a generic level in sub-tribe Asterinae. Aster albescens var. albescens, A. argyropholis and A. lavanduliifolius are representative of series Albescentes. Western China is the centre of diversity of this series, with six of the seven species being endemic to the region (the Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) exception being A. albescens which is distributed from western China to the southern Himalayas; Ling et al., 1985; Chen, 1988). Ling et al. (1985) established the series and placed it within Aster section Orthomeris. The series differs from others in the section with its shrubby habit, pinnate primary lateral leaf veins, relatively small heads, small rays and fourto six-veined, sub-cylindric achenes. Our studies (Figs 1A and 2) demonstrate that series Albescentes is a well-supported monophyletic taxon (clade 12: PP ¼ 1.00 and BS ¼ 98 in Fig. 1A; PP ¼ 1.00 and BS ¼ 100 in Fig. 2) and should be removed from Aster. Nesom (1994b) suggested that series Albescentes is closely related to the NA group, in which it would be positioned near NA Doellingeria. The present results provide no evidence to support this relationship, however. On the contrary, series Albescentes occurs in a polytomy (clade 18: PP ¼ 1.00 in Fig. 2) with Myriactis and other segregates of Aster s.s., implying that series Albescentes may belong to the Australasian lineages rather than to the NA clade (clade 20; Fig. 2). In the ITS analysis the series occurs at an unresolved position within a polytomy (Fig. 1A) in Astereae. Its systematics requires further investigation; however, series Albescentes should undoubtedly be removed from Aster and be considered for generic rank. According to Ling et al. (1985), A. auriculatus and A. panduratus belong to section Aster series Auriculati, and Aster pycnophyllus belongs to section Orthomeris series Sikkimenses. In the trees (Figs 1A and 2), the three species are well nested in a clade with Myriactis (clade 11: PP ¼ 1.00 and BS ¼ 97 in Fig.1A; PP ¼ 1.00 and BS ¼ 100 Fig. 2) and distantly related to Aster, suggesting that they should be removed from Aster. Although Myriactis is quite different from these three species with its two- to multiple-seriate ray florets, male disc florets and glandular collar, they do not form a sub-clade sister to Myriactis. Therefore, the relationships among the three species and Myriactis require further study. Of the 15 sampled species of Aster section Alpigenia (Appendix), seven fall in the Aster clade, and the other eight fall outside it (Figs 1A and 2). Of these eight species, six (A. asteroides, A. brachytrichus, A. diplostephioides, A. flaccidus, A. setchuenensis and A. yunnanensis) form a wellsupported clade (clade 10: PP ¼ 0.99 in Fig. 1A; PP ¼ 1.00 and BS ¼ 98 in Fig. 2), implying that these species might become a new genus. The systematic position of this group is unresolved, however. In the ITS tree (Fig. 1A) clade 10 falls within a big polytomy, and in the combined tree (Fig. 2) it belongs to clade 18, a polytomy, with Myriactis. Aster senecioides, the sole member of a monotypic series of section Alpigenia, forms a strongly supported clade (clade 15: PP ¼ 1.00 and BS ¼ 93 in Fig. 1; PP ¼ 1.00 and BS ¼ 100 in Fig. 2) with A. fuscescens, also the sole member of a monotypic series of section Aster (Ling et al., 1985). These two species are at an unresolved position within the big polytomy in the ITS tree (Fig. 1A) and belong to a polytomy (clade 18; Fig. 2) in the combined tree. Clade 15 might be treated as a separate genus. Similarly, in the ITS tree (Fig. 1A), A. batangensis (clade 16) occupies an unresolved position within the big polytomy of EA Astereae, and, in the combined tree (Fig. 2), clade 16 belongs to clade 18. Our phylogenetic trees (Figs. 1A and 2) show that A. batangensis seems to deserve the status of a monotypic genus. Thus, A. series 1351 Hersileoides, A. series Albescentes, a six-species group including A. asteroides, a group composed of A. senecioides and A. fuscescens, and A. batangensis should be elevated to generic level, and, together with the Aster clade, placed with the Australasian lineages. Nesom (2000) stated that Aster, even in its more restricted morphological definition, still encompasses a great deal of variation, and the description remains correspondingly general. Herein, Aster is expanded to include some segregates of Aster s.l. and other genera, making Aster more complex in some morphological features. For example, treating Sheareria as a section of Aster adds to Aster some new characters such as bract-like leaves, assimilating branches, small heads with only 5 – 8 florets, and functionally staminate disc flowers. Arctogeron brings to Aster such new features as caespitose herbs, narrow grass-like leaves with a scabrous ciliate margin and densely silvery pubescent cypselas. The high morphological diversity implies that Aster has undergone an evolutionary radiation since it originated. Aster displays a broad morphological variability in pappi (e.g. pappi are one- to four-seriate or absent, short or long, persistent or caducous) that, as mentioned above, has been used as a diagnostic character in delimiting some genera. Pappi are absent in clades 2 and 4 and in the M. piccolii – M. simplex clade of clade 3 (Figs 1B and 2), which implies that the disappearance of a pappus has evolved independently at least three times in Aster. Kalimeris, A. smithianus, A. dolichopodus and A. souliei share reduced pappi but occur in different sub-clades (Figs 1B and 2), suggesting convergent evolution toward pappus shortening. Dimorphic pappi (different lengths of pappi between ray and disc florets) are a diagnostic feature of Heteropappus, but dimorphic pappi are also found in A. homochlamydeus (W.-P. Li, unpubl. res.), which is another example of convergent evolution of pappi. Furthermore, no evolutionary relationships occur among dimorphic pappi, short pappi and absent pappi, i.e. no evolutionary series from dimorphic pappi to epappi exists. According to Ling et al. (1985), series Turbinati is characterized by four- to sevenseriate phyllaries, whereas our phylogenetic trees (Figs 1 and 2) show that three species (A. turbinatus, A. baccharoides and A. sampsonii), A. jishouensis (series Turbinati), with multiseriate phyllaries, and R. verticillatum, with two- to threeseriate phyllaries, form a clade, and this clade is not closely related to another species with multiseriate phyllaries, A. sampsonii. Therefore, the multiseriate phyllaries feature has arisen more than once in Aster. Noticeably, six species in one sub-clade of clade 7 (Figs 1B and 2) share a more or less shrubby habit, and clade 9 (sister to clade 8 in Fig. 2) is also characterized by a shrubby habit, which might mean that shrubby habit may represent a symplesiomorphy in clades 8 and 9. Whether EA Aster, with predominantly herbaceous perennials, originated from a woody ancestor is worth considering. Nonetheless, in clade 6 (Figs. 1B and 2), the shrubby habit of A. baccharoides and A. smithianus seems to be a convergence because these species occur within clades 1 and 3, respectively (Figs 1B and 2), and are not closely related to each other, and each of them is the only shrub in its clade. The morphology of Aster is so complex that further tracing of important morphological characters in the phylogenetic trees is necessary to reveal their phylogenetic significance. 1352 Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) Infrageneric classification of Aster After extensive changes in the generic delimitation of Aster, its infrageneric systematics should be reconstructed. Three infrageneric taxonomic schemes of EA Aster s.s. have been described. First, Ling et al. (1985) divided Chinese Aster s.s. into three sections [Aster, Orthomeris (a name based on an NA type in genus Oclemena) and Alpigenia] and 27 series. Next, Ito and Soejima (1995) recognized five sections of Japanese Aster: Tripolium (a monotypic section), Pseudocalimeris (largely equal to the genus Heteropappus), Teretiachaenium (including the taxa of Miyamayomena and eastern Asian Doellingeria), Asteromoea (similar to Kalimeris) and Aster (largely equal to Aster s.s.). Finally, Nesom (1994b) divided Aster into four sections and taxa incertae sedis. The former includes sections Aster, Alpigeni (including sub-sections Homochaeta, Heterochaeta and Senecioides), Ageratoides and Calimeridei, and the latter is a six-species group. The current study supports none of these taxonomic systems, however. We suggest clade 8 (Figs 1B and 2) as the genus Aster and clades 6 and 7 (Figs 1 and 2) as two subgenera of Aster. In clade 6, each of four sub-clades (clades 1 – 4; Figs 1B and 2) could be treated as a section. As mentioned above, M. angustifolius (clade 4; Figs 1B and 2) and S. nana (clade 2; Figs 1B and 2) may be treated as monotypic sections. Clade 1 is well supported in all analyses (PP ¼ 1.00 and BS ¼ 99 in Fig. 1B; PP ¼ 1.00 and PP ¼ 100 in Fig. 2) and could be regarded as section Ageratoides, typified by A. ageratoides (Nesom, 1994b). Section Ageratoides includes all taxa of section Pseudocalimeris and section Asteromoea and some members of section Teretiachaenium and section Aster (sensu Ito and Soejima, 1995); it corresponds more or less to sections Ageratoides of Nesom (1994b) and Orthomeris of Ling et al. (1985). Clade 3 is well supported only by BI (PP ¼ 0.96 in Fig. 1B; PP ¼ 1.00 in Fig. 2) but not by MP analysis. It might be treated as section Aster, typified by A. amellus. Although all previous schemes have recognized section Aster, their circumscriptions differ from ours. Some members (e.g. A. dolichopodus, A. mangshanensis, A. smithianus and A. vestitus) of section Aster of Ling et al. (1985) are nested in clade 1 (section Ageratoides) rather than in clade 3 (section Aster), and some members of section Orthomeris (e.g. A. sampsonii, A. turbinatus, A. baccharoides and A. jishouensis; Ling et al., 1985; Li and Liu, 2002) are nested in clade 3 rather than in clade 1. In fact, Nesom (1994b) agreed with Ling et al. (1985) in the circumscription of section Aster. As mentioned above, in Flora of Japan (Ito and Soejima, 1995) section Aster has a much wider definition than ours. Some species of section Alpigenia in the classifications of Ling et al. (1985) and Nesom (1994b) belong to clade 1 (section Ageratoides) or clade 3 (section Aster), and the others occur outside of the Aster clade, suggesting that section Alpigenia should be abandoned. Clade 7, the other sub-clade of clade 8, is moderately to well supported (PP ¼ 0.99 and BS ¼ 79 in Fig. 1B; PP ¼ 1.00 and BS ¼ 92 in Fig. 2) and could be treated as the other subgenus of Aster. The subgenus consists of three segregates (eastern Asian Doellingeria, Asterothamnus and Arctogeron) of Aster s.l. and three species (A. falcifolius, A. poliothamnus and A. sikuensis) of Aster s.s. As discussed above, eastern Asian Doellingeria, Asterothamnus and Arctogeron should be treated as three different sections. According to Ling et al. (1985), A. falcifolius is the only member of series Falcifolii of section Orthomeris, and A. poliothamnus and A. sikuensis belong to series Vestiti of section Aster. These three species have more or less woody stems that are similar to those of the other taxa of clade 7, Asterothamnus and Arctogeron. Aster falcifolius is characterized by solitary flowers and bracteole leaves that become denser until grading into phyllaries. It should be raised to the sectional level. Aster poliothamnus and A. sikuensis share some features, such as four- to fiveseriate phyllaries and the absence of rhizomes, and form a strongly to weakly supported clade (PP ¼ 1.00, BS ¼ 71; Fig. 2). These two species may deserve the status of a section. As a result, the subgenus (clade 7; Figs 1B and 2) would comprise five sections. According to Ling et al. (1985), the recircumscribed Aster has seven series with two or more species included in our analyses. None of these is monophyletic, however. All three species of series Vestiti (A. vestitus, A. poliothamnus and A. sikuensis; Ling et al., 1985) were sampled and occur in clades 6 and 7 (see Figs 1B and 2), and they should be placed in different subgenera. Aster alpinus, A. handelii, A. heterolepis and A. oreophilus are assigned to series Alpigenia (Ling et al., 1985) but occur in four clades of section Ageratoides (clade 1; Figs 1B and 2) and section Aster (clade 3; Figs 1B and 2). Although A. fanjingshanicus, A. tongolensis and A. souliei of series Tongolenses (Ling et al., 1985) belong to clade 1, they are not closely related to one another (Figs 1B and 2). All of the series (sensu Ling et al., 1985) of Aster must be re-evaluated. More than half the species of Aster (Tamamschyan, 1959; Grieson, 1975; Merxmüller and Schreiber, 1976; Ling et al., 1985; Czerepanov, 1995; Ito and Soejima, 1995) are not included in our study; therefore, a more extensive taxon sampling of molecular sequence data is necessary for a full phylogenetic reconstruction of Aster. Because more than half of the sampled species of section Alpigenia (sensu Ling et al., 1985) should be excluded from Aster, it is particularly important to collect molecular data for all the species. Because the combined analysis shows better resolution than that of the ITS phylogeny in Aster s.l., the combined data for the Australasian lineages are needed to resolve the origin and systematic position of Aster and its segregates. ACK N OW L E DG E M E N T S We thank the referees for their useful comments, suggestions and questions; Professor De-Yuan Hong, Liang-Bi Chen and Qiner Yang for their generous concern and help; the management departments of many of China’s National Nature Reserves; the faculty of the natural sciences departments at Shumen University; Professors Gong-Xi Chen and Dun-Yan Tan for field assistance; the curators of the PE, WUK, SZ, CDBI, HNNU, KUN, IBSC, IBK, NAS, HGAS, CCNU, HIB, HIMC and FUS herbaria for access to specimens; Professor Dong-Ping Li for kindly offering research facilities for our use; Professor Xiang Hu, Professor Ping Zhang, Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) Ming Tang and Feng-Ming Qian for technical assistance; and Professor Chengqi Ao for providing the leaves of M. angustifolius. 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GenBank accession number§ Present taxonomy* Unplaced taxa Doellingeria umbellata Eurybia sibirica Nannoglottis delavayi Sub-tribe Homochrominae Felicia filifolia Sub-tribe Hinterhuberinae Celmisia mackaui Chiliotrichum diffusum Phylogenetic lineages and infrageneric classification of Aster † Numbers, locations and altitudes of vouchers‡ ITS ETS trnL-F GU480699 NA NA BL – – – AF046966 AY772421 AY017167 NA AY772435 NA BL – FJ457937 NA NZ PSA – – AF422115 AF046945 NA DQ479128 AF452501 Continued Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) 1355 AP P E N D I X 1. Continued GenBank accession number§ Present taxonomy* Phylogenetic lineages and infrageneric classification of Aster † Numbers, locations and altitudes of vouchers‡ ITS ETS Madagaster madagascariensis Mairia hirsuta Olearia astroloba Olearia ballii Olearia calcarea Olearia ciliata Olearia cordata Olearia covenyi Olearia rudis Olearia tomentosa Oritrophium hieracioides Pleurophyllum hookeri Printzia polifolia Pteronia camphorata var. camphorata Remya kauaiensis Sub-tribe Brachyscominae Brachyscome rigidula Calotis hispidula Sub-tribe Bellidinae Bellis perennis Sub-tribe Grangeinae Grangea maderaspatana BL – DQ479031 NA BL AL AL AL AL AL NZ AL AL PSA NZ BL BL – – – – – – – – – – – – – FJ457929 AF497646 AF497662 AF497663 AF497667 AF497668 AF497711 AF497677 AF497650 DQ479116 HQ439864 FJ457927 DQ479118 NA NA NA NA NA NA NA NA NA NA NA NA NA AF497684 NA Dichrocephala auriculata Sub-tribe Lagenophorinae Myriactis nepalensis GR Myriactis wightii AL Rhynchospermum verticillatum Sheareria nana Sub-tribe Baccharidinae Baccharis neglecta Sub-tribe Podocominae Camptacra gracilis Kippistia suaedifolia Minuria integerrima Minuria macrorhiza Tetramolopium humile var. humile Sub-tribe Asterinae Arctogeron gramineum Aster Aster amellus Eurasian Aster s.s. Section Aster series Amelli Aster maackii Aster tataricus Section Aster series Macrocephali Section Aster series Macrocephali Aster fuscescens Section Aster series Fuscescentes Aster auriculatus Section Aster series Auriculati Aster panduratus Section Aster series Auriculati Aster mangshanensis Aster poliothamnus Aster sikuensis Section Aster series Auriculati Section Aster series Vestiti Section Aster series Vestiti AL trnL-F AL AL – – DQ478994 AB196597 NA NA BE LWP1003008; Changsha, cultivated JN315918 JN315942 JN315894 GR LWP0802034; Zhaoqing City, 200 m LWP0708234; Dali City, 2300 m JN315920 JN315944 JN315896 JN315919 JN315943 JN315895 JN315921 JN315945 JN315897 AL JN315922 JN315946 JN315898 AL LWP0509002; Kunming City, 2300 m LWP0509010; Kunming City, 2200 m LWP0607065; Mt. Emei, 1200 m JN543706 JN543707 JN543708 AL LWP0701001; Changsha City, 30m JN543703 JN543704 JN543705 SA – U97604 NA AL AL AL AL AL – – – – – AF247069 AF497660 AF046957 AF247076 DQ479040 NA NA NA SEA LWP0606014; Wulanhaote City, 300 m JN315928 JN315952 JN315904 LWP0408002; Shumen, Bulgaria, 400 m LWP0609043; Yichun City, 200 m LWP0108018; Xinglong County, 400 m YGS1007021; Gongshan County, 2500 m LWP0509059; Yongsheng County, 2200 m LWP1012067; Guiyang City, 1100 m LWP0511034; Mt. Mang, 1670 m LWP0506001; Zhang County, 500 m LWP0510025; Lueyang County, 300 m JN543742 JN543743 JN543744 JN543745 JN543748 JN543746 JN543749 JN543747 JN543750 JN543751 JN543752 JN543753 JN543754 JN543755 JN543756 JN543757 JN543758 JN543759 JN543760 JN543763 JN543766 JN543761 JN543764 JN543767 JN543762 JN543765 JN543768 NA Continued 1356 Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) AP P E N D I X 1. Continued GenBank accession number§ Present taxonomy* Phylogenetic lineages and infrageneric classification of Aster † Aster dolichopodus Section Aster series Vestiti Section Aster series Taliangshanensis Section Aster series Smithiani Aster smithianus Section Aster series Smithiani Aster ageratoides var. lasiocladus Aster homochlamydeus Aster falcifolius Aster baccharoides Aster jishouensis Aster sampsonii Aster turbinatus Aster alpinus Section Orthomeris series Ageratoides Section Orthomeris series Ageratoides Section Orthomeris series Hersileoides Section Orthomeris series Hersileoides Section Orthomeris series Albescentes Section Orthomeris series Albescentes Section Orthomeris series Albescentes Section Orthomeris series Sikkimenses Section Orthomeris series Falcifolii Section Orthomeris series Turbinati Section Orthomeris series Turbinati Section Orthomeris series Turbinati Section Orthomeris series Turbinati Section Alpinenia series Alpini Aster handelii Section Alpinenia series Alpini Aster heterolepis Section Alpinenia series Alpini Aster oreophilus Aster fanjingshanicus Aster tongolensis Section Alpinenia series Alpini Section Alpinenia series Tongolensis Section Alpinenia series Tongolensis Aster souliei Section Alpinenia series Tongolensis Aster brachytrichus Aster asteroides Section Alpinenia series Latibracteati Section Alpinenia series Asteroides Aster flaccidus Section Alpinenia series Asteroides Aster diplostephioides Section Alpinenia series Diplostephioides Section Alpinenia series Diplostephioides Section Alpinenia series Diplostephioides Section Alpinenia series Senecioides Section Alpinenia series Batangenses SEA Aster vestitus Aster taliangshanensis Aster hersileoides Aster nitidus Aster albescens var. albescens Aster argyropholis Aster lavanduliifolius Aster pycnophyllus Aster setchuenensis Aster yunnanensis Aster senecioides Aster batangensis Asterothamnus centrali-asiaticus Asterothamnus fruticosus SEA Callistephus chinensis Crinitina linosyris SEA SEA Crinitina villosa SEA Numbers, locations and altitudes of vouchers‡ ITS ETS trnL-F JN543769 JN543772 JN543770 JN543773 JN543771 JN543774 JN543775 JN543776 JN543777 JN543778 JN543779 JN543780 JN543781 JN543782 JN543783 JN543784 JN543785 JN543786 LWP0807002; Li County, 2100 m JN543787 JN543788 JN543789 LWP0505007; 660 m LWP0508123; 2010 m LWP0409045; 2500 m LWP0708053; 2720 m LWP0509091; Nanchuan County, JN543790 JN543791 JN543792 Baoxing County, JN543862 JN543863 JN543864 Maerkang City, JN543793 JN543794 JN543795 Yajiang county, JN543796 JN543797 JN543798 Dali City, 2800 m JN543799 JN543800 JN543801 JN543802 JN543805 JN543808 JN543811 JN543814 JN543817 JN543803 JN543806 JN543809 JN543812 JN543815 JN543818 JN543804 JN543807 JN543810 JN543813 JN543816 JN543819 JN543820 JN543821 JN543822 JN543823 JN543824 JN543825 JN543826 JN543829 JN543832 JN543827 JN543830 JN543833 JN543828 JN543831 JN543834 JN543835 JN543836 JN543837 JN543838 JN543839 JN543840 JN543841 JN543842 JN543843 JN543844 JN543845 JN543846 JN543847 JN543848 JN543849 JN543850 JN543851 JN543852 JN543853 JN543854 JN543855 JN543856 JN543859 JN543857 JN543860 JN543858 JN543861 Yinchuan City, JN315930 JN315954 JN315906 Wulumuqi City, JN315929 JN315953 JN315905 Anshan City, 340 m Shumen, Bulgaria, JN315931 JN315932 JN315955 JN315956 JN315907 JN315908 Shumen, Bulgaria, JN315933 JN315957 JN315909 LWP0509023; Lijiang City, 2610 m LWP0607056; Xichang City, 2800 m LWP0409060; Maerkang City, 2500 m LWP0508034; Maerkang City, 2600 m LWP0112018; Changsha City, 110 m LWP0508004; Li County, 2600 m LWP0410050; Mt. Huping, 400 m LWP0802001; Zhuhai City, 100 m LWP1012015; Jishou City, 600 m LWP0511060; Mt. Mang, 1100 m LWP0110029; Fenghua City, 60 m LWP0607020; Wulumuqi City, 2320 m LWP0708174; Zhongdian County, 3400 m LWP0507004; Jiuzhai County, 2600 m LWP0509016; Lijiang City, 3000 m LWP0606082; Mt. Fangjing, 2300 m LWP0708147; Xiangcheng County, 3300 m LWP0708084; Litang County, 4000 m LWP0607075; Xichang City, 2800 m LWP0708112; Daocheng County, 2780 m LWP0607026; Wulumuqi City, 3700 m LWP0507020; Jiuzhai County, 2600 m LWP0508007; Maerkang City, 2800 m LWP0508089; Kangding City, 3500 m LWP0708215; Lijiang City, 2800 m LWP0606039; Lijiang City, 2700 m LWP0607045; 1630 m LWP0607005; 950 m LWP0108021; LWP0408001; 400 m LWP0408009; 400 m Continued Li et al. — Molecular phylogenetics of Eurasian Aster (Asteraceae: Astereae) 1357 AP P E N D I X 1. Continued GenBank accession number§ Present taxonomy* Phylogenetic lineages and infrageneric classification of Aster † ITS ETS trnL-F LWP0108025; Anshan City, 350 m LWP0609047; Mt. A’er, Nei Mongol, 400 m LWP0506010; Zhang County, 600 m JN315934 JN315935 JN315958 JN315959 JN315910 JN315911 JN543709 JN543710 JN543711 LWP0409037; Maerkang City, 3200 m LWP0806017; Changsha City, 80 m LWP0609107; Tonghua County, 560 m LWP0609077; Mudanjiang City, 360 m LWP0508104; Baoxing County, 2600 m DBY9206; Yongjia County. 200 m JN543712 JN543713 JN543714 JN543715 JN543721 JN543716 JN543722 JN543717 JN543723 JN543718 JN543719 JN543720 JN315936 JN315960 JN315912 JN543736 JN543737 JN543738 LWP0510055; Mei County. 300 m LWP0508083; Kangding City, 2800 m LWP0607036; Wulumuqi City, 2620 m LWP0607012; Wulumuqi City, 1800 m LWP0311001; Varna, Bulgaria, 1 m LWP0609030; Daqin City, 150 m JN543730 JN543733 JN543731 JN543734 JN543732 JN543735 JN543727 JN543728 JN543729 JN543724 JN543725 JN543726 JN315937 JN543739 JN315961 JN543740 JN315913 JN543741 NA LWP0510116; Lichuan County, 1050 m JN204176 JN204177 JN204178 NA – AF046972 NA NA – AF477632 NA NA – AF477661 NA NA LWP0606002; Beijing, cultivated. JN315926 JN315950 JN315902 NA LWP1010007; Changsha City, 40 m JN315927 JN315951 JN315903 NA – AF046984 NA NA – GQ892729 NA AIS NA NA NA LWP0606032; Lijiang City, 2500 m LWP1009002; Changsha City, 35 m LWP1010009; Changsha City, 40 m LWP0606055; Lijiang City, 2500 m JN315938 JN315923 JN315924 JN315925 JN315962 JN315947 JN315948 JN315949 JN315914 JN315899 JN315900 JN315901 OG LWP1004010; Changsha, cultivated. JN315939 JN315963 JN315915 OG LWP1012002; Changsha City, 80 m JN315940 JN315964 JN315916 OG LWP1004006; Changsha, cultivated. JN315941 JN315965 JN315917 Doellingeria scaber Galatella dahurica SEA SEA Heteropappus altaicus var. millefolius Heteropappus crenatifoliu Kalimeris indica Kalimeris incisa SEA Kalimeris integrifolia SEA Kalimeris longipetiolata SEA Miyamayomena angustifolius Miyamayomena piccolii Miyamayomena simplex SEA Rhinactinidia eremophila SEA Rhinactinidia limoniifolia SEA Tripolium vulgare Turczaninowia fastigiata Sub-tribe Solidaginae Solidago decurrens SEA SEA Sub-tribe Pentachaetinae Pentachaeta aurea Sub-tribe Boltoniinae Boltonia asteroides Sub-tribe Machaerantherinae Machaeranthera tanacetifolia Sub-tribe Symphyotrichinae Symphyotrichum novi-belgii Symphyotrichum subulatum Sub-tribe Astranthiinae Astranthium integrifolium Sub-tribe Chrysopsidinae Chrysopsis mariana Sub-tribe Conyzinae Conyza japonica Conyza sumatrensis Erigeron annus Erigeron breviscapus Tribe Anthemideae Chrysanthemum coronarium Dendranthema indicum Tribe Calenduleae Calendula officinalis Numbers, locations and altitudes of vouchers‡ SEA SEA SEA SEA SEA * Generic circumscriptions and nomenclature of Astereae follow Nesom and Robinson (2007) except Turczaninowia which follows Ling et al. (1985) and Crinitina Soják is substituted for Crinitaria Cass. The name Aster setchuenensis follows the International Plant Names Index (IPNI). † Phylogenetic lineages: follows Brouillet et al. (2009b); infrageneric classification of Aster follows Ling et al. (1985). AIS, Astereae incertae sedis; AL, Australasian lineages; BE, Bellidinae; BL, early-branching lineages; ETS, external transcribed spacer; GR, Grangeinae; ITS, internal transcribed spacer; NA, North American lineage; NZ, New Zealand clade; OG, outgroup; PSA, palaeo South American clade; SA, South American lineages; SEA, segregates of Eurasian Aster s.l. ‡ Information is omitted for the accessions that were obtained from GenBank. Four species were collected from Bulgaria and the others from China. § One or two sequence (ETS, trnL-F) data unavailable.