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Molecular phylogeny of , and its allies (, ) as inferred from nuclear ribosomal ITS and chloroplast trn LF IGS sequences

ARTICLE in SYSTEMATICS AND EVOLUTION · FEBRUARY 2010 Impact Factor: 1.42 · DOI: 10.1007/s00606-009-0242-0

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All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Hongbo Zhao letting you access and read them immediately. Retrieved on: 02 December 2015 Plant Syst Evol (2010) 284:153–169 DOI 10.1007/s00606-009-0242-0

ORIGINAL ARTICLE

Molecular phylogeny of Chrysanthemum, Ajania and its allies (Anthemideae, Asteraceae) as inferred from nuclear ribosomal ITS and chloroplast trnL-F IGS sequences

Hong-Bo Zhao • Fa-Di Chen • Su-Mei Chen • Guo-Sheng Wu • Wei-Ming Guo

Received: 14 April 2009 / Accepted: 25 October 2009 / Published online: 4 December 2009 Ó Springer-Verlag 2009

Abstract To better understand the evolutionary history, positions of some ambiguous taxa were renewedly con- intergeneric relationships and circumscription of Chry- sidered. Subtribe Artemisiinae was chiefly divided into two santhemum and Ajania and the taxonomic position of groups, (1) one corresponding to Chrysanthemum, Arc- some small Asian genera (Anthemideae, Asteraceae), the tanthemum, Ajania, Opisthopappus and Elachanthemum sequences of the nuclear ribosomal internal transcribed (the Chrysanthemum group), (2) another to , spacer (nrDNA ITS) and the chloroplast trnL-F intergenic , and (the spacer (cpDNA IGS) were newly obtained for 48 taxa and Artemisia group). Within the Chrysanthemum group, combined with those already deposited in GenBank. Phy- Chrysanthemum and Ajania were closely logenies with an emphasis on Chrysanthemum, Ajania and related to each other, and the generic circumscription was its allies, by both maximum parsimony and Bayesian ambiguous; Phaeostigma was excluded from this group analysis, were constructed using either the ITS sequence that was also confirmed by the 6-bp insertion in the IGS alone, the IGS sequence alone or combined sequences. The sequence; radiate or rare discoid was IGS sequence was low phylogenetically informative, but excluded, and discoid Elachanthemum without ray florets some deletions and insertions were informative for inter- was added to this group; at the same time, Opisthopappus specific and intergeneric delimitations. The ITS and the in subtribe Tanacetinae should be transferred to subtribe ITS/IGS phylogenies both suggested the presence of two Artemisiinae and became one of the components of the major clades. The monophyly of subtribe Artemisiinae Chrysanthemum group. Based on the molecular phyloge- (clade A) could be retrieved when the phylogenetic netic framework, the evolution of pollen and capitulum characters was inferred.

Keywords Ajania Artemisiinae Chrysanthemum H.-B. Zhao F.-D. Chen (&) S.-M. Chen G.-S. Wu Floral characters Molecular phylogeny Radiate genera W.-M. Guo ITS trnL-F College of Horticulture, Nanjing Agricultural University, 210095 Nanjing, Jiangsu, e-mail: [email protected]; [email protected] S.-M. Chen Introduction e-mail: [email protected] G.-S. Wu There are two main groups (the Artemisia group and the e-mail: [email protected] Chrysanthemum group) in Artemisiinae (Anthemideae). W.-M. Guo According to Bremer and Humphries’s (1993) cladogram e-mail: [email protected] based on morphological characters, the Artemisia group includes Artemisia, along with eight minor Asian genera H.-B. Zhao [Ajaniopsis Shih, Crossostephium Less., Kitam., School of Agriculture and Food Science, Zhejiang Forestry College, 311300 Lin’an, Zhejiang, China Poljakov, Mausolea Bunge, Neopallasia Pol- e-mail: [email protected] jakov, Stilpnolepis H. Kraschen. (including Elachanthemum 123 154 H.-B. Zhao et al.

Ling et Y.R. Ling) and Turaniphytum Poljakov] and two branches and erect corolla lobes, with both of these North American genera (Picrothamnus Nutt., Sphaerome- characters, as well as the suffruticose habit and smooth ria Nutt.) characterized by their disciform or discoid, (non-spiny) pollen, being very similar to those of Artemisia commonly paniculate capitula, microechinate and thin- (Bremer and Humphries 1993). Pollen morphology sug- walled pollen, and ribless cypsela lacking a pappus. The gests that Phaeostigma is more related to Ajania than to Chrysanthemum group is comprised of Chrysanthe- Artemisia (Muldashev 1981, 1983), so Shih and Fu (1983) mum, some other radiate genera with solitary or laxly cor- reduced Phaeostigma to Ajania. ymbose capitula such as Arctanthemum (Tzvelev) Tzvelev, The sister group of Artemisiinae is to be found within Tridactylina (DC.) Schultz-Bip. and Brachanthemum DC., L., where there are some taxa such as T. par- and the disciform Ajania Poljakov and Phaeostigma Muld. thenium (L.) Schultz-Bip. and T. tatsienense (Bureau et with dense corymbose capitula, which were once included Franchet) Bremer et Humphries, which are very similar to within a broad concept of Chrysanthemum sharing the representatives of Chrysanthemum and Brachanthemum echinate exine pollen ornamentation. However, Oberprieler (Bremer and Humphries 1993). In addition, some genera et al. (2006) placed Ajaniopsis and Elachanthemum into the such as Tzvelev, Kitam Chrysanthemum group (i.e., Ajania group), and separated (subtribe Leucantheminae), Poljakov and the new genus Hulteniella Tzvelev from Arctanthemum.At Opisthopappus Shih (subtribe Tanacetinae) are closely the same time, they excluded Ajaniopsis, Kaschgaria, related to the Chrysanthemum group (Kondo and Abd Stilpnolepis and Turaniphytum from the Artemisia group El-Twab 2002; Zhao et al. unpublished). Both Leucan- that also was confirmed by the results of Watson et al. themella and Nipponanthemum are radiate, supporting a (2002) and Valle`s et al. (2003). radiate ancestry for the Artemisiinae (Watson et al. 2002). Chrysanthemum (formerly called Dendranthema) Oberprieler et al. (2007) transferred Leucanthemella, includes 41 species according to Oberprieler et al. (2006) Nipponanthemum and Hippolytia to subtribe Artemisiinae and is mainly distributed across eastern (China, based on ITS phylogeny and morphological evidence. , and ) (Shih and Fu 1983; Bremer and Previous molecular phylogenetic research on Artemis- Humphries 1993; Iwatsuki et al. 1997; Oberprieler et al. iinae was mainly focused on the phylogeny of Artemisia 2006). Based on capitulum characters, Chrysanthemum is and its allies (Kornkven et al. 1998, 1999; Torrell et al. divided into two sections (Sect. Chrysanthemum and Sect. 1999; Watson et al. 2002; D’Andrea et al. 2003; Valle`s Chlorochlamys) (Shih and Fu 1983). Arctanthemum (four et al. 2003; Sanz et al. 2008; Tkach et al. 2008a, b). The species) and Brachanthemum (ten species) were formerly results show the generic delimitation in this subtribe sensu classified within Chrysanthemum, but more recently they and the generic independence of several small genera such were segregated and reassembled into two new genera as Crossostephium, Filifolium, Neopallasia and Sphaer- (Shih and Fu 1983; Bremer and Humphries 1993; Iwatsuki omeria are questionable (Oberprieler et al. 2006). At the et al. 1997; Oberprieler et al. 2006). Tzvelev (in Bremer same time, probably due to fewer taxa, the phylogeny of and Humphries 1993) noted that Brachanthemum is prob- the Chrysanthemum group and intergeneric relationships of ably closely related to Chrysanthemum by its thin-walled, Chrysanthemum and allies remain poorly resolved. myxogenic fruits lacking a pappus. Arctanthemum com- We present here a reconstruction of the phylogeny of prises the rosulate herbs distributed across the Arctic, subtribe Artemisiinae and some allied or segregate taxa whereas most Chrysanthemum species are leafy herbs or from other subtribes endemic to eastern Asia, with an herbaceous perennials from China and Japan (Bremer and emphasis on Chrysanthemum, Ajania, some radiate genera Humphries 1993). and their allies based on sequence variation in both the Disciform Ajania including about 39 species (Oberpri- nuclear ribosomal DNA (ITS) and chloroplast DNA (the eler et al. 2006) was once treated as a new section of trnL-F IGS). The ITS and IGS have been successfully Chrysanthemum by Kitamura (1978) and Ohashi and used in phylogenetic studies of the Asteraceae and for Yonekura (2004). The relationship between Chrysanthe- Anthemideae specifically (Oberprieler and Vogt 2000; mum and Ajania is difficult to define. Some Chrysanthe- Oberprieler 2002; Watson et al. 2002; Valle`s et al. 2003). mum species may be more closely related to Ajania than to The main goals are: (1) to investigate the circumscription other Chrysanthemum species, and thus Chrysanthemum is of the Chrysanthemum group and intergeneric relation- even more plesiomorphic, and possibly paraphyletic with ships within this group and between the Chrysanthemum Ajania and its relatives (Bremer and Humphries 1993). group and its allies from other subtribes; (2) to reevaluate Ajania quercifolia (W. Smith) Ling et Shih, A. salicifolia the relationship between the Chrysanthemum group (Mattf.) Poljakov and A. variifolia (Chang) Tzvelev were and the Artemisia group; (3) to evaluate and infer the transferred to a new genus, Phaeostigma (three species), by evolution of the capitular and pollen morphology in Muldashev (1981) on the basis of their brownish style- Artemisiinae. 123 Molecular phylogeny of Chrysanthemum, Ajania, and its allies 155

Materials and methods of an initial incubation of 95°C/5 min, followed by 35 cycles of 94°C/1 min, 54°C/1 min and 72°C/1.5 min, and Taxa sampling terminated with an incubation of 72°C/5 min; that of IGS consisted of an initial incubation of 95°C/2 min, followed To reconstruction the molecular phylogeny, the respective by 30 cycles of 95°C/30 s, 54°C/1 min and 72°C/1 min, 48 sequences of ITS and IGS were newly generated, and terminated with an incubation of 72°C/2 min. Follow- including 17 species of Chrysanthemum, 11 species of ing PCR, the reaction products were concentrated five fold Ajania, 2 species of Phaeostigma, 1 species of Brachan- by vacuum centrifugation and separated by electrophoresis themum, Crossostephium, Elachanthemum, Neopallasia through a 1.5% agarose gel. Individual fragments were and 13 taxa from other subtribes, and 60 sequences of ITS excised from the gel and recovered using the TaKaRa from 4 species of Chrysanthemum, 15 species of Artemisia, Agarose Gel DNA Purification Kit v2.0, following the 2 species of Kaschgaria, 1 species of Arctanthemum, manufacturer’s instructions. The purified products were Brachanthemum, Stilpnolepis, Filifolium, Sphaeromeria sequenced directly by an ABI 3730 DNA sequencer and 9 species from other subtribes and the 12 sequences of (Applied Biosystems, USA) using the above-mentioned IGS from 2 species of Chrysanthemum, Artemisia, 1 spe- PCR amplification primers. cies of Arctanthemum, Ajania and 6 taxa from other sub- tribes were obtained from GenBank (Table 1). The Sequence analysis and phylogenetic reconstruction sampled taxa comprised 28 genera (85 species) covering all genera of the Chrysanthemum group except Tridactylina The boundaries of the target sequences were determined (DC.) Schultz-Bip. that included two sections of Chry- from Oberprieler and Vogt (2000), Oberprieler (2002) and santhemum, two sections (all four series) of Ajania, Arc- Watson et al. (2002). Sequence alignment was achieved tanthemum, Brachanthemum and Phaeostigma, most with ClustalX v1.83 (Thompson et al. 1997), with manual genera of the Artemisia group and the allied taxa related to correction. Maximum parsimony analyses were conducted the Chrysanthemum group from other subtribes. Because using PAUP* v4.0b10 (Swofford 2002, 2003). The heu- the Chrysanthemum group was mainly endemic to central ristic searches treated gaps as missing data or new-state and Asia and eastern Asia specifically, to better understand the single-site polymorphisms as uncertainties, and tree con- phylogeny, almost all taxa in ingroups were from the same struction was achieved via stepwise addition, using 1,000 distributed region (eastern Asia), and even some relatively replicates with random addition of sequences, tree-bisec- far-related genera were sampled from the same regions. tion-reconnection (TBR) branch swapping, with a maxi- Four African L. and Gaertner in mum of 500,000 parsimonious trees saved, with all subtribe Ursiniinae, three Aster L. species () and minimal trees found during branch swapping (MULTREES two L. and L. () option in effect) (Maddison 1991). The accelerated char- species were selected as outgroups according to both acter transformation (ACCTRAN) option was used. In all morphological (Bremer and Humphries 1993) and molec- analyses, nucleotide transformations within characters ular evidences (Kim and Jansen 1995; Watson et al. 2000). were equally weighted. Phylogenetic trees were rooted The classification of subtribes in Anthemideae was using the outgroup species. Support for monophyletic according to Bremer and Humphries (1993). groups was evaluated using 1,000 bootstrap replicates (Felsenstein 1985) with ACCTRAN, MULPARS and TBR DNA extraction, PCR amplification and amplicon options employed in a heuristic search using one random sequencing addition to the sequence. The consistency index (CI) (Kluge and Farris 1969), retention index (RI) (Farris 1989) Total DNA was isolated from fresh or silica-gel dried leaf and the rescaled consistency index were calculated as material using a modified CTAB procedure (Lassner et al. measures of homoplasy. MrBayes v3.1.2 (Ronquist and 1989). The ITS region including 5.8s gene of the nuclear Huelsenbeck 2003) was used for making Bayesian infer- ribosomal DNA was amplified by the primer pair 50-A ences, applying the best substitution model GTR ? G GAAATCGTAACAAGGTTTCCGTAGG-30 and 50-TCC obtained from MrModeltest v2.3 (Nylander 2004) and TCCGCTTATTGATATGC-30 (White et al. 1990) and the PAUP v4.0b10, with three parallel runs of four chains each, trnL-F IGS by 50-ATTTGAA CTGGTGACACGAG-30 and and default settings otherwise. One million generations 50-GGTTCAAGTCCCTCTATCCC-30 (Taberlet et al. were run, saving a tree every ten generations and dis- 1991) in 50-ll reactions containing *100 ng template, carding the first 25% for the construction of a consensus 0.2 lmol of each primer, 0.2 mM dNTP, 1U Ex Taq DNA tree (ensuring that equilibrium of chains had been reached). polymerase (TaKaRa, Japan) and 5 llof109 PCR buffer At the same time, the posterior probability of individual (TaKaRa). The temperature cycling regime of ITS consisted clades was calculated. Tree drawing was automated using 123 156 H.-B. Zhao et al.

Table 1 The sampled taxa, along with voucher information, their geographical origin, and the GenBank accession number of the DNA sequences used for phylogenetic analysis Taxon Source/voucher Distribution Accession number ITS (ITS1, ITS2) trnL-F

Ingroup Subtribe Artemisiinae Bremer & Humphries Chrysanthemum L. Sect. Chrysanthemum Chrysanthemum arisanense Hayata Kim et al., unpublished China (Taiwan) AB234740 (IGS) Chrysanthemum boreale (Makino) Makino Honshu, Japan/Chen0601 China, Korea, Japan EF577294 EF577347 Chrysanthemum chanetii H. Le´v. Beijing, China/Chen0005 China, Korea EF577295 EF577348 Chrysanthemum coreanum Nakai Francisco-Ortega et al. Korea L77802 1997 (ITS) Chrysanthemum crassum (Kitam.) Kitam. Kyushu, Japan/Chen0607 Japan EF577296 EF577349 Chrysanthemum dichrum (C.Shih) Neiqiu, , China/ China EF577297 EF577350 H.Ohashi & Yonek. Chen0003 Chrysanthemum indicum L. Nanjing, Jiangsu, China/ E. Asia EF577298 EF577355 Chen0004 Chrysanthemum japonense Nakai Kyushu, Japan/Chen0605 Japan EF577299 EF577356 Chrysanthemum japonicum Makino Honshu, Japan/Chen0602 Japan EF577300 EF577357 Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Beijing, China/Chen0002 China EF577301 EF577358 Makino Chrysanthemum 9 morifolium Ramat. Nanjing, Jiangsu, China/ China (cultivated) EF577311 EF577353 ‘Aoyunhuoju’ Zhao0503 Chrysanthemum naktongense Nakai H.-E. Zhao et al., China AF314595 unpublished (ITS) Chrysanthemum nankingense Hand.-Mazz Nanjing, Jiangsu, China/ China EF577302 EF577359 Chen0001 Chrysanthemum okiense Kitam. W. Honshu, Japan/ Japan EF577303 EF577360 Chen0603 Chrysanthemum oreastrum Hance Taibaishan, , China/ Far East, China, Korea EF577304 EF577361 Zhao.TB06-04 Chrysanthemum ornatum Hemsley Kyushu, Japan/Chen0606 Japan EF577305 EF577362 Chrysanthemum rhombifolium (Ling & C. Shih) H.-E. Zhao et al., China AF314603 H.Ohashi & Yonekura unpublished (ITS) Chrysanthemum vestitum (Hemsley) Stapf Tianzhushan, Anhui, China/ China EF577306 EF577363 Chen0006 Chrysanthemum yezoense Maekawa N. Honshu, Japan/ Japan EF577307 EF577365 Chen0611 Chrysanthemum zawadskii Herbich Huangshan, Anhui, China/ From E. through EF577309 EF577367 Chen0007 to China and Japan Sect. Chlorochlamys Shih Chrysanthemum mongolicum Y. Ling H.-E. Zhao et al., China, E. Siberia AF314593 AF314611 unpublished (ITS) Chrysanthemum yoshinaganthum Makino ex Kitam. Shikoku, Japan/Chen0604 Japan EF577308 EF577366 Arctanthemum (Tzvelev) Tzvelev Arctanthemum arcticum (L.) Tzvelev Francisco-Ortega et al. Far East, Arctic L77756 AB234734 1997 (ITS); Kim et al., America unpublished (IGS)

123 Molecular phylogeny of Chrysanthemum, Ajania, and its allies 157

Table 1 continued Taxon Source/voucher Distribution Accession number ITS (ITS1, ITS2) trnL-F

Ajania Poljakov Sect. Phaeoscyphus Shih Ser. Tibeticae Tzvelev Ajania breviloba (Franchet ex Hand.-Mazz.) Lijiang, , China/ China EF577271 EF577324 Ling et Shih Xie.LJ06-01 Ajania khartensis (Dunn) Shih Kangding, , China/ China EF577274 EF577327 Zhao.SC06-01 Ajania myriantha (Franchet) Ling ex Shih Jinchuan, Sichuan, China/ China EF577275 EF577328 Zhao.SC06-09 Ajania przewalskii Poljakov Ma’erkang, Sichuan, China/ China EF577279 EF577332 Zhao.SC06-14 Ajania remotipinna (Hand.-Mazz.) Taibaishan, Shaanxi, China/ China EF577280 EF577333 Ling ex Shih Zhao.TB06-01 Ajania shiwogiku Kitam. var. kinokuniense Tsukuba, Japan/Chen06121 Japan EF577282 EF577335 (Shimot. et Kitam.) Kitam. Sect. Ajania Ajania nematoloba (Hand.-Mazz.) Ling ex Shih Baiyin, , China/ China EF577276 EF577329 Zhao.GS06-05 Ajania fastigiata (Winkler) Poljakov Mulei, , China/ China, C. Asia EF577272 EF577325 Zhao.XJ06-07 Ajania fruticulosa (Ledeb.) Poljakov Helanshan, , China/ C. Asia, E. Siberia, EF577273 EF577326 Zhao.HL06-01 , China Ajania potaninii (H. Kraschen.) Poljakov Jinchuan, Sichuan, China/ China EF577278 EF577331 Zhao.SC06-06 Ajania pacifica (Nakai) Bremer et Humphries Nanjing, Jiangsu, China/ Japan EF577277 EF577330 Zhao0401 Ajania rupestris (Matsum. ex Koidz.) Muld. Kim et al., unpublished Japan AB234732 (IGS) Phaeostigma Muld. Phaeostigma salicifolium (Mattf.) Muld. Jingyuan, Gansu, China/ China EF577281 EF577334 Zhao.GS06-02 Phaeostigma variifolium (Chang) Muld. Taibaishan, Shaanxi, China/ China EF577283 EF577336 Zhao.TB06-02 Brachanthemum DC. Brachanthemum pulvinatum (Hand.-Mazz.) Jingyuan, Shaanxi, China/ China EF577291 EF577344 Shih Zhao.GS06-03 Brachanthemum titovii H. Kraschen. Valle`s et al. 2003 (ITS) China, Russia, AF504185, Kazakstan AF504158 Crossostephium Less. Crossostephium chinense (L.) Makino Xiamen, Fujian, China/ China, Japan, EF577293 EF577346 Zhao.XM05-01 Philippines Elachanthemum Ling et Y. R. Ling Elachanthemum intricatum (Franchet) Ling et Y. Helanshan, Ningxia, China/ China, Mongolia EF577313 EF577368 R. Ling Zhao.HL06-06 Stilpnolepis H. Kraschen. Stilpnolepis centiflora (Maxim.) H. Kraschen. Watson et al. 2002 (ITS) China, Mongolia AY127695, AY127696 Filifolium Kitam. Filifolium sibiricum (L.) Kitam. Watson et al. 2002 (ITS) China, Korea, Far East AY127687, AY127688

123 158 H.-B. Zhao et al.

Table 1 continued Taxon Source/voucher Distribution Accession number ITS (ITS1, ITS2) trnL-F

Kaschgaria Poljakov Kaschgaria brachanthemoides Valle`s et al. 2003 (ITS) China, C. Asia AF504189, (Winkler) Poljakov AF504162 Kaschgaria komarovii (H. Kraschen. Watson et al. 2002 (ITS) China, Mongolia, AY127689, et N. Rubtzov) Poljakov C. Asia AY127690 Neopallasia Poljakov Neopallasia pectinata Heshuo, Xinjiang, China/ China, C. Asia, EF577316 EF577372 (Pallas) Poljakov Zhao.XJ06-05 S. Siberia, Mongolia Artemisia L. Artemisia abrotanum L. Kornkven et al. 1998 (ITS) N. America AF060473, AF061390 Artemisia absinthium L. Kornkven et al. 1999 (ITS) N. America AF045394, AF079946 Artemisia annua L. Kornkven et al. 1999 (ITS) N. America AF045383, AF079935 Artemisia arborescens L. Kornkven et al. 1999 (ITS) Middle east N. Africa AF045393, AF079945 Artemisia arbuscula Nutt. Kornkven et al. 1998 (ITS) Western N. America AF060464, AF061380 Artemisia chamaemelifolia Villars. Kornkven et al. 1999 (ITS) C and S.W. Europe, AF045388, C. East AF079940 Artemisia fragrans Willd. Kornkven et al. 1999 (ITS) C. Asia AF045406, AF079957 Artemisia rigida (Nutt.) A. Gray Kornkven et al. 1998 (ITS) W. USA AF060465, AF061382 Artemisia rupestris L. Kornkven et al. 1998 China, C. Asia, AF060475, AJ298134 W. Siberia, AF061391 Mongolia, Canada Artemisia sericea G. Weber Nanjing, Jiangsu, China/ China, Russia, EF577290 EF577343 Chen0625 Siberia, Mongolia Artemisia sieberi Besser Kornkven et al. 1999 (ITS) C. East and C. Asia AF045407, AF079958 Artemisia splendens Willd. Kornkven et al. 1999 (ITS) C. East AF045396, AF079948 Artemisia sublessiniana (Kell.) Krasch. Kornkven et al. 1998 (ITS) C. Asia, Siberia and AF060478, ex Poljakov Mongolia AF061394 Artemisia tournefortiana Reichenb. Kornkven et al. 1999 (ITS) C. East and C.E. Asia AF045384, AF079936 Artemisia verlotorum Lamotte. Kornkven et al. 1999 (ITS) China, Africa, America AF045387, AF079939 Artemisia vulgaris L. Watson et al. 2002 (ITS) Eurasia, N. America, AJ296389 AJ296459 N. Africa AJ296424 Sphaeromeria Nutt. Sphaeromeria diversifolia (D.C. Eaton) Watson et al. 2002 (ITS) Western N. America AY127693, AY127694 Rydb. Subtribe Chrysantheminae Bremer & Humphries Cassini Ismelia carinata (Schousb.) Francisco-Ortega et al. Cultivated L77764 AJ748793 Schultz-Bip. 1997 (ITS); Oberprieler 2004 (IGS)

123 Molecular phylogeny of Chrysanthemum, Ajania, and its allies 159

Table 1 continued Taxon Source/voucher Distribution Accession number ITS (ITS1, ITS2) trnL-F

Glebionis Cassini coronaria (L.) Cassini ex Spach Nanjing, Jiangsu, China/ Cultivated EF577292 EF577345 Zhao0501 Webb ex Schultz-Bip. Argyranthemum frutescens (L.) Schultz-Bip. Tokyo, Japan/Chen0615 Cultivated EF577287 EF577340 Subtribe Tanacetinae Bremer & Humphries Opisthopappus Shih Opisthopappus taihangensis (Ling) Shih Yuntaishan, Henan, China/ China EF577318 EF577373 Zhao.HN06-03 Tanacetum L. Tanacetum cinerariifolium (Trevir.) Schultz-Bip. Kunming, Yunnan, China/ Cultivated EF577319 EF577374 Chen9905 Tanacetum parthenium (L.) Schultz-Bip. Chiba, Japan/Chen0621 Cultivated EF577320 EF577375 Tanacetum ptarmiciflorum (Webb & Berth.) Matsudo, Japan/Chen0620 EF577322 EF577377 Schultz-Bip. Tanacetum tatsienense (Bureau et Franchet) Kangding, Sichuan, China/ China EF577321 EF577376 Bremer et Humphries Zhao.SC06-02 Tanacetum vulgare L. Tsukuba, Japan/Chen0618 Europe, Asia EF577323 EF577378 Hippolytia Poljakov Hippolytia alashanensis (Ling) Shih Helanshan, Ningxia, China/ China EF577314 EF577369 Zhao.HL06-02 Hippolytia dolichophylla (Kitam.) Bremer et Oberprieler 2004 C. Asia AJ748784, AJ748785 AJ748791 Humphries Subtribe Leucantheminae Bremer & Humphries Cass. Coleostephus multicaulis (Desf.) Durieu Hiroshima, Japan/ Cultivated AJ296393, AJ296428 AJ296467 Chen0616 Leucanthemella Tzvelev (L.) Tzvelev Francisco-Ortega et al. E. Europe L77766 AJ748794 1997 (ITS); Oberprieler 2004 (IGS) Miller Lam. Beijing, China/Zhao0502 Cultivated EF577315 EF577370 Nipponanthemum Kitam. Nipponanthemum nipponicum Chiba, Japan/Chen0617 Japan EF577317 EF577371 (Franchet ex Maxim.) Kitam. Subtribe Matricariinae Bremer & Humphries L. Matricaria matricarioides Porter ex Britton Oberprieler and Vogt 2000 N. America AJ296412, AJ296447 AJ296487 Ruiz Lopez & Pavon Soliva anthemifolia R. Br. Yuan et al., unpublished S. USA AY947414 (ITS) Schultz-Bip. Tripleurospermum perforatum (Me´rat) Lainz Francisco-Ortega et al. Europe, Asia, America L77782 AJ296489 1997 (ITS); Oberprieler and Vogt 2000 (IGS) Subtribe Achilleinae Bremer & Humphries L. Achillea acuminata (Ledeb.) Schultz-Bip. Taibaishan, Shaanxi, China/ China, Mongolia, Japan, EF577284 EF577337 Zhao.TB06-08 W. Siberia, Far East

123 160 H.-B. Zhao et al.

Table 1 continued Taxon Source/voucher Distribution Accession number ITS (ITS1, ITS2) trnL-F

Achillea wilsoniana Heimerl ex Hand.-Mazz. Taibaishan, Shaanxi, China/ China EF577285 EF577338 Zhao.TB06-09 Anacylus L. Anacylus pyrethrum (L.) Lagasca var. Francisco-Ortega et al. W. Mediterranean L77778 depressus (Ball) Maire 1997 (ITS) Miller (L.) All. Watson et al. 2002 (ITS) S. Europe, N. Africa AY127671, AY127672 OUTGROUP Subtribe Ursiniinae Bremer & Humphries Athanasia crithmifolia (L.) L. Francisco-Ortega et al. S. Africa AF155241, AF155278 2001 (ITS) Athanasia juncea (D. C.) D. Dietr. Bayer and Cross 2003 S. Africa AF452497 (IGS) Ursinia anthemoides (L.) Poiret Francisco-Ortega et al. S. Africa AF155264, AF155301 2001 (ITS) Ursinia trifida (Thunb.) N. E. Br. Bayer and Cross 2003 S. Africa AF452507 (IGS) Tribe Astereae Aster glehnii F. Schmidt Hong et al., unpublished Far East, Japan, Korea AY722010 (ITS) Aster indicus L. Hsieh et al., unpublished China EF108396 (ITS) Aster novae-angliae L. Bayer and Starr 1998 (IGS) New England U82019 Tribe Calenduleae Calendula officinalis L. Wagstaff and Breitwieser Cultivated AF422114 U82021 2002 (ITS); Bayer and Starr 1998 (IGS) (L.) Norl. Wagstaff and Breitwieser W. USA AF422131 2002 (ITS) Osteospermum clandestinum (Less.) Norl. Bayer and Starr 1998 (IGS) U82049 Outgroup: The sequence of Athanasia L. was the combination of the sequences of A. crithmifolia (L.) L. and A. juncea (DC.) D. Dietr.; that of Ursinia Gaertner was the combination of the sequences of U. anthemoides (L.) Poiret and U. trifida (Thunb.) N. E. Br.; that of Aster L. was the combination of the sequences of A. glehnii F. Schmidt and A. novae-angliae L.; and that of Osteospermum L. was the combination of the sequences of O. fruticosum (L.) Norl. and O. clandestinum (Less.) Norl. in the reconstruction of the combination of ITS and IGS trees

TreeView (Page 1996) and further edited using Mega v4.0 consensus tree of all most-parsimonious trees (Fig. 1) shows (Tamura et al. 2007). two major clades. Clade A included all the subtribe Artemisiinae taxa, Hippolytia, Opisthopappus and one spe- cies of Tanacetum (subtribe Tanacetinae), Leucanthemella Results and Nipponanthemum (subtribe Leucantheminae). Two main subclades were recognizable, one subclade (a) com- Phylogeny based on the nrDNA ITS prising Chrysanthemum, Arctanthemum, Ajania, Opistho- pappus and Elachanthemum (96% bootstrap value, 88% The aligned ITS sequence was 447 bp in length, and 285 of posterior probability), and the other subclade (b) including these characters were phylogenetically informative. Artemisia, Crossostephium, Neopallasia and Sphaeromeria. Resulting phylogenetic trees obtained by maximum parsi- In addition, two small subclades (i.e., two species of Phae- mony (MP) and Bayesian analyses were almost consistent in ostigma and the Kaschgaria group) within clade A were also topology. Heuristic parsimonious research yielded 51,465 recognized. Clade B grouped all the Chrysantheminae taxa equally parsimonious trees with a length of 990 steps, a CI of along with most of the Leucantheminae, Tanacetinae, 0.5495, a RI of 0.7854 and a RC of 0.4316. The 50% strict Matricariinae and Achilleinae taxa. Soliva anthemifolia

123 Molecular phylogeny of Chrysanthemum, Ajania, and its allies 161

Fig. 1 The 50% strict consensus tree of equally most- parsimonious 51,465 trees based on nuclear ribosomal ITS sequence variation. Numbers followed by ‘%’ are bootstrap values derived from 1,000 replicates using parsimony criteria, and numbers followed by ‘p’ are posterior probabilities derived from Bayesian analysis using likelihood criteria (only the values above 50% were stated). Subtribal affinity of taxa is indicated by the following bar patterns: Artemisiinae (hatched box); Tanacetiinae (blank box); Achilleinae (grid box); Chrysantheminae (reverse hatched box); Leucantheminae (dotted box); Matricariinae (dense crossed box); Ursiniinae (black dotted box). A and B represent two clades; a and b represent two subclades within clade A

R. Br. was an outlier, indicating a distant relationship to the position 364 common to Phaeostigma indicated a close Anthemideae. relationship between these taxa, but the distinct difference from Ajania. Similarly, a 14-bp deletion observed at Phylogeny analysis based on the cpDNA trnL-F IGS position 104 was shared between Achillea acuminata (Ledeb.) Schultz-Bip. and Achillea wilsoniana Heimerl ex The aligned IGS sequence was 453 bp long, but only 50 of Hand.-Mazz. these characters were phylogenetically informative. The derived phylogenetic trees were not able to clearly resolve Combined phylogeny based on ITS and trnL-F IGS all phylogenetic relationships (Fig. 2), but some deletions and insertions were suggestive of interspecific and inter- The combined ITS and IGS alignment of 61 taxa was generic relationships. For example, a 6-bp insertion at 904 bp long with 306 phylogenetically informative

123 162 H.-B. Zhao et al.

Fig. 2 The 50% strict consensus tree of equally most- parsimonious 293,556 trees based on the chloroplast trnL-F IGS sequence variation. Notation is identical to that given in Fig. 1

characters. Similarly, the phylogenetic trees obtained by basal grade of the clade A according to our results, and maximum parsimony (MP) and Bayesian analyses were once were considered being sister relationships to subtribe almost consistent in topology. A heuristic research yielded Artemisiinae (Bremer and Humphries 1993) and even were 78,217 phylogenetic trees with a length of 935 steps, a CI transferred to subtribe Artemisiinae (Oberprieler et al. of 0.6610, a RI of 0.7960, and a RC of 0.5261. The 50% 2007). strict consensus tree of all most-parsimonious trees is presented in Fig. 3. The joint analysis was very similar to The Artemisia group that based on the ITS sequence alone, mainly reproducing clade A (further divided into two subclades a and b) and The Artemisia group represents the major part of the sub- clade B. tribe Artemisiinae, including the widespread Artemisia, some small Asian genera such as Neopallasia, Crosso- stephium, Stilpnolepis, Elachanthemum, Kaschgaria and Discussion Filifolium, and two small North American genera Picro- thamnus and Sphaeromeria (Bremer and Humphries 1993). Phylogeny of the subtribe Artemisiinae Watson et al. (2002) excluded Stilpnolepis, Elachanthe- mum and Kaschgaria from the Artemisia group based on The monophyly of subtribe Artemisiinae chiefly classified ITS phylogeny. The ITS phylogeny by Valle`s et al. (2003) into the Artemisia group and the Chrysanthemum group can showed the satellite genera Filifolium, Mausolea, Picro- be retrieved when the phylogenetic positions of some thamnus, Sphaeromeria and Turaniphytum are clearly unsolved taxa, i.e., two species of Hippolytia, one species placed within the Artemisia group, whereas other genera of Tanacetum (subtribe Tanacetinae) and Leucanthemella such as Brachanthemum, Elachanthemum, Hippolytia and and Nipponanthemum (subtribe Leucantheminae) are re- Kaschgaria fall outside this large group. The phylogeny newedly considered. Most of these taxa tend to lie at the results from nrDNA ETS and ITS sequences of Sanz et al. 123 Molecular phylogeny of Chrysanthemum, Ajania, and its allies 163

Fig. 3 The 50% strict consensus tree of equally most- parsimonious 78,217 trees based on the joint analysis of nuclear ribosomal ITS and the chloroplast trnL-F IGS sequence variation. Notation is identical to that given in Fig. 1

(2008) support that the Artemisia group comprises two Stilpnolepis is a monotypic genus distributed across main evolutionary lineages including (1) the subgenus northern and northwestern China and Mongolia (Shih and Dracunculus clade including various Artemisia species and Fu 1983; Ling 1987). Stilpnolepis and Elachanthemum are four Asian genera (Filifolium, Mausolea, Neopallasia and often treated as the single genus Stilpnolepis on account of Turaniphytum), and (2) the remaining subgenera of Arte- their common discoid and laxly corymbose capitula (Shih misia plus the two North American genera (Picrothamnus 1985; Bremer and Humphries 1993). Watson et al. (2002), and Sphaeromeria). Tkach et al. (2008a, b) also indicate however, treated them as two separate genera on the basis Kaschgaria is close to Artemisia and shows a sister group of ITS data, a conclusion that is consistent with ours. relationship to Artemisia, and Elachanthemum originated much later from within other genera. The conclusions were The Chrysanthemum group almost supported by the present results; however, we fur- ther suggest that Filifolium also should be removed from In the present study, Chrysanthemum, Ajania, Arctanthe- the Artemisia group. mum, Elachanthemum and Opisthopappus make up the Poljakov (1957) established the genus Kaschgaria by Chrysanthemum group (subclade a). However, some removing Kaschgaria brachanthemoides from Artemisia intergeneric relationships and generic delimitations within and Kaschgaria komarovii from Tanacetum mainly based this group are ambiguous. on the stellate nature of their corolla hairs. The monophyly The relationships among Chrysanthemum, Ajania, of Kaschgaria distributing in China and central Asia is well Phaeostigma and Arctanthemum. The genus Chrysanthe- supported by our ITS phylogeny. Bremer and Humphries mum is distributed across Asia (mainly China and Japan), (1993) suggested that Kaschgaria might be more closely with only one species [D. zawadskii (Herb.) Tzvelev] related to Sphaeromeria. And Sanz et al. (2008) supported extending into eastern Europe. The genus Ajania is mainly a sister group relationship between Kaschgaria and the distributed in central Asia and China (mainly northern and Artemisia group. Neither the former nor the latter propo- western China). Ajania distinctly differs from Chrysan- sition is supported by our analyses. themum by its smaller, disciform and densely corymbose

123 164 H.-B. Zhao et al. capitula (Shih and Fu 1983). Poljakov (1955) has specu- and Mongolia (Ling 1987; Ling and Ling 1978). Its phy- lated that Ajania is a very ancient group, sharing its logenetic status remains controversial, with Bremer and ancestors with Artemisia. All the examined Ajania species Humphries (1993) placing it as a Stilpnolepis species clustering within the Chrysanthemum group are closer to within the Artemisia group, while Watson et al. (2002) and Chrysanthemum and allied taxa than to Artemisia. Tkach et al. (2008a) excluded it from this group and treated Although Bremer and Humphries (1993) treated Chrysan- it as a separate genus. Our ITS phylogeny suggests that it themum and Ajania as distinct genera on the basis of belongs within the Chrysanthemum group, whereas Stilp- capitulum morphology, the boundary between these two nolepis is placed in the primitive position. genera was not definable by either ITS and/or IGS Placement of Opisthopappus. It is a derivative of sequences. Intergeneric hybridization between taxa Tanacetum (Bremer and Humphries 1993), within which belonging to these groups can be readily achieved (Abd there are two species endemic to the Henan, Hebei and El-Twab et al. 1999; Kondo and Abd El-Twab 2002; Yin provinces of China. It differs from Tanacetum and 2005;Li2006; Zhao et al. 2007), providing a further Spathipappus by its thickly ribbed cypsela and mainly indication of a close relationship among them. We specu- abaxial pappus of separate scales, rather than having a late that the divergence between Chrysanthemum and corona or an auricle (Shih 1979a). Here, O. taihangenesis Ajania was relatively recent, and there was a parallel (Ling) Shih was grouped within the Chrysanthemum group evolution among different sub-groups in this Chrysanthe- and located in the basal grade of this group, separated from mum–Ajania complex, a conclusion reinforced by AFLP subtribe Tanacetinae (Fig. 1). This shows its primitive results (Zhao et al. unpublished). character and position within the Chrysanthemum group. A few Ajania species were placed into Phaeostigma Thus, it appears reasonable to transfer it from subtribe (Muldashev 1981, 1983), and Bremer and Humphries (1993) Tanacetinae to subtribe Artemisiinae. thought the species A. pallasiana (Fischer ex Besser) Pol- jakov, A. latifolia Shih, A. rupestris (Matsum. ex Koidz.) The remaining taxa Muld. and A. shiwogiku (Kitam.) are all taxa similar to Phaeostigma. The present phylogeny suggests that a revision Tzvelev (1961) noted a close relationship between of this scheme is necessary, in which A. salicifolia and A. Brachanthemum and Chrysanthemum, emphasized also by variifolia should be transferred to Phaeostigma, which was the similar thin-walled, myxogenic fruits, lacking a pappus. monophyletic supported by ITS and IGS data. This is also It belongs to the Chrysanthemum group according to the proved by a 6-bp insertion of IGS sequences in Phaeostigma morphological cladogram (Bremer and Humphries 1993). salicifolium and Phaeostigma variifolium compared to the However, the phylogeny based on ITS alone and combined taxa of Ajania. Additionally, according to their morpholog- ITS and IGS suggests the exclusion of Brachanthemum ical characteristics, the three species endemic to China [A. from the Chrysanthemum group. The shrubby habit, few- quercifolia, A. latifolia and A. ramosa (Chang) Shih] should lobed leaves and rather small, cyathiform to urceolate also be transferred to Phaeostigma, forming a genus of five capitula of the species in this genus suggest a monophyletic species (series Quercifoliae and Variifoliae, Shih and Fu origin (Bremer and Humphries 1993); however, mono- 1983), while A. pallasiana, A. rupestris and A. shiwogiku phyly of this genus is not supported by the ITS data should be retained within Ajania. Further morphological and (Fig. 1). The simultaneous existence of radiate and discoid molecular investigations about these related taxa are needed capitulum characters also shows this genus is polypheletic. to clarify the delimitation of Phaeostigma. Phaeostigma and Leucanthemella and Nipponanthemum are likely to be Ajania were included in the Chrysanthemum group (Bremer closely related to the Chrysanthemum species (Bremer and and Humphries 1993), which was close to Chrysanthemum. Humphries 1993). A sister group relationship of Leucan- However, according to our results, Phaeostigma lays outside themella (eastern Europe and the Far East) and Nippo- of the Chrysanthemum group. nanthemum (Japan) to subtribe Artemisiinae has been The Arctanthemum found in Arctic Eurasia, eastern Asia revealed by molecular phylogeny (Francisco-Ortega et al. and Arctic North America formerly comprised a section of 1997; Watson et al. 2002; Oberprieler 2005). Successful Chrysanthemum (Ohashi and Yonekura 2004). The phy- intergeneric hybridizations between members of Leucan- logenetic analysis showed A. arcticum (L.) Tzvelev locates themella, Nipponanthemum and Chrysanthemum also within the Chrysanthemum–Ajania complex and is closely imply that there is a close relationship among them (Ohishi related to this group (Fig. 1). Intergeneric relationships et al. 1996; Kondo and Abd El-Twab 2002). Here, L. ser- remain unresolved, or the generic independence of otina (L.) Tzvelev and Nipponanthemum are included Arctanthemum should be reconsidered. within the Artemisiinae rather than being merely sister Placement of Elachanthemum. Elachanthemum is a groups of it. This finding is consistent with opinion of monotypic genus found in northern and northwestern China Oberprieler et al. (2007) where these two genera were 123 Molecular phylogeny of Chrysanthemum, Ajania, and its allies 165 transferred to subtribe Artemisiinae based on ITS phylog- the Artemisia group with microechinate pollen derived eny and morphological evidence. from the radiate Chrysanthemum group with echinate The genus Hippolytia was established for a number of exine pollens. Within this subtribe discoid capitula are central Asian discoid Tanacetum species based on discoid most parsimoniously interpreted as derived from disci- capitula and cypselas without pappus (Bremer and form, rather than from radiate capitula (Bremer and Humphries 1993). The composition of Hippolytia remains Humphries 1993). However, the reconstruction of capitula uncertain. Tzvelev removed some of Poljakov’s Hippolytia type evolution based on ITS and ETS sequences suggests species to Ajania, while Shih (1979b) revised and added a discoid-homogamous capitula as the ancestral condition in number of Chinese species. Tzvelev in Flora URSS (in subtribe Artemisiinae (Sanz et al. 2008). Our results Bremer and Humphries 1993) stated that Hippolytia is propose that the Chrysanthemum group includes radiate related to Chrysanthemum and Ajania, and is intermediate Chrysanthemum, Arctanthemum and Opisthopappus, dis- between these two genera. Moreover, Hippolytia and ciform Ajania and discoid Elachanthemum; the Artemisia Tanacetum were previously considered as sister groups to group includes discoid Artemisia, Neopallasia, Crosso- the Artemisiinae based on morphology (Bremer and stephium and Sphaeromeria; all the taxa of clade B and Humphries 1993). Oberprieler et al. (2007) also placed the outgroups are of radiate capitula (Fig. 5). Thus, Hippolytia in subtribe Artemisiinae based on ITS phylog- radiate capitula maybe the ancestral condition in subtribe eny. In our phylogenetic tree, two Hippolytia species and Artemisiinae, and then the reversal takes place multi- Tanacetum tatsienense are closer to the Artemisiinae rather times among discoid, disciform and radiate capitula dur- than to the other taxa within their various subtribes ing the evolution. (Fig. 1). So the placement of these taxa remains unre- solved, and further investigations in details should be made Geographical distribution in the Chrysanthemum group to better describe the phylogenetic positions and interge- neric relationships of these genera. The phylogeny based on the ndhF of Watson et al. (2000) revealed a basal grade of Southern Hemisphere genera that Evolution of floral characters are sister to a mostly Northern Hemisphere clade, and the latter consisted of a basal grade of predominantly Asian According to previous research about pollen morphology in genera, which includes generic assemblages related to Anthemideae (Chen and Zhang 1991; Jiang and Lin 1996; Artemisia and Chrysanthemum, sister to a monophyletic Wan et al. 1999; Martı´n et al. 2001, 2003; Jiang et al. 2005; group of Mediterranean and more widespread, northwest- Zhao et al. unpublished), all taxa within the Tanacetinae, ern Eurasian genera (Oberprieler et al. 2006). Leucantheminae, Matricariinae, Achilleinae, Chrysant- Under the current phylogenetic model, Stilpnolepis, heminae and outgroups, along with the Chrysanthemum Brachanthemum or their allies should be the primitive taxa group except for Elachanthemum and some remaining taxa or the ancestral taxa of subtribe Artemisiinae supported not share the echinate exine pollen ornamentation ( only by our data, but also the opinions of Watson et al. pollen type); the Artemisia group, Elachanthemum, and the (2002) and Sanz et al. (2008), and Opisthopappus locates other part of the remaining taxa have microechinate pollen the basal position of the Chrysanthemum group. Most of (Artemisia pollen type), while that of Phaeostigma and these taxa originated in central or western Asia and western Stilpnolepis has short spines, intermediate in length China. At the same time, the majority of taxa within the between the two formers in Artemisiinae (Fig. 4). This Chrysanthemum group such as Ajania, Chrysanthemum, shows the Anthemis pollen type with long spines is the Opisthopappus and Arctanthemum originated from western ancestral trait in Artemisiinae (Sanz et al. 2008). And the or central Asia, western China and Mongolia, to eastern evolution of Anthemis pollen type of the Chrysanthemum Asia, up to eastern Siberia and the Far East. Combining the group and Artemisia pollen type of the Artemisia group in phylogeny with geographic distribution, we propose that the recent events should be paraphyletic. the ancestors of the Chrysanthemum group originated in The capitulum morphology of all the examined taxa western or central Asia; that there was a gradual north- is presented in Fig. 5. Ajania, Artemisia, Kaschgaria, easterly movement into western China and Mongolia, fol- Filifolium, Neopallasia, Crossostephium and Phaeostigma lowed by an easterly spread into eastern Asia and capitula are disciform, and Elachanthemum, Stilpnolepis succeeded by an easterly and northeasterly migration into and Hippolytia capitula are discoid without ray florets; the eastern Siberia and the Far East; and finally that the species remaining taxa along with outgroups have radiate capit- were dispersed widely in every region, with geographical ula. According to the Bremer and Humphries (1993) barriers such as mountains and altiplanos creating bound- morphologically based phylogeny, the Artemisiinae lie in aries that encouraged the origin and evolution of new taxa an intermediate position within the Anthemideae, while such as Opisthopappus and Tridactylina. Tkach et al. 123 166 H.-B. Zhao et al.

Fig. 4 The evolution of pollen morphology in the eastern Asian Anthemideae mapped onto a maximum parsimony ITS tree. Blue, red and saffron boxes represent, respectively, echinate exine pollen, microechinate pollen and short-spiny pollen

(2008b) considered that, due to a conspicuous richness of Phylogeny and genetic improvement Artemisia species (eight species from four independent lineages) in outmost northeastern Asia, for Artemisia this Knowledge of phylogeny provides a guide for formulating region (perhaps including western parts of Alaska) seems genetic improvement strategies targeting interspecific and to have been the evolutionary center of two small radia- intergeneric hybridization. Like the chrysanthemum, not tions of three endemic species each; thus, it could addi- only have many interspecific wide crosses been used to tionally be characterized as a minor evolutionary hot spot improve the characteristics of commercial varieties (Fukai for Artemisia in the Arctic and, therefore, possibly also for et al. 2000; Fukai 2003; Li et al. 2008), but also a number other taxa. Some Chrysanthemum taxa and Arctanthemum of intergeneric crosses have been successfully performed should belong to those. (Ohishi et al. 1996; Abd El-Twab et al. 1999, Abd El-Twab

123 Molecular phylogeny of Chrysanthemum, Ajania, and its allies 167

Fig. 5 The evolution of capitulum morphology in the eastern Asian Anthemideae mapped onto the maximum parsimony ITS tree. Green, purple and saffron boxes represent, respectively, discord, disciform and radiate capitula

and Kondo 1999, 2001; Kondo and Abd El-Twab 2002, interesting as targets for intergeneric crossing aimed at the Kondo et al. 2003; Yin 2005;Li2006; Zhao et al. 2007). transfer of such traits to commercial varieties. The phylogenetic information reported here suggests that the probability of success in interspecific and intergeneric Acknowledgments The authors gratefully acknowledge Z.-Q. crosses would be higher between taxa within the same Yang, Y.-F. Wang, G.-B. Ren, J. Zhang, X.-Q. An and G.-L. Bai for their help in collecting plant materials and Dr. Hengbin Miao for his clade and between the taxa of subtribe Artemisiinae and technology support in experiments. The authors also thank Dr. Kare subtribe Tanacetinae. Some Anthemideae species possess a Bremer for his presentation copy ‘‘The generic monograph of the spectrum of potentially desirable traits, including silvery Asteraceae-Anthemideae.’’ This research was supported by the leaves, multiple branching, drought and cold tolerance, and National Natural Science Foundation of China and National Key Technology R&D Program of the Ministry of Science and Technol- insect resistance. These species are thus particularly ogy of the People’s Republic of China (grant no. 2006BAD01A1806)

123 168 H.-B. Zhao et al. and the Shanghai Key Science and Technology for Agriculture Kitamura S (1978) Dendranthema et Nipponanthemum. Acta Phyto- Promotion program (2006 no. 4-3). taxon Geobot 29:165–170 Kluge AG, Farris JS (1969) Quantitative phyletics and the evolution of anurans. Syst Zool 18:1–32 Kondo K, Abd El-Twab MH (2002) Analysis of inter- and intrs- References generic relationships sensu stricto among the members of Chrysanthemum sensu lato by using fluorescent in situ hybrid- Abd El-Twab MH, Kondo K (1999) Identification of nucleolar ization. Chromosome Science 6:87–100 organizing regions and parental chromosomes in F1 hybrid of Kondo K, Abd El-Twab MH, Idesawa R, Kimura S, Tanaka R (2003) Dendranthema japonica and Tanacetum vulgare simultaneously Genome phylogenetics in Chrysanthemum sensu lato. In: Sharma by fluorescence in situ hybridization and fluorescence genomic AK, Sharma A (eds) Plant genome––biodiversity and evolution, in situ hybridization. Chromosome Science 3:59–62 vol 1A. Phanerogams. Science Publishers, Inc., Plymouth, Abd El-Twab MH, Kondo K (2001) Genome territories of Dendrant- pp 117–200 hema horaimontana in mitotic nuclei of F1 hybrid between Kornkven A, Watson L, Estes J (1998) Phylogenetic analysis of D. horaimontana and Tanacetum parthenium. Chromosome Artemisia section Tridentatae (Asteraceae) based on sequences Science 5:63–71 from the internal transcribed spacers (ITS) of nuclear ribosomal Abd El-Twab MH, Kondo K, Hong DY (1999) Isolation of a DNA. Am J Bot 85:1787–1795 particular chromosome of Ajania remotipinna in a chromosome Kornkven A, Watson L, Estes J (1999) A molecular phylogeny of complement of an artificial F1 hybrid of Dendranthema lavan- Artemisia sect. Tridentatae (Asteraceae) based on chloroplast dulifolia 9 Ajania remotipinna by use of genomic in situ DNA restriction site variation. Syst Bot 24:69–84 hybridization. Chromosome Science 3:21–28 Lassner MW, Peterson P, Yoder JI (1989) Simultaneous amplification Bayer RJ, Cross EW (2003) A reassessment of tribal affinities of of multiple DNA fragments by polymerase chain reaction in the Cratystylis and Haegiela (Asteraceae) based on three chloroplast analysis of transgenic and their progeny. Plant Mol Biol DNA sequences. Plant Syst Evol 236:207–220 Rep 7:116–128 Bayer RJ, Starr JR (1998) Tribal phylogeny of the Asteraceae based Li W (2006) Preliminary studies on intergeneric hybridization within on two non-coding chloroplast sequences, the trnL intron and Chrysanthemum in broad sense. Dissertation, Beijing Forestry trnL/trnF intergenic spacer. Ann Missouri Bot Gard 85:242–256 University Bremer K, Humphries C (1993) The generic monograph of the Li XL, Chen FD, Zhao HB (2008) Compatibility of interspecific cross Asteraceae–Anthemideae. Bull Br Museum Bot Ser 23:71–177 in Dendranthema genus. Acta Hortic Sin 35:257–262 Chen SB, Zhang JT (1991) A study on pollen morphology of some Ling YR (1987) On the difference between Elachanthemum Y. Ling Chinese genera in tribe Amthemideae. Acta Phytotaxon Sin et Y.R. Ling and Stilpnolepis Krasch. Acta Phytotaxon Sin 29:246–251 25:390–392 D’Andrea S, Caramiello R, Ghignone S, Siniscalco C (2003) Ling Y, Ling YR (1978) Elachanthemum, genus novum familiae Systematic studies on some species of the genus Artemisia: compositarum. Acta Phytotaxon Sin 16:61–65 biomolecular analysis. Plant Biosyst 137:121–130 Maddison DR (1991) The discovery and importance of multiple Farris JS (1989) The retention index and homoplasy excess. Syst Zool islands of most parsimonious trees. Syst Zool 40:315–328 38:406–407 Martı´n J, Torrell M, Valle`s J (2001) Palynological features as a Felsenstein J (1985) Confidence limits on phylogenies: an approach systematic marker in Artemisia L. and related genera (Astera- using bootstrap. Evolution 39:783–791 ceae, Anthemideae). Plant Biol 3:372–378 Francisco-Ortega J, Santos-Guerra A, Hines A, Jansen RK (1997) Martı´n J, Torrell M, Korobkov AA, Valle`s J (2003) Palynological Molecular evidence for a Mediterranean origin of the Macaro- features as a systematic marker in Artemisia L. and related nesian endemic genus Argyranthemum (Asteraceae). Am J Bot genera (Asteraceae, Anthemideae), II: implications for subtribe 84:1595–1613 Artemisiinae delimitation. Plant Biol 5:85–93 Francisco-Ortega J, Barber JC, Santos-Guerra A, Febles-Hernandez Muldashev AA (1981) A new genus Phaeostigma (Asteraceae) from R, Jansen RK (2001) Origin and evolution of the endemic genera the . Botanischeskii Zhurnal 66:584–588 of Gonosperminae (Asteraceae: Anthemideae) from the Canary Muldashev AA (1983) A critical review of the genus Ajania Islands: evidence from nucleotide sequences of the internal (Asteraceae-Anthemideae). Botanischeskii Zhurnal 68:207–214 transcribed spacers of the nuclear ribosomal DNA. Am J Bot Nylander JAA (2004) MrModeltest v2. Program distributed by the 88:161–169 author. Evolutionary Biology Centre, Uppsala University Fukai S (2003) Dendranthema species as chrysanthemum genetic Oberprieler C (2002) A phylogenetic analysis of Chamaemelum Mill. resourses. Acta Hortic 620:223–230 (Compositae: Anthemideae) and related genera based upon Fukai S, Nagira T, Goi M (2000) Cross compatibility between nrDNA ITS and cpDNA trnL/trnF IGS sequence variation. Bot J chrysanthemum (Dendranthema grandiflorum) and Dendranthe- Linn Soc 138:255–273 ma species. Acta Hortic 508:337–340 Oberprieler C (2004) On the taxonomic status and the phylogenetic Iwatsuki K, Takasi Y, David EB, Hideaki O (1997) Flora of Japan. relationships of someunispecific Mediterranean genera of Com- III. Angiospermae Dicotyledoneae Sympetalae(b). Kodansha positae–Anthemideae. I. Brocchia, Endopappus and Heliocauta. Ltd, Tokyo Willdenowia 34:39–57 Jiang L, Lin YR (1996) Pollen morphology of Artemisia in China. Oberprieler C (2005) Temporal and spatial diversification of Circum- J Trop Subtrop Bot 4:1–14 Mediterranean Compositae–Anthemideae. Taxon 54:951–966 Jiang L, Wang Q, Ye LZ, Lin YR (2005) Pollen morphology of Oberprieler C, Vogt R (2000) The position of Vogt Artemisia L. and its systematic significance. J Wuhan Univ & Oberprieler and the phylogeny of Mediterranean Anthemideae (Natural Science Edition) 10:448–454 (Compositae) as inferred from nrDNA ITS and cpDNA trnL/ Kim KJ, Jansen RK (1995) ndhF sequence evolution and the major trnF IGS sequence variation. Plant Syst Evol 225:145–170 clades in the sunflower family. Proc Natl Acad Sci 92:10379– Oberprieler C, Vogt R, Watson LE (2006) Tribe Anthemideae Cass. 10383 In: Kadereit JW, Jeffrey C (eds) The families and genera of

123 Molecular phylogeny of Chrysanthemum, Ajania, and its allies 169

vascular plants VIII. Flowering plants. . . Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG Springer, Heidelberg, pp 342–374 (1997) The Clustal X windows interface: exible strategies for Oberprieler C, Himmelreich S, Vogt R (2007) A new subtribal multiple sequence alignment aided by quality analysis tools. classification of the tribe Anthemideae (Compositae). Willden- Nucleic Acids Res 25:4876–4882 owia 37:89–114 Tkach NV, Hoffmann MH, Ro¨ser M, Korobkov AA, von Hagen KB Ohashi H, Yonekura K (2004) New combinations in Chrysanthemum (2008a) Temporal patterns of evolution in the Arctic explored in (Compositae–Anthemideae) of Asia with a list of Japanese Artemisia L. (Asteraceae) lineages of different age. Plant Ecol species. J Jpn Bot 79:186–195 Divers 1:161–169 Ohishi K, Hasegawa T, Itakura N, Kawai A (1996) Morphological Tkach NV, Hoffmann MH, Ro¨ser M, Korobkov AA, von Hagen KB characteristics and fertility of F1 hybrid of Dendranthema (2008b) Parallel evolutionary patterns in multiple lineages of grandiflora and Nipponanthemum nipponicum. J Jpn Soc Hortic arctic Artemisia L. (Asteraceae). Evolution 62:184–198 Sci 65:510–511 Torrell M, Garcia-Jacas N, Susanna A, Valle`s J (1999) Infrageneric Page RDM (1996) TREEVIEW: an application to display phyloge- phylogeny of the genus Artemisia L. (Asteraceae, Anthemideae) netic trees on personal computers. Comput Appl Biosci 12:357– based on nucleotide sequences of nuclear ribosomal DNA 358 internal transcribed spacers (ITS). Taxon 48:721–736 Poljakov PP (1955) Duo genere novae fam. Compositae. Botaniches- Tzvelev NN (1961) Brachanthemum. In: Komarov VL (ed) Compos- kie Materialy Gerbariia Botanicheskogo Instituta imeni V. L. itae: Anthemideae–Arctotideae. Flora URSS 26. Academy of Komarova Akademii Nauk SSSR 17:418–431 Science, Leningrad, pp 642–653 Poljakov PP (1957) De genere novo Kaschgaria Polj. Botanicheskie Valle`s J, Torrell M, Garnatje T, Garcia-Jacas N, Vilatersana R, Materialy Gerbariia Botanicheskogo Instituta imeni V. L. Susanna A (2003) The genus Artemisia and its allies: phylogeny Komarova Akademii Nauk SSSR 18:285–290 of the subtribe Artemisiinae (Asteraceae, Anthemideae) based on Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phyloge- nucleotide sequences of nuclear ribosomal DNA internal tran- netic inference under mixed models. Bioinformatics 19:1572– scribed spacers (ITS). Plant Biol 5:274–284 1574 Wagstaff SJ, Breitwieser I (2002) Phylogenetic relationships of New Sanz M, Vilatersana R, Hidalgo O, Garcia-Jacas N, Susanna A, Zealand Asteraceae inferred from ITS sequences. Plant Syst Schneeweiss GM, Valle`s J (2008) Molecular phylogeny and Evol 231:203–224 evolution of floral characters of Artemisia and allies (Anthemi- Wan T, Wei ZJ, Yang J, Zhao C, Yan L, Zhang EH (1999) Pollen deae, Asteraceae): evidence from nrDNA ETS and ITS morphology of mordern plants in grassland of . sequences. Taxon 57:1–13 China Agriculture Press, Beijing, pp 25–33 (see also pp 91–105) Shih C (1979a) Opisthopappus Shih––a new genus of Compositae Watson LE, Evans TM, Boluarte T (2000) Molecular phylogeny and from China. Acta Phytotaxon Sin 17:110–112 biogeography of tribe Anthemideae (Asteraceae), based on Shih C (1979b) A taxonomic study of the genus Hippolytia Poljakov. chloroplast gene ndhF. Mol Phylogenet Evol 15:59–69 Acta Phytotaxon Sin 17:62–71 Watson LE, Bates PL, Evans TM, Unwin MM, Estes JR (2002) Shih C (1985) A new combination of the Compositae–Anthemideae Molecular phylogeny of subtribe Artemisiinae (Asteraceae), from China. Acta Phytotaxon Sin 23:470–472 including Artemisia and its allied and segregate genera. BMC Shih C, Fu GX (1983) Angiospermae, Dicotyledoneae, Compositae Evol Biol 2:17–28 (3) Anthemideae, Angiospermae. Flora Republicae Popularis White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct Sinicae 76(1). Science Press, Beijing sequencing of fungal ribosomal RNA genes for phylogenetics. Swofford DL (2002) PAUP*: phylogenetic analysis using parsimony In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR (*and other methods). Sinauer, Sunderland protocols: a guide to methods and application. Academic Press, Swofford DL (2003) PAUP*: Phylogenetic analysis using parsimony San Diego, pp 315–322 (* and other methods), version 4.0b10. Sinauer Associates, Yin JL (2005) Preliminary studies on intergeneric hybridization in Sunderland Asteraceae–Anthemideae–Chrysantheminae tribe. Dissertation, Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for Beijing Forestry University, Beijing amplification of three noncoding regions of chloroplast DNA. Zhao HB, Chen FD, Guo WM, Miao HB, Li C, Fang WM (2007) Plant Mol Biol 17:1105–1109 Intergeneric hybrid of Dendranthema 9 grandiflorum ‘‘Aoyun- Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular huoju’’ and Ajania pacifica and its taxonomic implications. Acta evolutionary genetics analysis (MEGA) software version 4.0. Phytotaxon Sin 45:661–669 Mol Biol Evol 24:1596–1599

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