ISSN 1346-7565 INSTRUCTIONS TO AUTHORSActa Phytotax. Geobot. 70 (3): 141–147 (2019) doi: 10.18942/apg.201903 Acta Phytotaxonomica et Geobotanica (Revised October, 2017)

Editor-in-Chief Members of the Japanese Society for Systematics Matsumura, J. 1912. Index Plantarum Japonicarum, vol. TAMURA, Minoru N. (Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan) are encouraged to submitKaryological manuscripts pertinent and to plant Phylogenetic 2. Maruzen, Analyses Tokyo. of taxonomy, systematicAster botany, koshikiensis phytogeography andKitamura close- Ridder-Numan, (, J. 1997. ), The continuing story of Spatho- Editors ly related disciplines for publication in Acta Phytotaxo- lobus (Leguminosae-Papilionoideae) and its allies. In: BOUFFORD, David E. (Boston, U.S.A.) NAIKI, Akiyo (Taketomi, Japan) nomica et GeobotanicaEndemic. Contribution to the Koshiki from non-members Islands, KagoshimaDransfield, J., (Kyushu,M. J. E. Coode Japan)& D. A. Simpson (eds.), abroad is also welcome, and if accepted, may be entitled Plant Diversity in Malesia III, pp. 205–217. The Royal EBIHARA, Atsushi (Tsukuba, Japan) TAKAMIYA, Masayuki (Kumamoto, Japan) free of charge for publication. Botanic Gardens, Kew. FUSE, Shizuka (Kyoto, Japan) TAKAYAMA, Koji (Kyoto, Japan) Wain, R. P., W. T. Haller & D. F. Maktin. 1985. Isozymes IKEDA, Hiroshi (Tokyo, Japan) TSUBOTA, Hiromi (Hiroshima, Japan) Manuscripts should be written in English. in studies of aquatic . J. Aquatic Pl. Managem. KUROSAWA, Takahide (Fukushima, Japan) WATANO, Yasuyuki (Chiba, Japan) Full original papers are limited to 20 printed pages1,* in 23: 42–45. 2 kiko oejima and atsuya amashima MAKI, Masayuki (Sendai, Japan) length including tables andA figures. S Short communi - TTables should H be included the heading, a lucid legend to cations are limited to 4 printed pages. With the approv- explain the meaning of the content. 1 al of theDepartment Editorial ofBoard, Biological additional Science, pages Faculty may be of pub Advanced- Figures Science should and Technology, be prepared Kumamotoas follows: University,(a) Size for figures Editorial Board 2 lishedKumamoto only at 860-8555, the author’s Japan. expense *[email protected] (3,000 Yen per as(author published for correspondence); should be less than Faculty 135 ×of 180Science, mm for a AZUMA, Takayuki (Sapporo, Japan) NISHIDA, Sachiko (Nagoya, Japan) page). Monographs exceedingKumamoto 30 printed University, pages may Kumamoto whole 860-8555, page. (b) Japan Legends for figures should be grouped FUJII, Shinji (Okazaki, Japan) OHMURA, Yoshihito (Tsukuba, Japan) be compiled in supplementary issues. Reviews (not on a separate sheet. Original figures should be kept at KAWAKUBO, Nobumitsu (Gifu, Japan) PAK, Jae-hong (Taegu, Korea) more than 20 printed pages) and short reviews (not hand until requested. Figures can be color-printed at MURAKAMI, Noriaki (Hachioji, Japan) POOMA, Rachun (Bangkok, Thailand) moreThe than hypothesized 10 pages) are hybridalso considered origin of for publication. koshikiensis (Asteraceae,the author’s Astereae), expense (18,000 a species Yen endemic per page). to the NAGAMASU, Hidetoshi (Kyoto, Japan) YAMADA, Toshihiro (Katano, Japan) PreparationKoshiki of Islands,Manuscripts: Japan, was tested using karyological featuresWhether and or molecular not the manuscript phylogenetic is analyses.accepted Mo and- the or- NAKADA, Takashi (Yokohama, Japan) YANG, Yong-Ping (Kunming, China) Cover sheetlecular should phylogenetic contain analyses(a) full name(s)suggested of thatauthor(s) A. koshikiensis der ofis a publication member of isthe to A. beleiophyllus decided bycomplex, the Editorial while a karyological analysis indicated its close relationship with A. satsumensis, a species on southern NAKATA, Masashi (Toyama, Japan) YONEKURA, Koji (Sendai, Japan) and address, (b) the corresponding author’s name, un- Committee after peer reviews by two or more referees. abbreviatedKyushu, completeJapan. address, phone and facsimile The positions and scale of figures and tables in pub- NISHIDA, Harufumi (Tokyo, Japan) number and e-mail address, (c) title, (d) numbers of fig- lished page may be changed from the author’s designa- ures andKey tables,words: and Aster (e), endemicrunning titlespecies, including karyotype author’s analysis, Koshikition. Islands, phylogeny Acta Phytotaxonomica et Geobotanica (APG) is an international peer-reviewed journal published family name with less than 50 characters. Galley proof will be sent to the corresponding author. three times a year by the Japanese Society for Plant Systematics. It is the continuation of the jour- Type in double-space on one side of A4 size papers with The authors are responsible for reading the first galley nal Societas Phytogeographica Kyoto, Japan. APG publishes research manuscripts in the fields of tax- margin at least 2.5 cm wide. Each typewritten page proof. No change of the content of the manuscript is onomy, phylogeny, evolution, conservation, biogeography of plants (including algae and fungi) and usually consists of 25 lines. permitted on the galley proof. If the Editors recognize related disciplines. Contributions devoted to new taxa, nomenclature, monographs, floristic studies Arrange the manuscript in the order listed below and the necessity for a change, this change may be made at numberAster all koshikiensis pages consecutively, Kitam. including (Asteraceae, tables As and- andthe involucral author’s expense. bracts. The basal leaves are spatu- and genetic variation are also invited. Instructions to Authors are on cover page 3. All manuscripts tereae),figures. a perennial herb grows in open places, lateOffprints. to narrowly The authors lanceolate will receive and freethe ofcauline charge leaves50 off- should be sent to the Editor-in-Chief. suchTitle ofas paperon slopes, name(s) along of author(s)roads and and on affiliation rocky sea with- areprints. spatulate. Additional The heads copies are can 3.5–4 be obtained cm in atdiameter author’s coasts,addresses. is endemic to the Koshiki Islands, Ka- withcost. pale purple or rarely white ligules. It blooms Application of admission for membership of the society should be addressed to the Treasurer, Dr. Sa- Abstract should be a lucid digest of the paper, not ex- Copyright. The articles published in APG are subject to toshi Koi (Botanical Gardens, Faculty of Science, Osaka City University, 2000 Kisaichi, Katano, Osa- goshima,ceeding 200Japan. words It forhas a fulldense, paper. long, soft hairs on in copyright. October All and rights November. are reserved The by the other Japanese putative Soci- ka 576-0004, Japan) and other correspondences the Secretary, Dr. Atsushi Ebihara (Department of stoutKey words stems, not more leaves, than and 10 words involucral are arranged bracts. in an The al- parent,ety for A. Plant microcephalus Systematics. The var. authors ovatus should is also submit pe- Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan). leavesphabetical are ovateorder. to oblong-lanceolate and defi- rennial.the agreement It occurs that widely the copyright from Hokkaido for their article to Ky is- The annual fee is 7,000 Yen for Japanese members, 3,000 Yen for student members and 3,000 Yen for nitelyText containing petiolate. Introduction The heads (without are 1.5–2.5 heading), cm Materi in di- ushutransferred in open to places the Japanese such Societyas at the for margin Plant Systemat of for- foreign members resided in abroad. APG is available by subscription for 8,000 Yen per year by the ameterals and and Methods, the ligules Results, are Discussion, white or and sometimes Acknowl- ests,ics when boundaries the article of is paddyaccepted fields to publication. and on slopes edgments is suggested. Scientific names are written in Submit all manuscripts by an e-mail or a registered mail Treasurer. purple-tinged.Italic. Scientific Anthesis names that is appear in October firstly in and each No para- alongto Editor-in-Chief, roads. It has short, Dr. Minoru rigid N.hairs Tamura, on the Graduate stems vember.graph shouldAster koshikiensis be unabbreviated. was postulated Voucher specimens to be a andSchool lanceolate of Science, to ovate Kyoto leaves. University, The heads Sakyo, are Kyoto 1.5– Reprographic Reproduction outside Japan hybridshould between be cited. A.Abbreviations spathulifolius of herbaria Maxim. should and fol A.- 2.5606-8502, cm in diameter Japan. with pale purple ligules. Flow- Making a copy of this publication microcephaluslow Index Herbariorum. (Miq.) Franch. et Sav. var. ovatus ering E-mail: is from [email protected] September to November. Please obtain permission from the following Reproduction Rights Organizations (RROs) to which the copyright (Franch.References et cited Sav.) in Soejimathe text should et Mot. be arranged Ito (syn. alphabet A. ag- According to Kitamura (1970), A. koshikien- holder has consigned the management of the copyright regarding reprographic reproduction. ically according to the name(s) of author(s). Text refer- Obtaining permission to quote, reproduce; translate, etc. eratoidesence should Turcz. be made subsp. by the ovatus author’s (Franch. names followed et Sav.) by sis is similar to A. spathulifolius in having long, Please contact the Secretary of the society. Kitam.)the year by ofKitamura publication. in 1970. [for example: Matsumura dense, soft hairs, while in other characteristics, it (1912),Aster or spathulifolius (Matsumura 1912)]., one If of the the number putative of authors par- resembles A. microcephalus var. ovatus. Al- Users in countries and regions where there is a local RRO under bilateral contact with Japan Academic Associa- ents,is three is a orperennial more, use herbTakamiya on rocks et al. (1997),along orthe (Taka sea- though Kitamura (1970) noted that the two spe- tion for Copyright Clearance (JAACC). shoremiya of et Japanal. 1997). (Japan Each Seareference side ofshould western be given Honshu in the cies had different chromosome numbers (n = 9 for Users in countries and regions of which RROs are listed on the following website are requested to contact the re- following form: and Kyushu), Korea, and Russia (Primorskiy). It A. spathulifolius and n = 18 of A. microcephalus spective RROs directly to obtain permission. Sheh, M.-L. 1992. Peucedanum. In: Shan, R.-H. & M.-L. hasSheh long (eds.), soft Florahairs, Reipublicae mixed with Popularis long, sticky, Sinicae, glan vol.- var. ovatus), which is generally accepted as a bar- Japan Academic Association for Copyright Clearance (JAACC) dular55 (3), hairs pp. 123–175. that densely Science cover Press, theBeijing stems, (in Chinese). leaves, rier to hybridization, he considered A. koshikien- Address 9-6-41 Akasaka, Minato-ku, Tokyo 107-0052 Japan Website http://www.jaacc.jp/ E-mail [email protected] Fax: +81-3-3475-5619 142 Acta Phytotax. Geobot. Vol. 70

Table 1. Collection data of the taxa used in this study. Accession no. taxa Locality Voucher ITS trnL-trnF trnH-psbA Sect. Aster A. fastigiatus Fisch. Kobe C., Hyogo Soejima 1091 LC482289 LC482637 LC484269 A. koshikiensis Kitam. Shimo-Koshiki Is. Kagoshima Soejima et al. 141110-1 LC482290 LC482638 LC484270 A. lucens Kitam. Iriomote Is., Okinawa Naiki 141009 LC482291 LC482639 LC484271 A. leiophyllus Franch. et Sav. var. Mt. Kiyosumi, Chiba Soejima s.n. LC482293 LC482641 LC484273 leiophyllus A. maackii Regel Kokonoe T., Oita Soejima 1123 LC482292 LC482640 LC484272 A. miquelianus H.Hara Kikuchi Gorge, Kumamoto Soejima 1064 LC482302 LC482650 LC484282 A. satsumensis Soejima Ichikikushikino C., Kagoshima Soejima et al. 141111-64 LC482294 LC482642 LC484274 A. semiamplexicaulis Makino Kawanishi C., Hyogo Soejima 130923 LC482303 LC482651 LC484283 A. sugimotoi Kitam. Mt. Akiwa, Shizuoka Soejima s.n. LC482295 LC482643 LC484275 A. yoshinaganus (Kitam.) Mot. Ito Mt. Yokogura, Kochi Soejima 130730 LC482296 LC482644 LC484276 et Soejima Sect. Teretiachenium A. rugulosus Maxim. var. shibu- Shinshiro C., Aichi Nishino shibukawa 141 LC482297 LC482645 LC484277 kawaensis Kitam. et Murata A. savatieri Makino var. savatieri Miyama T., Kyoto Soejima 1053 LC482298 LC482646 LC484278 A. scaber Thunb. Nishihara V., Kumamoto Soejima s.n. LC482299 LC482647 LC484279 Sect. Asteromoea A. iinumae Kitam. Mennoki Pass, Aichi Soejima 140805 LC482300 LC482648 LC484280 Sect. Pseudocalimeris A. spathulifolius Maxim. Hirado C., Nagasaki Soejima 1060 LC482301 LC482649 LC484281 Outgroup Erigeron thunbergii A. Gray Mt. Chokai, Yamagata Soejima 140613 LC497318 LC497319 LC497317 subsp. thunbergii sis to be their hybrid, since these three species co- be allotetraploid (Tara 1977, Matoba et al. occur on Shimo-Koshiki Island. In this study, 2005, 2007), it was not included in the molecu- through observations of karyological features and lar phylogenetic analysis. Erigeron thunbergii molecular phylogenetic analyses, we investigated A. Gray var. thunbergii was chosen as an out- the hypothesis that Aster koshikiensis was of hybrid group because of its close relationship with As- origin. ter.

Karyological observation Materials and Methods Twenty-two plants from five populations of Aster koshikiensis were cultivated and used for Plant materials karyological observations. The root tips were Twenty-two plants of Aster koshikiensis were kept in ice water for 24 h prior to fixation with collected from five populations on Shimo-Koshiki an ethanol and acetic acid (3:1) solution. A solu- Island (Fig. 1A). The aerial parts were pressed to tion of 1% aceto-orcein and 1% HCl (9:1) was generate herbarium specimens and the underground applied for more than 12 h to stain the chromo- parts were planted in pots at Kumamoto University. somes before squashing. Other samples used in the phylogenetic analysis represented four sections of Japanese Aster (Table DNA extraction, amplification, and sequencing 1). Although A. leiophyllus var. leiophyllus, A. sa- DNA was extracted from silica gel-dried vatieri, A. sugimotoi, and A. yoshinaganus are leaves of all the species by using the CTAB known to include polyploids (triploids, tetraploids, method (Doyle & Doyle 1987). The nuclear and hexaploids) (cf. Ito & Soejima 1995), the mate- rDNA region, which included two internal rials used in this study were all diploids. Moreover, transcribed spacers (ITS), was amplified by us- because A. microcephalus var. ovatus is known to ing the pair of primers ITS4 and ITS5 (White et October 2019 Soejima & Hamashima – Phylogeny of Island Endemic, Aster koshikiensis 143

A B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

C

Fig. 1. Aster koshikiensis Kitam. A. Flowering shoot with petiolate cauline leaves; B–C. Somatic metaphase chromosomes at mitosis (2n = 18). Thin arrows indicate satellites on short arms of chromosomes 1 and 2, thick arrows indicate satellites on long arms of chromosomes 3 and 4. Scale bars: 25 µm. al. 1990). PCR conditions comprised an initial Phylogenetic analyses denaturation of 2 min at 95 °C, followed by 30 The nucleotide sequences of the nuclear ITS cycles of 1 min at 94 °C, 1 min at 52 °C, and 2 and chloroplast trnL-trnF and trnH-psbA were min at 72 ºC, with a final extension of 5 min at 72 aligned separately using ClustalW (Thompson et ºC. In the chloroplast DNA, two intergenic spac- al. 1994). Gaps were treated as missing data; ers of the trnL-trnF and trnH-psbA regions were however, three informative gaps in the ITS align- amplified using primers trnL and trnF (Taberlet ment were included in the analysis after being et al. 1991), and trnH (GUG) and psbA (Hamilton transformed into binary data (Table 2). We per- 1999), respectively. PCR conditions comprised an formed separate Bayesian phylogenetic analyses initial denaturation of 2 min at 95 °C, followed by for the ITS dataset, the dataset of chloroplast re- 35 cycles of 2 min at 94 °C, 2 min at 52 °C, 2 min gions, and the concatenated dataset of ITS and at 72 °C, with a final extension of 5 min at 72 °C. chloroplast regions using MrBayes v.3.2.6 (Ron- All the sequences were read in both directions. quist et al. 2012). In each analysis, the same pa- 144 Acta Phytotax. Geobot. Vol. 70 rameters were used. Bootstrap analyses (1,000 Table 2. Informative ITS indels included in the phyloge- netic analyses. pseudoreplicates) were conducted to evaluate 455 610-611 656 clade credibility under maximum likelihood Aster fastigiatus - - - - (ML) criteria using PAUP* 4.0b10 (Swofford A. iinumae - TG - 2003). Bayesian tree inferences were computed A. koshikiensis - TG - using MrBayes v.3.2.6 (Ronquist et al. 2012). A. leiophyllus var. leiophyllus - TG - Each Metropolis-coupled Markov chain Monte A. lucens - - - - Carlo analysis was performed for 10,000,000 A. maackii - - - - generations. The sampled trees and models from A. miquelianus T TG G the first half of the generations were discarded as A. rugulosus var. shibukawaensis T TG G A. satsumensis - TG - burn-in and a majority-rule consensus tree was A. savatieri var. savatieri - - - - constructed from the sampled trees from the sec- A. scaber T TG G ond half of the generations. A partition homoge- A. semiamplexicaulis - TG - neity test was conducted to examine the extent of A. spathulifolius - - - - conflict between the two data sets using the in- A. sugimotoi - TG - congruence length difference (ILD) test (Farris et A. yoshinaganus - TG - al. 1995) as implemented in PAUP* 4.0b10 (Swof- Erigeron thunbergii subsp. thunbergii T TG - ford 2003) (simple addition-sequence, tree bisec- tion-reconnection, heuristic search option with 1,000 replicates analyzed). Table 3. Karyological features of each chromosome. Chromosome no. Arm ratio Length proportion* 1 0.16: 0.32: 0.52 6.76 2 0.18: 0.31: 0.51 6.65 Results 3 0.40: 0.41: 0.18 5.83 4 0.41: 0.42: 0.17 5.90 Karyotype analysis 5 0.47: 0.53 6.00 The chromosome number of Aster koshikien- 6 0.47: 0.53 6.00 sis was determined to be 2n = 18 (Fig. 1B, C). 7 0.47: 0.53 5.32 8 0.46: 0.54 5.98 Chromosome lengths varied continuously from 9 0.45: 0.55 5.51 long to short (Fig. 1C, Table 3). The shortest chro- 10 0.44: 0.56 5.26 mosome was approximately 67% as long as the 11 0.44: 0.56 4.89 longest one. A satellite was present at the end of 12 0.44: 0.56 5.18 the short arm on the longest pair of chromosomes 13 0.39: 0.61 5.47 and at the end of the long arm on the second-lon- 14 0.36: 0.64 6.10 gest pair. 15 0.41: 0.59 4.97 16 0.42: 0.58 5.06 17 0.45: 0.55 4.69 Phylogenetic analyses 18 0.43: 0.57 4.52 The sequences used in the analyses were 402– * proportion between length of each chromosome and total 505 bp, 224–284 bp, and 711–796 bp long for the length of all chromosomes trnL-trnF, trnH-psbA, and ITS regions, respec- tively. No heterozygous mutations were observed in the ITS region. In both chloroplast and ITS roplast tree and PP = 1.00 and BP = 100% in the trees (Fig. 2), A. koshikiensis was clustered with ITS tree. Results of the partition homogeneity five other species A.( leiophyllus, A. satsumensis, test were not significant (P = 1.000), indicating A. semiamplexicaulis, A. sugimotoi, and A. yoshi- that the two datasets could be combined. In the naganus), with PP (posterior probability) = 0.98 combined tree (Fig. 3), the same five species were and BP (bootstrap percentage) = 64% in the chlo- clustered with A. koshikiensis (PP = 1.00 and BP = 100%). October 2019 Soejima & Hamashima – Phylogeny of Island Endemic, Aster koshikiensis 145

A. leiophyllus

A B A. koshikiensis A. leiophyllus A. satsumensis A. koshikiensis A. semiamplexicaulis 64 A. satsumensis 0.98 A. semiamplexicaulis A. sugimotoi

A. sugimotoi A. yoshinaganus

A. yoshinaganus A. iinumae - 1.00 A. iinumae A. maackii

A. maackii A. fastigiatus A. fastigiatus A. savatieri A. savatieri A. lucens A. lucens A. spathulifolius A. spathulifolius A. scaber A. scaber A. rugulosus A. miqueliana var. shibukawaensis A. miqueliana A. rugulosus var. shibukawaensis E. thunbergii E. thunbergii

Fig. 2. Trees resulting from Bayesian analysis. A. chloroplast DNA regions; B. nuclear ITS. Values above and below branch- es indicate bootstrap percentages (> 50%) and Bayesian inference posterior probability (> 0.5), respectively.

A. leiophyllus

A. koshikiensis

A. satsumensis

A. semiamplexicaulis

A. sugimotoi

A. yoshinaganus

A. iinumae

A. maackii

A. fastigiatus

A. savatieri

A. lucens

A. spathulifolius

A. scaber

A. miqueliana

A. rugulosus var. shibukawaensis E. thunbergii

Fig. 3. A trees resulting from Bayesian analysis of chloroplast DNA regions + ITS. Values above and below branches indicate bootstrap percentages (> 50%) and Bayesian inference posterior probability (> 0.5), respectively.

diploid with 18 chromosomes somewhat equal in Discussion length (Huziwara 1954, 1957a). Aster microceph- alus var. ovatus, on the other hand, is tetraploid Rejection of the hybrid hypothesis by karyologi- with 36 chromosomes and has extreme differenc- cal analysis es in size between the shortest and longest chro- Aster spathulifolius has been reported to be mosomes: the shortest chromosome is less than 146 Acta Phytotax. Geobot. Vol. 70

50% as long as the longest one (Huziwara 1953, koshikiensis the second longest pair of chromo- 1957b, Tara 1977, Matoba et al. 2005, 2007). We somes also has satellites on their long arm. The found that Aster koshikiensis was diploid with 18 same additional satellites were also observed in chromosomes somewhat equal in length; the A. satsumensis (Soejima 1993). shortest chromosome was approximately 67% as Aster satsumensis has not been found in the long as the longest one (Fig. 1C, Table 3). There- Koshiki Islands, but it grows in southern Kyushu. fore, based on chromosome numbers and karyo- It differs from A. koshikiensis in leaf shape; logical features, it is clear that A. koshikiensis is A. satsumensis has lanceolate to oblong-lanceo- not a hybrid between A. spathulifolius and A. mi- late, sessile leaves, whereas A. koshikiensis has crocephalus var. ovatus. round to ovate, distinctly petiolate leaves. In con- trast, they are similar in their long, dense, soft Phylogenetic position of A. koshikiensis hairs on the stems and leaves. Although the phy- Although the karyological features of A. ko- logenetic resolution of the clade forming the A. shikiensis resemble those of A. spathulifolius, the leiophyllus complex was not sufficient to draw two species were clearly separated in the phylo- firm conclusions, the morphological resemblance, genetic trees (Figs. 2 & 3). The combined tree geographic approximation, and karyological sim- strongly suggests the existence of a clade includ- ilarity suggest that A. koshikiensis may be de- ing A. koshikiensis, A. leiophyllus, A. satsumen- rived from A. satsumensis. It is noteworthy that sis, A. semiamplexicaulis, A. sugimotoi, and A. this endemic species, which may have originated yoshinaganus. The latter five species have been in the Koshiki Islands, is an important constitu- considered to be closely related in previous stud- ent of the flora of these islands. ies, and different names have been proposed for them: A. ageratoides subsp. amplexifolius com- plex (Irifune 1990), A. ageratoides subsp. leio- More than ten years ago, the late Dr. M. Hotta sent phyllus group (Matsuda & Shinohara 1985, Mat- A. Soejima, one of the authors, several plants of A. koshi- kiensis. He was suspicious about the hybrid hypothesis suda & Suyama 1980), A. leiophyllus complex proposed by Kitamura (1970) and asked for an investiga- (Soejima 1992, 1993), and A. ageratoides com- tion of the origin of this species. We hope to have an- plex (Soejima & Peng 1998, Soejima et al. 1998, swered his question in the study that resulted in this short 1999, 2005). The members of this group [hereaf- article. We are grateful to Mr. Y. Inoue for providing sam- ter designated as the A. leiophyllus complex, fol- ples of A. koshikiensis, which he grew in his nursery, and to Mr. M. Igari for useful information about the natural lowing the latest nomenclatural treatment by habitat of these plants. We thank Drs. M. Ito, A. Naiki, T. Soejima (2017)], are similar in having mostly Nishino, and H. Setoguchi for their kind assistance in the white but sometimes purple-tinged ligules in collection of plants for our phylogenetic analyses, and Dr. small heads 1–2 cm in diameter, trinerved leaves, H. Matoba for his advice in the karyotype investigation. and long stolons, which are also features of A. ko- shikiensis. These morphological similarities and the results of the molecular phylogenetic analyses References indicate that A. koshikiensis is a member of this group. Doyle, J. J. & J. L. Doyle. 1987. A rapid DNA isolation According to previous studies, diploids of the procedure for small quantities of fresh leaf tissue. Phytochem. Bulletin 19: 11–15. A. leiophyllus complex are similar in their karyo- Farris, J. S., M. Källersjö, A. G. Kluge & C. Bult. 1995. types (Huziwara 1953, 1957b, Irifune 1990, Constructing a significance test for incongruence. Soejima 1992, 1993). They have 18 chromo- Syst. Biology 44: 570–572. somes, mostly similar in length, with the longest Hamilton, M. B. 1999. Four primer pairs for the amplifica- pair in each species having a satellite on its short tion of chloroplast intergenic regions with intraspe- cific variation. Molec. Ecol. 8: 521–523. arm. The karyotype of A. koshikiensis is similar Huziwara, Y. 1953. Karyotype analysis in Aster I. Bot. to the other species in this group. However, in A. Mag. (Tokyo) 66: 263–268. October 2019 Soejima & Hamashima – Phylogeny of Island Endemic, Aster koshikiensis 147

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Received January 15, 2019; accepted February 19, 2019