J Plant Res (2004) 117:109–120 © The Botanical Society of Japan and Springer-Verlag Tokyo 2004Digital Object Identifier (DOI) 10.1007/s10265-003-0133-3

Springer-VerlagTokyo102650918-94401618-086030669031Journal of Plant ResearchJ Plant Res13310.1007/s10265-003-0133-3

Molecular phylogeny of monocotyledons inferred from combined analysis of plastid matK and rbcL gene sequences

ORIGINAL ARTICLE

Received: August 21, 2003 / Accepted: November 18, 2003 / Published online: January 27, 2004

Minoru N. Tamura • Jun Yamashita • Shizuka Fuse •

Masatake Haraguchi

M. N. Tamura (*) · J. Yamashita · S. Fuse1 · M. HaraguchiBotanical Gardens, Graduate School of Science, Osaka City University, 2000 Kisaichi, Katano-shi, Osaka 576-0004, Japane-mail: nmtamura@sci.osaka-cu.ac.jp

Present address:1Museum of Nature and Human Activities, Hyogo, Japan

Abstract Using matK and rbcL sequences (3,269 bp intotal) from 113 genera of 45 families, we conducted a com-bined analysis to contribute to the understanding of majorevolutionary relationships in the monocotyledons. Treesresulting from the parsimony analysis are similar to thosegenerated by earlier single or multiple gene analyses, buttheir strict consensus tree provides much better resolutionof relationships among major clades. We find that Acorus(Acorales) is a sister group to the rest of the monocots,which receives 100% bootstrap support. A clade comprisingAlismatales is diverged as the next branch, followed succes-sively by Petrosaviaceae, the Dioscoreales–Pandanalesclade, Liliales, Asparagales and commelinoids. All of theseclades are strongly supported (with more than 90% boot-strap support). The sister-group relationship is also stronglysupported between Alismatales and the remaining mono-cots (except for Acorus) (100%), between Petrosaviaceaeand the remaining monocots (except for Acorus andAlismatales) (100%), between the clade comprisingDioscoreales and Pandanales and the clade comprisingLiliales, Asparagales and commelinoids (87%), andbetween Liliales and the Asparagales–commelinoids clade(89%). Only the sister-group relationship between Aspar-agales and commelinoids is weakly supported (68%).Results also support the inclusion of Petrosaviaceae in itsown order Petrosaviales, Nartheciaceae in Dioscoreales andHanguanaceae in Commelinales.

Electronic Supplementary Material Supplementary mate-rial is available in the online version of this article athttp://dx.doi.org/10.1007/s10265-003-0133-3

Key words Hanguanaceae · matK · Molecular phylogeny ·Monocotyledons · Petrosaviaceae · rbcL

Introduction

Over the past 10 years, several papers have been publishedon the phylogenetic relationships within the monocotsbased on DNA sequence data. They include, for instance,the articles published by Chase et al. (1993, 1995, 2000),Nadot et al. (1995), Bharathan and Zimmer (1995), Soltiset al. (1997, 2000), Davis et al. (1998), Savolainen et al.(2000), and Fuse and Tamura (2000). The studies were basedon one-, two- or three-gene analyses of plastid (rbcL, rps4,atpB, matK), mitochondrial (atpA), and nuclear (18SrDNA) genes. The most comprehensive study of the mono-cot phylogeny by three-gene analyses as well as a classifica-tion based on a synthesis of molecular analyses by variousresearchers was published by Chase et al. (2000). Accordingto this study, the monocots are monophyletic, with Acorales(Acorus) at the basal-most position and with Alismatales asthe next branch; the rest of the monocots are split into fourlarge clades leading to Pandanales, Dioscoreales, Liliales,and Asparagales–commelinoids. Although all of these largeclades have at least some bootstrap support, their inter-relationships receive less than 50% bootstrap support.Additional, morphological and molecular data are in needto clarify relationships within the monocots (Chase et al.2000).

We have tested an analysis using a combined data set ofrbcL and matK sequences, and found that the combinedanalysis provides a much better resolution of relationshipswithin the monocots than analysis of the combined data setsof rbcL, atpB and 18S rDNA sequences (Chase et al. 2000;Soltis et al. 2000) and also better than analysis of rbcL(Chase et al. 1993, 1995) or matK sequences (Fuse andTamura 2000) separately. The combined analysis of rbcLand matK sequences generated many fewer polytomies thanthe analysis of either rbcL or matK sequences alone, andeven more distinct clades (107 in the strict consensus tree),

110

most of which received higher bootstrap support (thoughexact data are not presented).

In this paper we present the results of the combinedanalysis of rbcL and matK sequences as another insight intounderstanding the exact relationships within the monocots.The matK sequences contained many indels (insertion/deletion) throughout the monocots but, when properlyaligned for the analysis, provided useful and significant datafor molecular analyses of higher-level taxa. Based on theresults, we discuss systematic positions of several taxa thathave been unclear in earlier classifications.

Materials and methods

Plant materials and DNA sequence data

A total of 113 genera (114 operational taxonomic units[OTUs]) representing 45 families of the monocots wereused in the present study (Table 1). Among them, 28 genera(29 OTUs) and 34 genera (35 OTUs) were analyzed withrespect to their matK and rbcL sequences, respectively.Also, the matK sequences of the 14 genera as well as the

rbcL sequences of three genera that were analyzed byFuse and Tamura (2000) and Yamashita and Tamura (2000),respectively, were revised. Data on matK and rbcLsequences of the remaining genera were obtained from theDNA Data Bank of Japan (DDBJ).

DNA extraction and PCR amplification

Total genomic DNA was extracted from fresh leaves or fromleaves dried with silica gel using the cetyltrimethylammo-nium bromide (CTAB) method of Kawahara et al. (1995)and Tamura et al. (1997). The matK and rbcL gene regionson the plastid DNA were amplified using the polymerasechain reaction (PCR) with a programmable temperature-control system PC-700 (Astec) or GeneAmp PCR System9600 (Perkin Elmer). The amplification reaction mixturewas prepared using TaKaRa Ex Taq DNA polymerase fol-lowing its instruction manual (Takara Shuzo). We used 11and 4 PCR primers (Table 2) for the amplification of matKand rbcL regions, respectively, and the following PCR pro-file for both regions: a 37-cycle reaction with denaturationat 94°C for 1 min, annealing at 52°C for 1 min, and extensionat 72°C for 1–3 min, in addition to an initial denaturation at

Table 1. Reference, accession number and voucher data for taxa included in DNA analysis

Taxona Reference Accessionnumber(matK)

Accessionnumber(rbcL)

Voucherb

Dicotyledons (outgroup)Magnoliales

MagnoliaceaeMagnolia pseudokobus Abe & Akasawa Fuse and Tamura (2000) AB040152 Magnolia hypoleuca Siebold & Zucc. Qiu et al. (1993) L12655

PiperalesPiperaceae

Piper nigrum L. Fuse and Tamura (2000) AB040153c M. N. Tamura andS. Fuse 10016

Piper betle L. Qiu et al. (1993) L12660 Monocotyledons (ingroup)

Not rankedPetrosaviaceae

Japonolirion osense Nakai Fuse and Tamura (2000) AB040161 Japonolirion osense The present study AB088835 M. N. Tamura and

S. Sugata 10009 Petrosavia sakuraii (Makino) J. J. Sm. ex Steenis Fuse and Tamura (2000) AB040156 Petrosavia sakuraii The present study AB088839 H. Takahashi and

M. N. Tamura 7101Acorales

AcoraceaeAcorus calamus L. Fuse and Tamura (2000) AB040154 Acorus calamus Shinwari et al. (1994) D28865

AlismatalesAlismataceae

Alisma canaliculatum A. Braun & Bouche exSamuelsson

Fuse and Tamura (2000) AB040179

Alisma plantago-aquatica L. Chase et al. (1993) L08759 Aponogetonaceae

Aponogeton fenestralis Hook. f. The present study AB088779 AB088808 M. Haraguchi 9Araceae

Amorphophallus campanulatus Blume ex Decne. The present study AB088777 M. N. Tamura 14308Amorphophallus rivieri Dur. ex Riviere Cho and Palmer (1999) AJ005630 Gymnostachys anceps R. Br. Fuse and Tamura (2000) AB040177

111

Gymnostachys anceps The present study AB088806 M. N. Tamura 8037Zantedeschia aethiopica (L.) K. Spreng. Fuse and Tamura (2000) AB040178 Zantedeschia aethiopica Piques et al. (1998) AF065474

HydrocharitaceaeBlyxa echinosperma Hook. f. The present study AB088781 AB088810 M.N. Tamura et al.

13546Juncaginaceae

Triglochin maritimum L. The present study AB088782 AB088811 M.N. Tamura et al.13563

LimnocharitaceaeLimnocharis flava Buchen. The present study AB088778 AB088807 M. N. Tamura 14311

PotamogetonaceaePotamogeton distinctus A. Benn. The present study AB088780 AB088809 M. Haraguchi 14

TofieldiaceaeTofieldia nuda Maxim. Fuse and Tamura (2000) AB040159 Tofieldia nuda The present study AB088840 S. Fuse 8020

AsparagalesAgavaceae

Camassia cusickii S. Watson Ito et al. (1999) AB017319 Camassia leichtlinii S. Watson Fay and Chase (1996) Z69238 Chlorophytum comosum (Thunb.) Jacques Yamashita and Tamura (2000) AB029806 Chlorophytum comosum Duvall et al. (unpublished) L05031 Hosta sieboldii (Paxton) J. W. Ingram Ito et al. (1999) AB017317 Hosta rectifolia Nakai Duvall et al. (1993) L10253 Polianthes tuberosa L. Ito et al. (1999) AB017305 Polianthes geminiflora Rose Fay and Chase (1996) Z69227 Yucca aloifolia L. The present study AB088788 AB088819 M. Haraguchi 17Yucca australis Trel. The present study AB088789 AB088820 M. N. Tamura 14305

AlliaceaeAllium grayi Regel Ito et al. (1999) AB017307 Allium cepa L. Katayama and Ogihara (1996) D38294 Nothoscordum fragrans Kunth Ito et al. (1999) AB017311 Nothoscordum bivalve Britton Fay and Chase (1996) Z69202 Tulbaghia sp. Ito et al. (1999) AB017312 Tulbaghia violacea Harv. Fay and Chase (1996) Z69203

AmaryllidaceaeAmaryllis belladonna L. Ito et al. (1999) AB017274 Amaryllis belladonna Fay and Chase (1996) Z69219 Clivia miniata Regel Ito et al. (1999) AB017278 Clivia miniata Duvall et al. (unpublished) L05032 Galanthus elwesii Hook. f. Ito et al. (1999) AB017285 Galanthus plicatus Bieb. Fay and Chase (1996) Z69218 Leucojum aestivum L. Ito et al. (1999) AB017289 Leucojum autumnale L. Rudall et al. (1997) Z77256 Stenomesson variegatum (Ruiz & Pav.) Macbridge Ito et al. (1999) AB017299 Stenomesson pearcei Baker Fay and Chase (1996) Z69217

AsparagaceaeAsparagus cochinchinensis Merr. Yamashita and Tamura (2000) AB029804 AB029849c M. N. Tamura 14302

BlandfordiaceaeBlandfordia punicea Sweet Ito et al. (1999) AB017315 Blandfordia punicea de Bruijn et al. (unpublished) Z73694

DoryanthaceaeDoryanthes excelsa Correa The present study AB088785 M. N. Tamura 14304Doryanthes excelsa de Bruijn et al. (unpublished) Z73697

HemerocallidaceaeDianella ensifolia DC. The present study AB088787 M. N. Tamura 14301Dianella ensifolia Eguiarte et al. (1992) M96960 Hemerocallis fulva L. Ito et al. (1999) AB017318 Hemerocallis fulva Duvall et al. (unpublished) L05036

HyacinthaceaeBowiea volubilis Harv. ex Hook. f. The present study AB088790 M. Haraguchi 6Bowiea volubilis Fay and Chase (1996) Z69237 Scilla scilloides (Lindl.) Druce Ito et al. (1999) AB017323 Scilla scilloides Shinwari et al. (1994) D28161

Taxona Reference Accessionnumber(matK)

Accessionnumber(rbcL)

Voucherb

Table 1. Continued

112

HypoxidaceaeCurculigo capitulata Kuntze The present study AB088783 M. Haraguchi 2Curculigo capitulata de Bruijn et al. (unpublished) Z73701 Rhodohypoxis baurii (Baker) Nel Ito et al. (1999) AB017324 Rhodohypoxis milloides (Baker) Hilliard & B. L.

BurttRudall et al. (1997) Z77280

IridaceaeIris japonica Thunb. The present study AB088786 M. Haraguchi 15Iris ensata Thunb. Kawanod D28332

IxioliriaceaeIxiolirion tataricum Herb. Ito et al. (1999) AB017327 Ixiolirion tataricum de Bruijn et al. (unpublished) Z73704

LaxmanniaceaeCordyline cf. stricta Endl. The present study AB088784 AB088818 M. N. Tamura 14309

OrchidaceaePhaius tancarvilleae (Banks ex L’ Her.) Blume Fuse and Tamura (2000) AB040205c M. N. Tamura and

M. Tamura 2801Phaius minor Blume Cameron et al. (1999) AF074210 Spiranthes sinensis Ames Fuse and Tamura (2000) AB040206c S. Fuse 8063Spiranthes cernua (L.) Rich. Cameron et al. (1999) AF074229

RuscaceaeAspidistra elatior Blume Yamashita and Tamura (2000) AB029780 Aspidistra elatior Yamashita and Tamura (2004) AB089631 Campylandra siamensis Yamashita & M. N.

TamuraYamashita and Tamura (2000) AB029773 AB029835

Comospermum yedoense (Maxim. ex Franch. &Sav.) Rauschert

Yamashita and Tamura (2000) AB029808

Comospermum yedoense The present study AB088821 M. N. Tamura 14313Convallaria majalis L. Yamashita and Tamura (2000) AB029771 Convallaria majalis Yamashita and Tamura (2004) AB089627 Dasylirion serratifolium Zucc. Yamashita and Tamura (2000) AB029800 AB029847 Disporopsis longifolia Craib Yamashita and Tamura (2000) AB029766 AB029833 Dracaena draco L. Yamashita and Tamura (2000) AB029803 AB029848 Heteropolygonatum pendulum (Z. G. Liu & X. H.

Hu) M. N. Tamura & OgisuYamashita and Tamura (2000) AB029764 AB029831

Liriope platyphylla F. T. Wang & T. Tang Yamashita and Tamura (2000) AB029784 Liriope platyphylla Rudall et al. (1997) Z77271 Maianthemum dilatatum A. Nelson & Macbride Yamashita and Tamura (2000) AB029770 Maianthemum dilatatum Yamashita et al. (submitted) AB089915 Nolina recurvata Hemsl. Yamashita and Tamura (2000) AB029799 AB029846c M. N. Tamura 14310Ophiopogon jaburan Lodd. Yamashita and Tamura (2000) AB029788 AB029840 Peliosanthes campanulata (Baill.) L. Rodrigues Yamashita and Tamura (2000) AB029798 AB029845 Pleomele thalioides N. E. Br. The present study AB088791 AB088823 M. Haraguchi 4Polygonatum involucratum (Franch. & Sav.)

Maxim.Yamashita and Tamura (2000) AB029762 AB029829

Reineckia carnea Kunth Yamashita and Tamura (2000) AB029772 AB029834 Rohdea japonica Roth Yamashita and Tamura (2000) AB029774 AB029836 Ruscus aculeatus L. Yamashita and Tamura (2000) AB029801 Ruscus aculeatus The present study AB088822 M. N. Tamura 14306Smilacina japonica A. Gray Yamashita and Tamura (2000) AB029768 Smilacina japonica Yamashita et al. (submitted) AB089916 Tricalistra ochracea Ridl. Yamashita and Tamura (2000) AB029777 AB029839 Tupistra albiflora K. Larsen Yamashita and Tamura (2000) AB029775 AB029837c M. N. Tamura 14303

ThemidaceaeBessera elegans Schult. f. Ito et al. (1999) AB017308 Bessera elegans Fay and Chase (1996) Z69215 Brodiaea uniflora Engl. Ito et al. (1999) AB017309 Brodiaea coronaria (Salisb.) Hort. Fay and Chase (1996) Z69210 Milla biflora Cav. Ito et al. (1999) AB017310 Milla biflora Fay and Chase (1996) Z69216

ArecalesArecaceae

Butia yatay Becc. The present study AB088794 AB088827 M. Haraguchi 11Phoenix dactylifera L. Fuse and Tamura (2000) AB040211 Phoenix reclinata Jacq. Gaut et al. (1992) M81814

Taxona Reference Accessionnumber(matK)

Accessionnumber(rbcL)

Voucherb

Table 1. Continued

113

CommelinalesHaemodoraceae

Anigozanthos flavidus DC. The present study AB088796 AB088829 M. Haraguchi 8Hanguanaceae

Hanguana malayana Merr. The present study AB088800 AB088830 H. Nagamasu s. n.Pontederiaceae

Eichhornia crassipes Solms Fuse and Tamura (2000) AB040212c S. Fuse 8065Eichhornia crassipes Graham et al. (1998) U41574 Monochoria korsakowii Regel & Maack The present study AB088795 AB088828 M. Haraguchi 12

DioscorealesDioscoreaceae

Dioscorea alata L. Fuse and Tamura (2000) AB040208 Dioscorea bulbifera L. Kato et al. (1995) D28327 Tacca sp. The present study AB088792 AB088824 M. N. Tamura 14312

NartheciaceaeAletris spicata (Thunb.) Franch. Fuse and Tamura (2000) AB040174 Aletris spicata The present study AB088834 S. Fuse 8021Lophiola aurea Ker Gawl. Fuse and Tamura (2000) AB040176c W. B. Zomlefer 719Lophiola aurea The present study AB088836 W. B. Zomlefer 719Metanarthecium luteo-viride Maxim. Fuse and Tamura (2000) AB040163 Metanarthecium luteo-viride The present study AB088837 M. N. Tamura 8009Narthecium asiaticum Maxim. Fuse and Tamura (2000) AB040162 Narthecium asiaticum The present study AB088838 M. N. Tamura 8012

LilialesAlstroemeriaceae

Alstroemeria sp. Ito et al. (1999) AB017328 Alstroemeria sp. Rudall et al. (1997) Z77254

ColchicaceaeColchicum speciosum Steven Fuse and Tamura (2000) AB040181c M. N. Tamura 3428Colchicum speciosum Chase et al. (1993) L12673 Disporum sessile D. Don Fuse and Tamura (2000) AB040182c M. N. Tamura 2049 Disporum sessile Shinwari et al. (1994) D17376 Uvularia grandiflora Sm. Hayashi et al. (1998) AB024395 Uvularia grandiflora Kawano and Katod AB009950

LiliaceaeAmana edulis (Miq.) Honda Hayashi et al. (1998) AB024388 AB024385 Clintonia borealis Raf. Hayashi et al. (1998) AB024542 Clintonia borealis Shinwari et al. (1994) D17372 Erythronium japonicum Decne. Hayashi et al. (1998) AB024387 Erythronium japonicum Shinwari et al. (1994) D28156 Fritillaria koidzumiana Ohwi Hayashi et al. (1998) AB024390 Fritillaria agrestis Greene. Baysdorferd AF013233 Lilium regale E. H. Wilson Fuse and Tamura (2000) AB040200 Lilium regale The present study AB088816 M. N. Tamura 98116 Medeola virginiana L. Hayashi et al. (1998) AB024547 Medeola virginiana Shinwari et al. (1994) D28158 Nomocharis saluenensis Balf. f. Hayashi et al. (1998) AB024391 Nomocharis pardanthina Franch. Rudall et al. (1997) Z77295 Scoliopus bigelovii Torr. Hayashi et al. (1998) AB024394 Scoliopus bigelovii Shinwari et al. (1994) D28162 Streptopus parviflorus Franch. Fuse and Tamura (2000) AB040203c M. N. Tamura 9835Streptopus parviflorus The present study AB088817 M. N. Tamura 9835Tricyrtis hirta Hook. ¥ T. formosana Baker Fuse and Tamura (2000) AB040202 Tricyrtis affinis Makino Shinwari et al. (1994) D17382 Tulipa sp. Fuse and Tamura (2000) AB040201 Tulipa kolpakowskiana Regel Rudall et al. (1997) Z77292

LuzuriagaceaeDrymophila moorei Baker Fuse and Tamura (2000) AB040180c M. N. Tamura 8023Drymophila moorei The present study AB088812 M. N. Tamura 8023

MelanthiaceaeChionographis japonica Maxim. Fuse and Tamura (2000) AB040198 Chionographis japonica The present study AB088813 M. N. Tamura and

J. Yamashita 9904Daiswa polyphylla Raf. Kazempour Osaloo and

Kawano (1999)AB018828

Taxona Reference Accessionnumber(matK)

Accessionnumber(rbcL)

Voucherb

Table 1. Continued

114

Table 1. Continued

aThe classification follows APG II (2003)bVoucher specimens are preserved in the herbarium of the Botanical Gardens, Osaka City UniversitycDNA sequence data, which had been already submitted to the DNA Data Bank of Japan (DDBJ) by Fuse and Tamura (2000) or Yamashitaand Tamura (2000), were revised in the present studydDirectly submitted to the DDBJ/EMBL/GenBank database

Daiswa polyphylla Kato et al. (1995) D28155 Heloniopsis orientalis C. Tanaka Fuse and Tamura (2000) AB040188c M. N. Tamura 10014Heloniopsis orientalis The present study AB088814 M. N. Tamura 10014Kinugasa japonica (Franch. & Sav.) Tatew. & Suto Kazempour Osaloo and

Kawano (1999)AB018831

Kinugasa japonica Kato et al. (1995) D28157 Paris thibetica Franch. Fuse and Tamura (2000) AB040199 Paris tetraphylla A. Gray Kato et al. (1995) D28159 Trillium undulatum Willd. Kazempour Osaloo et al.

(1999)AB017413

Trillium undulatum Kazempour Osaloo andKawano (1999)

AB018848

Veratrum maackii Regel Kazempour Osaloo et al.(1999)

AB017417

Veratrum maackii Kazempour Osaloo andKawano (1999)

AB018849

Ypsilandra thibetica Franch. Fuse and Tamura (2000) AB040185c M. N. Tamura 9818Ypsilandra thibetica The present study AB088815 M. N. Tamura 9818

PandanalesCyclanthaceae

Carludovica palmata Ruiz & Pav. The present study AB088793 M. Haraguchi 5Carludovica palmata Qiu et al. (1999) AF197596

PandanaceaeFreycinetia formosana Hemsl. Fuse and Tamura (2000) AB040209c M. N. Tamura et al. 9029Freycinetia formosana The present study AB088825 M. N. Tamura et al. 9029

StemonaceaeStemona japonica Franch. & Sav. Fuse and Tamura (2000) AB040210c M. N. Tamura and

S. Fuse 10020Stemona japonica The present study AB088826 M. N. Tamura and

S. Fuse 10020Poales

CyperaceaeScirpodendron ghaeri (Goertn.) Merr. The present study AB088804 AB088832 M. Haraguchi 10

FlagellariaceaeFlagellaria indica L. Fuse and Tamura (2000) AB040214c M. N. Tamura et al. 9032Flagellaria indica Chase et al. (1993) L12678

JuncaceaeJuncus effusus L. The present study AB088803 M. Haraguchi 16Juncus effusus Chase et al. (1993) L12681

PoaceaeHordeum vulgare L. Ems et al. (1995) X64129 Hordeum jubatum L. Seberg and Linde-Laursen Z49841 Phyllostachys bambusoides Siebold. & Zucc. The present study AB088805 AB088833 M. N. Tamura 14307

TyphaceaeSparganium stoloniferum Buch.-Ham. The present study AB088802 AB088831 M. Haraguchi 18Typha latifolia L. The present study AB088801 M. Haraguchi 13Typha latifolia Smith et al. (1993) L05464

ZingiberalesZingiberaceae

Globba curtisii Holttum The present study AB088797 M. Haraguchi s. n.Globba curtisii Smith et al. (1993) L05449 Hedychium flavum Roxb. The present study AB088798 M. Haraguchi s. n.Hedychium flavum Smith et al. (1993) L05450 Zingiber gramineum Noronha The present study AB088799 M. Haraguchi s. n.Zingiber gramineum Smith et al. (1993) L05465

Taxona Reference Accessionnumber(matK)

Accessionnumber(rbcL)

Voucherb

115

94°C for 2.5 min and final extension at 72°C for 7 min. Wepurified the PCR products with MicroSpin S-300 HR Col-umns (Pharmacia Biotech) to remove the primers andremaining deoxynucleoside triphosphates.

DNA sequencing

We sequenced matK genes ranging from 1,515 bp (as inEichhornia) to 1,575 bp (as in Iris). Tricyrtis has the longestsequence, i.e., 1,590 bp (Fuse and Tamura 2000). On theother hand, the exact length of the rbcL gene is unclearbecause the most-downstream region was not sequenced.Overall, the lengths of aligned sequences of matK and rbcLwere 1,879 bp and 1,390 bp, respectively.

We used 25 primers for the DNA sequencing of matKand 6 primers for rbcL (Table 2) following two sequencingreaction profiles: (1) a 25-cycle reaction (30 s at 95°C, 30 sat 50°C, and 30 s at 72°C) with an initial denaturation at

95°C for 2 min and final extension at 72°C for 5 min; and(2) a 25-cycle reaction (10 s at 96°C, 5 s at 50°C, and 4 minat 60°C) with an initial denaturation at 96°C for 3 min butwithout a final extension. In most cases, both the sensestrand and antisense strand of the matK and rbcL regionswere sequenced, but in a few cases only a single strand wassequenced. The DNA sequences were aligned manually.

Phylogenetic analysis

We employed the maximum-parsimony method for phylo-genetic reconstruction, using PAUP* version 4.0 beta 10(Swofford 2002). The heuristic search options of randomaddition sequence (100 replications), tree bisection andreconnection (TBR) swapping algorithm and multiple par-simonious trees (MULPARS) were used to find the mostparsimonious trees. Only the base substitutions weretreated as characters for phylogenetic reconstruction, and

Table 2. Name, direction and reference/DNA sequence for PCR and sequence primers used in the present study

aUsed only for PCR amplificationbUsed only for DNA sequencing; primers that were not used only for PCR amplification and were not used only for DNA sequencing were usedboth for PCR amplification and for DNA sequencingcNewly designeddDesigned by K. Abe and K. Ueda (unpublished)eDesigned by N. Fujii (personal communication)fDesigned by Y. Mori and H. Okada (unpublished)

Primer name Direction Reference/DNA sequence

For sequencing the matK genetrnK-11a Sense Liston and Kadereit (1995)trnK-300Fb Sense Yamashita and Tamura (2000)trnK-710F Sense Johnson and Soltis (1995)trnK-780Fb Sense Yamashita and Tamura (2004)matK-AF Sense Ooi et al. (1995)matK-960Fc Sense 5¢-TCGAATGTATCAACAGAATTATTTG-3¢matK-1080Fb Sense Yamashita and Tamura (2000)matK-1100Fb Sense Fuse and Tamura (2000)matK-1150Fb Sense Fuse and Tamura (2000)matK-1164Rbc Antisense 5¢-TTGAATGAATAGATCGTAAATT-3¢matK-1235Fb Sense Johnson and Soltis (1995)matK-1235R Antisense Johnson and Soltis (1995)matK-1300Fb Sense Fuse and Tamura (2000)matK-1412F Sense Johnson and Soltis (1995)matK-1470F Sense Johnson and Soltis (1995)matK-1470R Antisense Johnson and Soltis (1995)matK-1649Fbc Sense 5¢-TCTTTTGATGAAGAAATGGA-3¢matK-1696Rbd Antisense 5¢-CATTGCCAAAAATTGACAAAGT-3¢matK-1700Rbc Antisense 5¢-ACCAAAAGTGAAAATAATATTGCCA-3¢matK-1900Rb Antisense Fuse and Tamura (2000)matK-1940Fb Sense Yamashita and Tamura (2000)matK-1987Fbe Sense 5¢-GTCAGATTCTGAGATTCTCAATCG-3¢matK-8F Sense Steele and Vilgalys (1994)matK-8R Antisense Ooi et al. (1995)FM-05Fbf Sense 5¢-CATGTGCTAGAACTTTGGCCCG-3¢trnK-2621 Antisense Liston and Kadereit (1995)

For sequencing the rbcL generbcLN¢ Sense Terauchi et al. (1987)rbcL-590Rb Antisense Yamashita and Tamura (2000)rbcL-630Fb Sense Terauchi et al. (1987)rbcL-840F Sense Yamashita and Tamura (2000)rbcL-840R Antisense Yamashita and Tamura (2000)DBRBAS2 Antisense Shinwari et al. (1994)

116

all kinds of base substitutions were equally weighted. Gapswere treated as missing values. The insertion/deletion of aDNA fragment into/from matK was postulated based on thegaps. Neither insertions nor deletions were found in therbcL region.

In the combined analysis of matK and rbcL sequences,the sequence data for matK and rbcL of the same genuswere united in tandem even though they were obtainedfrom different species. The consistency index (CI) andretention index (RI) were calculated with PAUP. The datadecisiveness (DD [Goloboff 1991]) was calculated withPAUP and MacClade version 3.02 (Maddison and Maddi-son 1992). The CI, RI, and DD values were calculatedincluding uninformative characters. A bootstrap analysiswith 100 replications (heuristic; 100 random; TBR) wasperformed using PAUP.

The sister group for the monocots is yet to be identifiedbut a comprehensive study of angiosperms using a com-bined data set of rbcL, atpB and 18S rDNA sequencesshowed that the monocots join the eumagnoliids togetherwith Magnoliales, Laurales, Winterales, Piperales and Chlo-ranthales (Soltis et al. 2000). For the present study, we usedMagnolia (Magnoliaceae) and Piper (Piperaceae) as theoutgroup for the monocots, which represent two rather dis-tantly related orders (Chase et al. 2000).

Results

Of the 1,879 bp of matK sequences that were aligned forthe analysis, 1,319 bp (70.2%) were variable, and 1,043 bp(55.5%) phylogenetically informative. On the other hand,of the 1,390 bp of rbcL sequences, 661 bp (47.6%) werevariable, and 468 bp (33.7%) phylogenetically informative.

Values of sequence divergence for matK range from0.3% (between Paris and Daiswa) to 28.9% (between Piperand Hordeum) for all the taxa examined in the presentstudy, and to 27.2% (between Limnocharis and Juncus)within the monocots. Those of rbcL range from 0.1%(between Lilium and Nomocharis and between Smilacinaand Maianthemum) to 13.8% (between Limnocharis andPhyllostachys).

We found 124 insertions or deletions (indels) in the matKgene region. Among them, 111 indels are multiples of 3 bp,while the remaining 13 indels are not and are restricted tothe 3¢ terminal region of the gene, i.e. at positions 1,830–1,871. Accordingly, the latter indels result in a shift of framein each most-downstream region of matK and the successiveshift of the stop codon position.

The combined analysis resulted in 15 most parsimonioustrees. The CI is 0.34, the RI 0.65, and the DD 0.60. The strictconsensus tree of the 15 most parsimonious trees supportsthe monophyly of the monocots (with 98% bootstrap sup-port) (Fig. 1). Of the 107 distinct clades, 67 receive morethan 95% bootstrap support, 82 more than 80% bootstrapsupport, and 95 more than 60% bootstrap support.

The strict consensus topology (Fig. 1) shows that Acorus(Acoraceae) is a sister group to the rest of the monocots,

which receives 100% bootstrap support. A clade comprisingAlismatales is diverged as a next branch, followed succes-sively by Petrosaviaceae, the clade of Dioscoreales andPandanales, Liliales, Asparagales and commelinoids. All ofthese clades receive more than 90% bootstrap support. Thesister-group relationship is also strongly supported betweenAlismatales and the remaining monocots (except for Aco-raceae) (100%), between Petrosaviaceae and the remainingmonocots (except for Acoraceae and Alismatales) (100%),between the clade of Dioscoreales and Pandanales and theclade of Liliales, Asparagales and commelinoids (87%), andbetween Liliales and the clade of Asparagales and commeli-noids (89%). Only the sister-group relationship betweenAsparagales and commelinoids is weakly supported (68%).

The first large clade comprising Alismatales is dividedinto two subclades: one comprising Araceae, and theother comprising Aponogetonaceae, Alismataceae, Hydro-charitaceae, Juncaginaceae, Limnocharitaceae, Potamo-getonaceae and Tofieldiaceae. Based on three generaexamined, the former subclade receives 100% bootstrapsupport, with Gymnostachys placed as a sister group to aAmorphophallus–Zantedeschia clade. The latter subclade ismoderately supported (77%), but, if Tofieldia is excluded,the rest including Alismataceae, Aponogetonaceae,Hydrocharitaceae, Juncaginaceae, Limnocharitaceae, andPotamogetonaceae receives strong support (100%).

The second clade comprising only Petrosaviaceae, whichis strongly supported (96%), includes two Asian genera,Japonolirion and Petrosavia.

The third large clade comprising Dioscoreales andPandanales is divided into two strongly (97% and 100%)supported subclades: one comprising Dioscoreales(Dioscoreaceae and Nartheciaceae), and the other compris-ing Pandanales (Cyclanthaceae, Pandanaceae and Stemo-naceae). In the former, Nartheciaceae are a sister group toDioscoreaceae, while in the latter, Stemonaceae are posi-tioned basal and adjacent to a Pandanaceae–Cyclanthaceaeclade (with 100% bootstrap support).

The fourth clade comprising Liliales is divided into twosubclades: one (with 100% bootstrap support) comprisingMelanthiaceae, and the other (with 81% bootstrap support)including Alstroemeriaceae, Colchicaceae, Liliaceae andLuzuriagaceae. The latter is further subdivided into twostrongly (100%) supported subclades. One of these com-prises Liliaceae, and the other includes Alstroemeriaceae,Colchicaceae and Luzuriagaceae.

In the remaining large Asparagales–commelinoids cladethat is weakly supported (68%), both the Asparagales cladeand the commelinoid clade are strongly supported (95%and 99%).

Within the Asparagales clade, Orchidaceae are posi-tioned basal and adjacent to the rest of the order which,however, receives weak support (74%). Subsequently, thereis divergence of a Blandfordiaceae–Hypoxidaceae clade(with 90% bootstrap support) from the remainder (with100% bootstrap support), a Doryanthaceae–Ixioliriaceaeclade (with 81% bootstrap support) from the remainder(with 98% bootstrap support), and Iridaceae from a clade(with 100% bootstrap support) including a majority of

117

Asparagales. The terminal clade in the Asparagales, whichincludes Hyacinthaceae, Themidaceae, Alliaceae, Amarylli-daceae, Hemerocallidaceae, Agavaceae, Asparagaceae,Laxmanniaceae and Ruscaceae, may be divided into two ormore large subclades, which however receive less than 50%bootstrap support. Nevertheless, nearly all of the familiesincluded receive 100% bootstrap support.

Within the commelinoids, the strongly (100%) supportedArecales (Arecaceae) diverges first from the remainder

(with 84% bootstrap support). The latter is divided intotwo subclades: one including Commelinales (Haemodora-ceae, Hanguanaceae and Pontederiaceae), and Zingi-berales (Zingiberaceae), and the other including Poales(Typhaceae, Cyperaceae, Juncaceae, Flagellariaceae andPoaceae). Relationships among the families are notresolved well in the former, but resolved well in the latter.Typhaceae are placed basally as a sister group to the restof the Poales (with 100% bootstrap support), where a

Fig. 1. Strict consensus of the 15 equally most parsimonious trees resulting from the combined analysis of matK and rbcL DNA sequence data of the monocotyledons. Numbers above the branches indicate bootstrap values that are expressed as percentages of 100 replicates. The circumscription of orders and families follow APG II (2003)

Dracaena

Aspidistra

Hordeum

Typha

Globba

ZingiberHedychium

Phyllostachys

Sparganium

ScirpodendronJuncus

Flagellaria

EichhorniaMonochoria

Butia

Anigozanthos

Hanguana

Phoenix

PolygonatumHeteropolygonatum

LeucojumGalanthus

StenomessonClivia

AlliumNothoscordumTulbaghia

Bessera

BrodiaeaMilla

ScillaBowieaIris

DoryanthesIxiolirion

CurculigoRhodohypoxisBlandfordiaPhaiusSpiranthes

DisporopsisSmilacina

Dasylirion

Peliosanthes

Maianthemum

Nolina

LiriopeOphiopogon

TricalistraTupistra

CampylandraRohdea

ReineckeaConvallaria

PleomeleRuscus

ComospermumCordylineAsparagusYucca1Yucca2PolianthesCamassiaHostaChlorophytum

HemerocallisDianella

Amaryllis

Colchicum

DisporumUvularia

DrymophilaAlstroemeriaLiliumNomocharisFritillaria

TulipaAmanaErythronium

MedeolaClintoniaTricyrtisStreptopusScoliopusParisDaiswaKinugasaTrillium

Chionographis

HeloniopsisYpsilandra

Veratrum

Stemona

FreycinetiaCarludovica

DioscoreaTacca

LophiolaNartheciumMetanartheciumAletris

PetrosaviaJaponolirion

Tofieldia

PotamogetonTriglochinAponogeton

AlismaLimnocharis

Blyxa

ZantedeschiaAmorphophallus

Gymnostachys

Acorus

PiperMagnolia

10092

100100

100

100

73100

100

51<50

100

100

99

100

<50

<50<50

<50

100

95

8266

100

91

69

97

66

94

77

<50100

10092

<5059

100

<50

100

98

100

<50

79100

98

10099

99100

78100 63

100

68

74

95

89

87

100

81

100100 100

99

100

96

100

99100

100

97

71

100

100

100

64100

8187

96100

90

100

97

100

100

56100

100

96

100

100

84

98

95

100100

77

10096

98100

92

81

90100

100

Acoraceae

Tofieldiaceae

Petrosaviaceae

Nartheciaceae

Araceae

HydrocharitaceaeAlismataceaeLimnocharitaceaeAponogetonaceaeJuncaginaceaePotamogetonaceae

Dioscoreaceae

PandanaceaeCyclanthaceaeStemonaceae

Melanthiaceae

Liliaceae

AlstroemeriaceaeLuzuriagaceae

Colchicaceae

Orchidaceae

HypoxidaceaeBlandfordiaceae

DoryanthaceaeIxioliriaceaeIridaceae

Amaryllidaceae

Alliaceae

Themidaceae

Hemerocallidaceae

Agavaceae

AsparagaceaeLaxmanniaceae

Ruscaceae

Hyacinthaceae

Arecaceae

Haemodoraceae

Pontederiaceae

Zingiberaceae

Hanguanaceae

Typhaceae

JuncaceaeCyperaceae

FlagellariaceaePoaceae

Zingiberales

Commelinales

Asparagales

Pandanales

Dioscoreales

Alismatales

Acorales

Poales

Arecales

Liliales

118

Cyperaceae–Juncaceae clade is sister to a Flagellariaceae–Poaceae clade.

Discussion

Phylogenetic relationships at higher level within the monocotyledons

Like nearly all previous molecular analyses (e.g., Chaseet al. 2000; Fuse and Tamura 2000), the present analysisbased on a combined data set of matK plus rbcL sequencesshows that the monocots are strongly supported (98%) asmonophyletic. As regards relationships within the mono-cots, the analysis of the combined data set of matK plusrbcL sequences further shows a topology highly similar tothat obtained based on matK sequences alone (Fuse andTamura 2000) and three-gene (rbcL plus atpB plus 18SrDNA) sequences (Chase et al. 2000). However, the topol-ogy is much better resolved in the combined analysis ofmatK plus rbcL sequences than in the other analyses. Infact, major clades, which received less than 50% bootstrapsupport in the previous studies (Fuse and Tamura 2000;Chase et al. 2000) but more than 85% bootstrap support inthe present study, include the clade (87%) containing all themonocots except for Acorus, Alismatales and Petrosavi-aceae, and the clade (89%) comprising Liliales, Asparagalesand commelinoids.

Noticeably, the relationships among Petrosaviaceae,Dioscoreales, Pandanales, Liliales, Asparagales and com-melinoids that formed polytomy in the earlier study (Chaseet al. 2000) are clearly resolved by the combined analysis ofmatK plus rbcL sequences. Petrosaviaceae, whose positionhas been unclear (APG II 2003), are placed as a sistergroup to the well-supported clade (87%) comprising Dios-coreales, Pandanales, Liliales, Asparagales and commeli-noids. Because of its distinct position, Petrosaviaceae canbe placed in its own order, Peterosaviales. Dioscorealesand Pandanales have a sister-group relationship; Lilialesare related to Asparagales and commelinoids, formingtogether the well-supported clade (89%). Asparagales area sister group to commelinoids, although their commonclade receives weak support (68%).

Relationships among and within families

The present study shows clear relationships among andwithin some families whose systematic position has beenunclear in morphology-based comparisons and even in ear-lier molecular phylogenetic analyses (Fuse and Tamura2000; Chase et al. 2000).

For instance, the position of Nartheciaceae (sensustricto) in Dioscoreales has never been strongly supportedbefore (see APG II 2003), but a common clade withDioscoreaceae, receives 97% bootstrap support in thepresent study.

As regards relationships within Asparagales, Chase et al.(2000) on the basis of a three-gene analysis, showed that

Orchidaceae, Hypoxidaceae and Blandfordiaceae formed aclade (<50%) at the basal portion of Asparagales, statingthat the basal portion of Asparagales requires some furtherattention. The present study, however, showed thatOrchidaceae are a sister group to a clade (74%) containingthe rest of the Asparagales including Hypoxidaceae andBlandfordiaceae.

The position of Hanguana (Hanguanaceae) distributedin Sri Lanka, southeast Asia, Micronesia and Australia hasbeen unclear. Traditionally, the genus had been placed inFlagellariaceae (Poales) (for review see Takhtajan 1997).Tillich (1996) suggested that Hanguana is related to Com-melinaceae in seedling morphology. Rudall et al. (1999) alsosuggested that Hanguana is related to Zingiberales insharing spinulate inaperturate pollen, plasmodial-tapetumtype, mucilage-secreting intra-ovarian trichomes andmodified septal nectaries. Based on an analysis of rbcLsequences, Chase et al. (1995) showed that Hanguana is asister group to the Commelinales–Zingiberales clade. Chaseet al. (2000) further showed on the basis of a three-geneanalysis (rbcL plus atpB plus 18S rDNA) that Hanguana isembedded in the Commelinales clade, and more recentlyAPG II (2003) moved the genus to Commelinales. How-ever, since Chase et al. (2000) did not determine the 18SrDNA sequence of Hanguana, the position of Hanguanawas given only with the rbcL and atpB data set. Thebootstrap support for the inclusion of Hanguana in theCommelinales clade was less than 50%.

In contrast, the present study showed that Hanguana isnot only embedded in Commelinales but also forms a well-supported (100%) clade with Zingiberaceae (Zingiberales).Although Hanguana does not necessarily form a well-supported clade with Eichhornia and Monochoria(Pontederiaceae in Commelinales), the genus shares withMonochoria (uncertain in Eichhornia) an insertion of 5 bpin the region just below the stop codon of the matK gene(positions 1,853–1,857 [see electronic supplementary mate-rial]). It seems very likely that the 5-bp insertion is one ofthe synapomorphies for the clade of Hanguana and Ponte-deriaceae (Eichhornia, Monochoria). Molecular evidencethus more strongly supports the inclusion of Hanguana inCommelinales.

Acknowledgements We express our sincere gratitude to Hiroshi Tobefor helpful comments and critical reading of the manuscript, to WendyB. Zomlefer and Hidetoshi Nagamasu for supplying us with plantmaterials used in this study, to Pu Fading of Chengdu Institute ofBiology, Academia Sinica, China and staff members of WawushanNational Forest Park, China and Teshio Experimental Forest,Hokkaido University, Japan for their assistance in various ways infieldwork, and to Moritoshi Iino for allowing us to use his ABI auto-sequencer freely. The study was supported in part by Grants-in-Aid forScientific Research (11640701 and 14405012) from the Ministry ofEducation, Science and Culture, Japan.

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