Springer-VerlagTokyo102650918-94401618-086030669031Journal of Plant Research J Plant Res13310.

1007/s10265-003-0133-3

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

ORIGINAL ARTICLE

Minoru N. Tamura • Jun Yamashita • Shizuka Fuse •

Masatake Haraguchi

Molecular phylogeny of monocotyledons inferred from combined analysis of

plastid matK and rbcL gene sequences

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

Abstract Using matK and rbcL sequences (3,269 bp in Key words Hanguanaceae · matK · Molecular phylogeny ·
total) from 113 genera of 45 families, we conducted a com- Monocotyledons · Petrosaviaceae · rbcL
bined analysis to contribute to the understanding of major
evolutionary relationships in the monocotyledons. Trees
resulting from the parsimony analysis are similar to those
generated by earlier single or multiple gene analyses, but Introduction
their strict consensus tree provides much better resolution
of relationships among major clades. We find that Acorus Over the past 10 years, several papers have been published
(Acorales) is a sister group to the rest of the monocots, on the phylogenetic relationships within the monocots
which receives 100% bootstrap support. A clade comprising based on DNA sequence data. They include, for instance,
Alismatales is diverged as the next branch, followed succes- the articles published by Chase et al. (1993, 1995, 2000),
sively by Petrosaviaceae, the Dioscoreales–Pandanales Nadot et al. (1995), Bharathan and Zimmer (1995), Soltis
clade, Liliales, Asparagales and commelinoids. All of these et al. (1997, 2000), Davis et al. (1998), Savolainen et al.
clades are strongly supported (with more than 90% boot- (2000), and Fuse and Tamura (2000). The studies were based
strap support). The sister-group relationship is also strongly on one-, two- or three-gene analyses of plastid (rbcL, rps4,
supported between Alismatales and the remaining mono- atpB, matK), mitochondrial (atpA), and nuclear (18S
cots (except for Acorus) (100%), between Petrosaviaceae rDNA) genes. The most comprehensive study of the mono-
and the remaining monocots (except for Acorus and cot phylogeny by three-gene analyses as well as a classifica-
Alismatales) (100%), between the clade comprising tion based on a synthesis of molecular analyses by various
Dioscoreales and Pandanales and the clade comprising researchers was published by Chase et al. (2000). According
Liliales, Asparagales and commelinoids (87%), and to this study, the monocots are monophyletic, with Acorales
between Liliales and the Asparagales–commelinoids clade (Acorus) at the basal-most position and with Alismatales as
(89%). Only the sister-group relationship between Aspar- the next branch; the rest of the monocots are split into four
agales and commelinoids is weakly supported (68%). large clades leading to Pandanales, Dioscoreales, Liliales,
Results also support the inclusion of Petrosaviaceae in its and Asparagales–commelinoids. Although all of these large
own order Petrosaviales, Nartheciaceae in Dioscoreales and clades have at least some bootstrap support, their inter-
Hanguanaceae in Commelinales. relationships receive less than 50% bootstrap support.
Additional, morphological and molecular data are in need
Electronic Supplementary Material Supplementary mate- to clarify relationships within the monocots (Chase et al.
rial is available in the online version of this article at 2000).
http://dx.doi.org/10.1007/s10265-003-0133-3 We have tested an analysis using a combined data set of
rbcL and matK sequences, and found that the combined
analysis provides a much better resolution of relationships
within the monocots than analysis of the combined data sets
of rbcL, atpB and 18S rDNA sequences (Chase et al. 2000;
M. N. Tamura (*) · J. Yamashita · S. Fuse1 · M. Haraguchi Soltis et al. 2000) and also better than analysis of rbcL
Botanical Gardens, Graduate School of Science, Osaka City (Chase et al. 1993, 1995) or matK sequences (Fuse and
University, 2000 Kisaichi, Katano-shi, Osaka 576-0004, Japan
e-mail: nmtamura@sci.osaka-cu.ac.jp Tamura 2000) separately. The combined analysis of rbcL
and matK sequences generated many fewer polytomies than
Present address: the analysis of either rbcL or matK sequences alone, and
1
Museum of Nature and Human Activities, Hyogo, Japan even more distinct clades (107 in the strict consensus tree),
110

most of which received higher bootstrap support (though
exact data are not presented).
In this paper we present the results of the combined
analysis of rbcL and matK sequences as another insight into
understanding the exact relationships within the monocots.
The matK sequences contained many indels (insertion/
deletion) throughout the monocots but, when properly
aligned for the analysis, provided useful and significant data
for molecular analyses of higher-level taxa. Based on the
results, we discuss systematic positions of several taxa that
have 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 were
used in the present study (Table 1). Among them, 28 genera
(29 OTUs) and 34 genera (35 OTUs) were analyzed with
respect 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 by
Fuse and Tamura (2000) and Yamashita and Tamura (2000),
respectively, were revised. Data on matK and rbcL
sequences of the remaining genera were obtained from the
DNA Data Bank of Japan (DDBJ).
DNA extraction and PCR amplification
Total genomic DNA was extracted from fresh leaves or from
leaves 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 regions
on the plastid DNA were amplified using the polymerase
chain reaction (PCR) with a programmable temperature-
control system PC-700 (Astec) or GeneAmp PCR System
9600 (Perkin Elmer). The amplification reaction mixture
was prepared using TaKaRa Ex Taq DNA polymerase fol-
lowing its instruction manual (Takara Shuzo). We used 11
and 4 PCR primers (Table 2) for the amplification of matK
and rbcL regions, respectively, and the following PCR pro-
file for both regions: a 37-cycle reaction with denaturation
at 94°C for 1 min, annealing at 52°C for 1 min, and extension
at 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 Accession Accession Voucherb
number number
(matK) (rbcL)

Dicotyledons (outgroup)
Magnoliales
Magnoliaceae
Magnolia pseudokobus Abe & Akasawa Fuse and Tamura (2000) AB040152
Magnolia hypoleuca Siebold & Zucc. Qiu et al. (1993) L12655
Piperales
Piperaceae
Piper nigrum L. Fuse and Tamura (2000) AB040153c M. N. Tamura and
S. Fuse 10016
Piper betle L. Qiu et al. (1993) L12660
Monocotyledons (ingroup)
Not ranked
Petrosaviaceae
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 7101
Acorales
Acoraceae
Acorus calamus L. Fuse and Tamura (2000) AB040154
Acorus calamus Shinwari et al. (1994) D28865
Alismatales
Alismataceae
Alisma canaliculatum A. Braun & Bouche ex Fuse and Tamura (2000) AB040179
Samuelsson
Alisma plantago-aquatica L. Chase et al. (1993) L08759
Aponogetonaceae
Aponogeton fenestralis Hook. f. The present study AB088779 AB088808 M. Haraguchi 9
Araceae
Amorphophallus campanulatus Blume ex Decne. The present study AB088777 M. N. Tamura 14308
Amorphophallus rivieri Dur. ex Riviere Cho and Palmer (1999) AJ005630
Gymnostachys anceps R. Br. Fuse and Tamura (2000) AB040177
 111

Table 1. Continued
Taxona Reference Accession Accession Voucherb
number number
(matK) (rbcL)

Gymnostachys anceps The present study AB088806 M. N. Tamura 8037

Zantedeschia aethiopica (L.) K. Spreng. Fuse and Tamura (2000) AB040178
Zantedeschia aethiopica Piques et al. (1998) AF065474
Hydrocharitaceae
Blyxa echinosperma Hook. f. The present study AB088781 AB088810 M.N. Tamura et al.
13546
Juncaginaceae
Triglochin maritimum L. The present study AB088782 AB088811 M.N. Tamura et al.
13563
Limnocharitaceae
Limnocharis flava Buchen. The present study AB088778 AB088807 M. N. Tamura 14311
Potamogetonaceae
Potamogeton distinctus A. Benn. The present study AB088780 AB088809 M. Haraguchi 14
Tofieldiaceae
Tofieldia nuda Maxim. Fuse and Tamura (2000) AB040159
Tofieldia nuda The present study AB088840 S. Fuse 8020
Asparagales
Agavaceae
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 17
Yucca australis Trel. The present study AB088789 AB088820 M. N. Tamura 14305
Alliaceae
Allium 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
Amaryllidaceae
Amaryllis 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
Asparagaceae
Asparagus cochinchinensis Merr. Yamashita and Tamura (2000) AB029804 AB029849c M. N. Tamura 14302
Blandfordiaceae
Blandfordia punicea Sweet Ito et al. (1999) AB017315
Blandfordia punicea de Bruijn et al. (unpublished) Z73694
Doryanthaceae
Doryanthes excelsa Correa The present study AB088785 M. N. Tamura 14304
Doryanthes excelsa de Bruijn et al. (unpublished) Z73697
Hemerocallidaceae
Dianella ensifolia DC. The present study AB088787 M. N. Tamura 14301
Dianella ensifolia Eguiarte et al. (1992) M96960
Hemerocallis fulva L. Ito et al. (1999) AB017318
Hemerocallis fulva Duvall et al. (unpublished) L05036
Hyacinthaceae
Bowiea volubilis Harv. ex Hook. f. The present study AB088790 M. Haraguchi 6
Bowiea volubilis Fay and Chase (1996) Z69237
Scilla scilloides (Lindl.) Druce Ito et al. (1999) AB017323
Scilla scilloides Shinwari et al. (1994) D28161
112

Table 1. Continued
Taxona Reference Accession Accession Voucherb
number number
(matK) (rbcL)

Hypoxidaceae
Curculigo capitulata Kuntze The present study AB088783 M. Haraguchi 2
Curculigo capitulata de Bruijn et al. (unpublished) Z73701
Rhodohypoxis baurii (Baker) Nel Ito et al. (1999) AB017324
Rhodohypoxis milloides (Baker) Hilliard & B. L. Rudall et al. (1997) Z77280
Burtt
Iridaceae
Iris japonica Thunb. The present study AB088786 M. Haraguchi 15
Iris ensata Thunb. Kawanod D28332
Ixioliriaceae
Ixiolirion tataricum Herb. Ito et al. (1999) AB017327
Ixiolirion tataricum de Bruijn et al. (unpublished) Z73704
Laxmanniaceae
Cordyline cf. stricta Endl. The present study AB088784 AB088818 M. N. Tamura 14309
Orchidaceae
Phaius tancarvilleae (Banks ex L’ Her.) Blume Fuse and Tamura (2000) AB040205c M. N. Tamura and
M. Tamura 2801
Phaius minor Blume Cameron et al. (1999) AF074210
Spiranthes sinensis Ames Fuse and Tamura (2000) AB040206c S. Fuse 8063
Spiranthes cernua (L.) Rich. Cameron et al. (1999) AF074229
Ruscaceae
Aspidistra elatior Blume Yamashita and Tamura (2000) AB029780
Aspidistra elatior Yamashita and Tamura (2004) AB089631
Campylandra siamensis Yamashita & M. N. Yamashita and Tamura (2000) AB029773 AB029835
Tamura
Comospermum yedoense (Maxim. ex Franch. & Yamashita and Tamura (2000) AB029808
Sav.) Rauschert
Comospermum yedoense The present study AB088821 M. N. Tamura 14313
Convallaria 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. Yamashita and Tamura (2000) AB029764 AB029831
Hu) M. N. Tamura & Ogisu
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 14310
Ophiopogon 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 4
Polygonatum involucratum (Franch. & Sav.) Yamashita and Tamura (2000) AB029762 AB029829
Maxim.
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 14306
Smilacina 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
Themidaceae
Bessera 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
Arecales
Arecaceae
Butia yatay Becc. The present study AB088794 AB088827 M. Haraguchi 11
Phoenix dactylifera L. Fuse and Tamura (2000) AB040211
Phoenix reclinata Jacq. Gaut et al. (1992) M81814
 113

Table 1. Continued
Taxona Reference Accession Accession Voucherb
number number
(matK) (rbcL)

Commelinales
Haemodoraceae
Anigozanthos flavidus DC. The present study AB088796 AB088829 M. Haraguchi 8
Hanguanaceae
Hanguana malayana Merr. The present study AB088800 AB088830 H. Nagamasu s. n.
Pontederiaceae
Eichhornia crassipes Solms Fuse and Tamura (2000) AB040212c S. Fuse 8065
Eichhornia crassipes Graham et al. (1998) U41574
Monochoria korsakowii Regel & Maack The present study AB088795 AB088828 M. Haraguchi 12
Dioscoreales
Dioscoreaceae
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
Nartheciaceae
Aletris spicata (Thunb.) Franch. Fuse and Tamura (2000) AB040174
Aletris spicata The present study AB088834 S. Fuse 8021
Lophiola aurea Ker Gawl. Fuse and Tamura (2000) AB040176c W. B. Zomlefer 719
Lophiola aurea The present study AB088836 W. B. Zomlefer 719
Metanarthecium luteo-viride Maxim. Fuse and Tamura (2000) AB040163
Metanarthecium luteo-viride The present study AB088837 M. N. Tamura 8009
Narthecium asiaticum Maxim. Fuse and Tamura (2000) AB040162
Narthecium asiaticum The present study AB088838 M. N. Tamura 8012
Liliales
Alstroemeriaceae
Alstroemeria sp. Ito et al. (1999) AB017328
Alstroemeria sp. Rudall et al. (1997) Z77254
Colchicaceae
Colchicum speciosum Steven Fuse and Tamura (2000) AB040181c M. N. Tamura 3428
Colchicum 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
Liliaceae
Amana 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 9835
Streptopus parviflorus The present study AB088817 M. N. Tamura 9835
Tricyrtis 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
Luzuriagaceae
Drymophila moorei Baker Fuse and Tamura (2000) AB040180c M. N. Tamura 8023
Drymophila moorei The present study AB088812 M. N. Tamura 8023
Melanthiaceae
Chionographis japonica Maxim. Fuse and Tamura (2000) AB040198
Chionographis japonica The present study AB088813 M. N. Tamura and
J. Yamashita 9904
Daiswa polyphylla Raf. Kazempour Osaloo and AB018828
Kawano (1999)
114

Table 1. Continued

Taxona Reference Accession Accession Voucherb

number number
(matK) (rbcL)

Daiswa polyphylla Kato et al. (1995) D28155

Heloniopsis orientalis C. Tanaka Fuse and Tamura (2000) AB040188c M. N. Tamura 10014
Heloniopsis orientalis The present study AB088814 M. N. Tamura 10014
Kinugasa japonica (Franch. & Sav.) Tatew. & Suto Kazempour Osaloo and AB018831
Kawano (1999)
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. AB017413
(1999)
Trillium undulatum Kazempour Osaloo and AB018848
Kawano (1999)
Veratrum maackii Regel Kazempour Osaloo et al. AB017417
(1999)
Veratrum maackii Kazempour Osaloo and AB018849
Kawano (1999)
Ypsilandra thibetica Franch. Fuse and Tamura (2000) AB040185c M. N. Tamura 9818
Ypsilandra thibetica The present study AB088815 M. N. Tamura 9818
Pandanales
Cyclanthaceae
Carludovica palmata Ruiz & Pav. The present study AB088793 M. Haraguchi 5
Carludovica palmata Qiu et al. (1999) AF197596
Pandanaceae
Freycinetia formosana Hemsl. Fuse and Tamura (2000) AB040209c M. N. Tamura et al. 9029
Freycinetia formosana The present study AB088825 M. N. Tamura et al. 9029
Stemonaceae
Stemona japonica Franch. & Sav. Fuse and Tamura (2000) AB040210c M. N. Tamura and
S. Fuse 10020
Stemona japonica The present study AB088826 M. N. Tamura and
S. Fuse 10020
Poales
Cyperaceae
Scirpodendron ghaeri (Goertn.) Merr. The present study AB088804 AB088832 M. Haraguchi 10
Flagellariaceae
Flagellaria indica L. Fuse and Tamura (2000) AB040214c M. N. Tamura et al. 9032
Flagellaria indica Chase et al. (1993) L12678
Juncaceae
Juncus effusus L. The present study AB088803 M. Haraguchi 16
Juncus effusus Chase et al. (1993) L12681
Poaceae
Hordeum 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
Typhaceae
Sparganium stoloniferum Buch.-Ham. The present study AB088802 AB088831 M. Haraguchi 18
Typha latifolia L. The present study AB088801 M. Haraguchi 13
Typha latifolia Smith et al. (1993) L05464
Zingiberales
Zingiberaceae
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
a
The classification follows APG II (2003)
b
Voucher specimens are preserved in the herbarium of the Botanical Gardens, Osaka City University
c
DNA sequence data, which had been already submitted to the DNA Data Bank of Japan (DDBJ) by Fuse and Tamura (2000) or Yamashita
and Tamura (2000), were revised in the present study
d
Directly submitted to the DDBJ/EMBL/GenBank database
 115

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

For sequencing the matK gene

trnK-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 gene
rbcLN¢ 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)
a
Used only for PCR amplification
b
Used only for DNA sequencing; primers that were not used only for PCR amplification and were not used only for DNA sequencing were used
both for PCR amplification and for DNA sequencing
c
Newly designed
d
Designed by K. Abe and K. Ueda (unpublished)
e
Designed by N. Fujii (personal communication)
f
Designed by Y. Mori and H. Okada (unpublished)

94°C for 2.5 min and final extension at 72°C for 7 min. We
purified the PCR products with MicroSpin S-300 HR Col-
umns (Pharmacia Biotech) to remove the primers and
remaining deoxynucleoside triphosphates.
DNA sequencing
We sequenced matK genes ranging from 1,515 bp (as in
Eichhornia) to 1,575 bp (as in Iris). Tricyrtis has the longest
sequence, i.e., 1,590 bp (Fuse and Tamura 2000). On the
other hand, the exact length of the rbcL gene is unclear
because the most-downstream region was not sequenced.
Overall, the lengths of aligned sequences of matK and rbcL
were 1,879 bp and 1,390 bp, respectively.
We used 25 primers for the DNA sequencing of matK
and 6 primers for rbcL (Table 2) following two sequencing
reaction profiles: (1) a 25-cycle reaction (30 s at 95°C, 30 s
at 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 min
at 60°C) with an initial denaturation at 96°C for 3 min but
without a final extension. In most cases, both the sense
strand and antisense strand of the matK and rbcL regions
were sequenced, but in a few cases only a single strand was
sequenced. 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 random
addition sequence (100 replications), tree bisection and
reconnection (TBR) swapping algorithm and multiple par-
simonious trees (MULPARS) were used to find the most
parsimonious trees. Only the base substitutions were
treated as characters for phylogenetic reconstruction, and
116
all kinds of base substitutions were equally weighted. Gaps
were treated as missing values. The insertion/deletion of a
DNA fragment into/from matK was postulated based on the
gaps. Neither insertions nor deletions were found in the
rbcL region.
In the combined analysis of matK and rbcL sequences,
the sequence data for matK and rbcL of the same genus
were united in tandem even though they were obtained
from different species. The consistency index (CI) and
retention index (RI) were calculated with PAUP. The data
decisiveness (DD [Goloboff 1991]) was calculated with
PAUP and MacClade version 3.02 (Maddison and Maddi-
son 1992). The CI, RI, and DD values were calculated
including uninformative characters. A bootstrap analysis
with 100 replications (heuristic; 100 random; TBR) was
performed using PAUP.
The sister group for the monocots is yet to be identified
but a comprehensive study of angiosperms using a com-
bined data set of rbcL, atpB and 18S rDNA sequences
showed that the monocots join the eumagnoliids together
with Magnoliales, Laurales, Winterales, Piperales and Chlo-
ranthales (Soltis et al. 2000). For the present study, we used
Magnolia (Magnoliaceae) and Piper (Piperaceae) as the
outgroup 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 for
the 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%) were
variable, and 468 bp (33.7%) phylogenetically informative.
Values of sequence divergence for matK range from
0.3% (between Paris and Daiswa) to 28.9% (between Piper
and Hordeum) for all the taxa examined in the present
study, and to 27.2% (between Limnocharis and Juncus)
within the monocots. Those of rbcL range from 0.1%
(between Lilium and Nomocharis and between Smilacina
and Maianthemum) to 13.8% (between Limnocharis and
Phyllostachys).
We found 124 insertions or deletions (indels) in the matK
gene region. Among them, 111 indels are multiples of 3 bp,
while the remaining 13 indels are not and are restricted to
the 3¢ terminal region of the gene, i.e. at positions 1,830–
1,871. Accordingly, the latter indels result in a shift of frame
in each most-downstream region of matK and the successive
shift of the stop codon position.
The combined analysis resulted in 15 most parsimonious
trees. The CI is 0.34, the RI 0.65, and the DD 0.60. The strict
consensus tree of the 15 most parsimonious trees supports
the monophyly of the monocots (with 98% bootstrap sup-
port) (Fig. 1). Of the 107 distinct clades, 67 receive more
than 95% bootstrap support, 82 more than 80% bootstrap
support, 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 comprising
Alismatales is diverged as a next branch, followed succes-
sively by Petrosaviaceae, the clade of Dioscoreales and
Pandanales, Liliales, Asparagales and commelinoids. All of
these clades receive more than 90% bootstrap support. The
sister-group relationship is also strongly supported between
Alismatales and the remaining monocots (except for Aco-
raceae) (100%), between Petrosaviaceae and the remaining
monocots (except for Acoraceae and Alismatales) (100%),
between the clade of Dioscoreales and Pandanales and the
clade of Liliales, Asparagales and commelinoids (87%), and
between Liliales and the clade of Asparagales and commeli-
noids (89%). Only the sister-group relationship between
Asparagales and commelinoids is weakly supported (68%).
The first large clade comprising Alismatales is divided
into two subclades: one comprising Araceae, and the
other comprising Aponogetonaceae, Alismataceae, Hydro-
charitaceae, Juncaginaceae, Limnocharitaceae, Potamo-
getonaceae and Tofieldiaceae. Based on three genera
examined, the former subclade receives 100% bootstrap
support, with Gymnostachys placed as a sister group to a
Amorphophallus–Zantedeschia clade. The latter subclade is
moderately supported (77%), but, if Tofieldia is excluded,
the rest including Alismataceae, Aponogetonaceae,
Hydrocharitaceae, Juncaginaceae, Limnocharitaceae, and
Potamogetonaceae receives strong support (100%).
The second clade comprising only Petrosaviaceae, which
is strongly supported (96%), includes two Asian genera,
Japonolirion and Petrosavia.
The third large clade comprising Dioscoreales and
Pandanales 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 to
Dioscoreaceae, while in the latter, Stemonaceae are posi-
tioned basal and adjacent to a Pandanaceae–Cyclanthaceae
clade (with 100% bootstrap support).
The fourth clade comprising Liliales is divided into two
subclades: one (with 100% bootstrap support) comprising
Melanthiaceae, and the other (with 81% bootstrap support)
including Alstroemeriaceae, Colchicaceae, Liliaceae and
Luzuriagaceae. The latter is further subdivided into two
strongly (100%) supported subclades. One of these com-
prises Liliaceae, and the other includes Alstroemeriaceae,
Colchicaceae and Luzuriagaceae.
In the remaining large Asparagales–commelinoids clade
that is weakly supported (68%), both the Asparagales clade
and 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, there
is divergence of a Blandfordiaceae–Hypoxidaceae clade
(with 90% bootstrap support) from the remainder (with
100% bootstrap support), a Doryanthaceae–Ixioliriaceae
clade (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

Fig. 1. Strict consensus of 92

100 Hordeum Poaceae
Phyllostachys
the 15 equally most parsimonious 100 Flagellaria Flagellariaceae
trees resulting from the 100
100 Juncus
Scirpodendron
Juncaceae
Cyperaceae Poales
combined analysis of matK and 100 Typha
Typhaceae
Sparganium
rbcL DNA sequence data of the 84
73
100 Zingiber
monocotyledons. Numbers above Hedychium
Globba
Zingiberaceae Zingiberales
the branches indicate bootstrap 100 100 Eichhornia
99 <50 Monochoria Pontederiaceae
values that are expressed as 51 Hanguana Hanguanaceae Commelinales
percentages of 100 replicates.
Anigozanthos Haemodoraceae
The circumscription of orders 100 Butia
and families follow APG II Phoenix Arecaceae Arecales
(2003) <50 <50
Polygonatum
Heteropolygonatum
<50 Disporopsis
100 Smilacina
Maianthemum
100 Dracaena
Pleomele
Ruscus
82 Ophiopogon
66 Liriope
100 Peliosanthes Ruscaceae
Convallaria
91 66 Reineckea
77 Campylandra
69 Rohdea
<50 94 Tupistra
97 Tricalistra
Aspidistra
95 Nolina
68 Dasylirion
Comospermum
<50 Cordyline Laxmanniaceae
Asparagus Asparagaceae
100 Yucca1
59 Yucca2
<50 Polianthes
100
92 Camassia Agavaceae Asparagales
Hosta
<50 Chlorophytum
100 Dianella
100 Hemerocallis Hemerocallidaceae
100 Galanthus
99 Leucojum
99
100 Stenomesson Amaryllidaceae
Clivia
79 Amaryllis
98 98 Tulbaghia
100 Nothoscordum Alliaceae
<50 Allium
63 Brodiaea
100 Milla Themidaceae
100 78 Bessera
100 Scilla
89 Bowiea Hyacinthaceae
74 Iris Iridaceae
81 Ixiolirion Ixioliriaceae
Doryanthes Doryanthaceae
95 100 Curculigo
90 Rhodohypoxis Hypoxidaceae
Blandfordia Blandfordiaceae
100 Phaius
Spiranthes Orchidaceae
100 Disporum
100
100 Uvularia Colchicaceae
Colchicum
99 Drymophila Luzuriagaceae
Alstroemeria Alstroemeriaceae
97 Lilium
100 Nomocharis
81 99 Fritillaria
71 Erythronium
87 100 100
Amana
Tulipa Liliaceae
96 100 Medeola
100 Clintonia Liliales
Tricyrtis
100 100 Streptopus
Scoliopus
87 Paris
81 Daiswa
100 Kinugasa
64 Trillium
100 Melanthiaceae
100 96 Heloniopsis
Ypsilandra
Chionographis
100 Veratrum
100 Freycinetia Pandanaceae
100 Carludovica Cyclanthaceae Pandanales
Stemona Stemonaceae
90 100 Dioscorea
Tacca Dioscoreaceae
97
100 Lophiola Dioscoreales
56 Narthecium
100 Nartheciaceae
100 Metanarthecium
Aletris
96 Petrosavia
Japonolirion Petrosaviaceae

Tofieldia Tofieldiaceae
77 92 Potamogeton Potamogetonaceae
98 96 Triglochin Juncaginaceae
100 Aponogeton Aponogetonaceae
100 Limnocharis Limnocharitaceae
95 98 Alisma Alismataceae Alismatales
Blyxa Hydrocharitaceae
100 Amorphophallus
100 Zantedeschia Araceae
Gymnostachys

Acorus Acoraceae Acorales

Piper
Magnolia

Asparagales. The terminal clade in the Asparagales, which
includes Hyacinthaceae, Themidaceae, Alliaceae, Amarylli-
daceae, Hemerocallidaceae, Agavaceae, Asparagaceae,
Laxmanniaceae and Ruscaceae, may be divided into two or
more large subclades, which however receive less than 50%
bootstrap support. Nevertheless, nearly all of the families
included receive 100% bootstrap support.
Within the commelinoids, the strongly (100%) supported
Arecales (Arecaceae) diverges first from the remainder
(with 84% bootstrap support). The latter is divided into
two subclades: one including Commelinales (Haemodora-
ceae, Hanguanaceae and Pontederiaceae), and Zingi-
berales (Zingiberaceae), and the other including Poales
(Typhaceae, Cyperaceae, Juncaceae, Flagellariaceae and
Poaceae). Relationships among the families are not
resolved well in the former, but resolved well in the latter.
Typhaceae are placed basally as a sister group to the rest
of the Poales (with 100% bootstrap support), where a
118

Cyperaceae–Juncaceae clade is sister to a Flagellariaceae– Orchidaceae, Hypoxidaceae and Blandfordiaceae formed a

Poaceae clade. clade (<50%) at the basal portion of Asparagales, stating
that the basal portion of Asparagales requires some further
attention. The present study, however, showed that
Orchidaceae are a sister group to a clade (74%) containing
Discussion
the rest of the Asparagales including Hypoxidaceae and
Blandfordiaceae.
Phylogenetic relationships at higher level within
The position of Hanguana (Hanguanaceae) distributed
the monocotyledons
in Sri Lanka, southeast Asia, Micronesia and Australia has
been unclear. Traditionally, the genus had been placed in
Like nearly all previous molecular analyses (e.g., Chase
Flagellariaceae (Poales) (for review see Takhtajan 1997).
et al. 2000; Fuse and Tamura 2000), the present analysis
Tillich (1996) suggested that Hanguana is related to Com-
based on a combined data set of matK plus rbcL sequences
melinaceae in seedling morphology. Rudall et al. (1999) also
shows that the monocots are strongly supported (98%) as
suggested that Hanguana is related to Zingiberales in
monophyletic. As regards relationships within the mono-
sharing spinulate inaperturate pollen, plasmodial-tapetum
cots, the analysis of the combined data set of matK plus
type, mucilage-secreting intra-ovarian trichomes and
rbcL sequences further shows a topology highly similar to
modified septal nectaries. Based on an analysis of rbcL
that obtained based on matK sequences alone (Fuse and
sequences, Chase et al. (1995) showed that Hanguana is a
Tamura 2000) and three-gene (rbcL plus atpB plus 18S
sister group to the Commelinales–Zingiberales clade. Chase
rDNA) sequences (Chase et al. 2000). However, the topol-
et al. (2000) further showed on the basis of a three-gene
ogy is much better resolved in the combined analysis of
analysis (rbcL plus atpB plus 18S rDNA) that Hanguana is
matK plus rbcL sequences than in the other analyses. In
embedded in the Commelinales clade, and more recently
fact, major clades, which received less than 50% bootstrap
APG II (2003) moved the genus to Commelinales. How-
support in the previous studies (Fuse and Tamura 2000;
ever, since Chase et al. (2000) did not determine the 18S
Chase et al. 2000) but more than 85% bootstrap support in
rDNA sequence of Hanguana, the position of Hanguana
the present study, include the clade (87%) containing all the
was given only with the rbcL and atpB data set. The
monocots except for Acorus, Alismatales and Petrosavi-
bootstrap support for the inclusion of Hanguana in the
aceae, and the clade (89%) comprising Liliales, Asparagales
Commelinales clade was less than 50%.
and commelinoids.
In contrast, the present study showed that Hanguana is
Noticeably, the relationships among Petrosaviaceae,
not only embedded in Commelinales but also forms a well-
Dioscoreales, Pandanales, Liliales, Asparagales and com-
supported (100%) clade with Zingiberaceae (Zingiberales).
melinoids that formed polytomy in the earlier study (Chase
Although Hanguana does not necessarily form a well-
et al. 2000) are clearly resolved by the combined analysis of
supported clade with Eichhornia and Monochoria
matK plus rbcL sequences. Petrosaviaceae, whose position
(Pontederiaceae in Commelinales), the genus shares with
has been unclear (APG II 2003), are placed as a sister
Monochoria (uncertain in Eichhornia) an insertion of 5 bp
group to the well-supported clade (87%) comprising Dios-
in the region just below the stop codon of the matK gene
coreales, Pandanales, Liliales, Asparagales and commeli-
(positions 1,853–1,857 [see electronic supplementary mate-
noids. Because of its distinct position, Petrosaviaceae can
rial]). It seems very likely that the 5-bp insertion is one of
be placed in its own order, Peterosaviales. Dioscoreales
the synapomorphies for the clade of Hanguana and Ponte-
and Pandanales have a sister-group relationship; Liliales
deriaceae (Eichhornia, Monochoria). Molecular evidence
are related to Asparagales and commelinoids, forming
thus more strongly supports the inclusion of Hanguana in
together the well-supported clade (89%). Asparagales are
Commelinales.
a sister group to commelinoids, although their common
clade receives weak support (68%).
Acknowledgements We express our sincere gratitude to Hiroshi Tobe
for helpful comments and critical reading of the manuscript, to Wendy
B. Zomlefer and Hidetoshi Nagamasu for supplying us with plant
Relationships among and within families materials used in this study, to Pu Fading of Chengdu Institute of
Biology, Academia Sinica, China and staff members of Wawushan
The present study shows clear relationships among and National Forest Park, China and Teshio Experimental Forest,
within some families whose systematic position has been Hokkaido University, Japan for their assistance in various ways in
fieldwork, and to Moritoshi Iino for allowing us to use his ABI auto-
unclear in morphology-based comparisons and even in ear- sequencer freely. The study was supported in part by Grants-in-Aid for
lier molecular phylogenetic analyses (Fuse and Tamura Scientific Research (11640701 and 14405012) from the Ministry of
2000; Chase et al. 2000). Education, Science and Culture, Japan.
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