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Phylogeny and classification of Ranunculales: Evidence from four molecular loci and morphological data

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Previous phylogenetic analyses of Ranunculales, which have mostly been focused on an individual family and were based on molecular data alone, have recovered three main clades within the order. However, support for relationships among these three clades was weak. Earlier hypotheses were often hampered by limited taxon sampling; to date less than one-tenth of the genera in the order have been sampled. In this study, we used a greatly enlarged taxon sampling (105 species, representing 99 genera of all seven families in the order). Our study is, furthermore, the first to employ morphology (65 characters) in combination with sequence data from four genomic regions, including plastid rbcL, matK and trnL-F, and nuclear ribosomal 26S rDNA to reconstruct phylogenetic relationships within Ranunculales. Maximum parsimony and Bayesian inference were performed on the individual and combined data sets. Our analyses concur with those of previous studies, but in most cases provide stronger support and better resolution for relationships among the three main clades retrieved. The first, comprised solely of the monogeneric family Eupteleaceae, is the earliest-diverging lineage. The second clade is composed exclusively of taxa of Papaveraceae, which is sister to the third clade, the core Ranunculales, comprising the other five families of the order. Circaeasteraceae and Lardizabalaceae form a strongly supported clade. Pteridophyllum is supported as sister to Hypecoum, contradicting the viewpoint that the former is the earliest-diverging genus in Papaveraceae. Glaucidium is basalmost in Ranunculaceae. Within this phylogenetic framework, the evolution of selected characters is inferred and diagnostic morphological characters at different taxonomic levels are identified and discussed. Based on both morphological and molecular evidence, a classification outline for Ranunculales is presented, including the proposal of two new subfamilies, Menispermoideae and Tinosporoideae in Menispermaceae and a new tribe, Callianthemeae, for the genus Callianthemum (Ranunculaceae).
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Perspectives
in Plant Ecology,
Evolution and
Systematics
Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–110
Phylogeny and classification of Ranunculales: Evidence from four
molecular loci and morphological data
Wei Wang
a
, An-Ming Lu
a
, Yi Ren
b
, Mary E. Endress
c
, Zhi-Duan Chen
a,
a
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun,
Xiangshan, Beijing 100093, PR China
b
Key Laboratory of Medicinal Plant Resource and Natural Pharmaceutical Chemistry of Ministry of Education,
College of Life Sciences, Shaanxi Normal University, Xi’an 710062, PR China
c
Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland
Received 30 June 2008; received in revised form 21 November 2008; accepted 5 January 2009
Abstract
Previous phylogenetic analyses of Ranunculales, which have mostly been focused on an individual family and were
based on molecular data alone, have recovered three main clades within the order. However, support for relationships
among these three clades was weak. Earlier hypotheses were often hampered by limited taxon sampling; to date less
than one-tenth of the genera in the order have been sampled. In this study, we used a greatly enlarged taxon sampling
(105 species, representing 99 genera of all seven families in the order). Our study is, furthermore, the first to employ
morphology (65 characters) in combination with sequence data from four genomic regions, including plastid rbcL,
matK and trnL-F, and nuclear ribosomal 26S rDNA to reconstruct phylogenetic relationships within Ranunculales.
Maximum parsimony and Bayesian inference were performed on the individual and combined data sets. Our analyses
concur with those of previous studies, but in most cases provide stronger support and better resolution for
relationships among the three main clades retrieved. The first, comprised solely of the monogeneric family
Eupteleaceae, is the earliest-diverging lineage. The second clade is composed exclusively of taxa of Papaveraceae,
which is sister to the third clade, the core Ranunculales, comprising the other five families of the order.
Circaeasteraceae and Lardizabalaceae form a strongly supported clade. Pteridophyllum is supported as sister to
Hypecoum, contradicting the viewpoint that the former is the earliest-diverging genus in Papaveraceae. Glaucidium is
basalmost in Ranunculaceae. Within this phylogenetic framework, the evolution of selected characters is inferred and
diagnostic morphological characters at different taxonomic levels are identified and discussed. Based on both
morphological and molecular evidence, a classification outline for Ranunculales is presented, including the proposal of
two new subfamilies, Menispermoideae and Tinosporoideae in Menispermaceae and a new tribe, Callianthemeae, for
the genus Callianthemum (Ranunculaceae).
r2009 Ru¨ bel Foundation, ETH Zu¨ rich. Published by Elsevier GmbH. All rights reserved.
Keywords: Bayesian inference; Combined molecular and morphological data; Diagnostic characters; Parsimony analysis;
Phylogenetic relationships of Ranunculales
ARTICLE IN PRESS
www.elsevier.de/ppees
1433-8319/$ - see front matter r2009 Ru¨ bel Foundation, ETH Zu¨ rich. Published by Elsevier GmbH. All rights reserved.
doi:10.1016/j.ppees.2009.01.001
Corresponding author. Tel.: +86 10 62836434; fax: +86 10 62590843.
E-mail address: zhiduan@ibcas.ac.cn (Z.-D. Chen).
Introduction
The order Ranunculales is recognized in many
classification systems (e.g., Hutchinson, 1973;Dahlgren,
1983;Cronquist, 1988;Kubitzki et al., 1993;Takhtajan,
1997;APG, 1998;Wu et al., 2002;APG II, 2003;
Thorne, 2007); however, its circumscription has long
been controversial. Dahlgren’s (1983) Ranunculales
included Ranunculaceae (excluding Glaucidium), Lardi-
zabalaceae, Menispermaceae, Berberidaceae, Circaeast-
eraceae, and several segregate families, such as
Kingdoniaceae, Sargentodoxaceae, Fumariaceae, and
Glaucidiaceae. In addition to the nine families above,
Cronquist (1988) added Sabiaceae and Coriariaceae,
and Kubitzki et al. (1993) and Thorne (2007) merged
Dahlgren’s (1983) Papaverales into the order. In
contrast, Takhtajan’s (1997) and Wu et al.’s (2002)
Ranunculales contained only the family Ranunculaceae
(excluding Hydrastis and Glaucidium).
Molecular phylogenetics has contributed greatly to
the delimitation of the Ranunculales over the past
decade. Based on chloroplast rbcL sequences, Chase
et al. (1993) first recovered a clade (labeled ‘‘Ranuncu-
lids’’), which contained Berberidaceae, Eupteleaceae,
Lardizabalaceae, Menispermaceae, Papaveraceae sensu
lato (including Fumariaceae), and Ranunculaceae. The
inclusion of Euptelea in the ranunculids was surprising,
as it has traditionally been placed among the ‘‘lower’’
hamamelids. However, the same ‘‘Ranunculids’’ clade
was recovered by 18S rDNA sequence analysis (Soltis
et al., 1997), and this in turn was congruent with results
from a study using three gene data by Hoot and Crane
(1995), which also added Circaeasteraceae (including
Kingdonia) to the clade. Subsequent analyses have
supported seven families in Ranunculales: Berberidaceae
(including Nandinaceae), Circaeasteraceae (including
Kingdoniaceae), Eupteleaceae, Lardizabalaceae (includ-
ing Sargentodoxaceae), Menispermaceae, Papaveraceae
(including Fumariaceae and Pteridophyllaceae), and
Ranunculaceae (including Glaucidiaceae and Hydrasti-
daceae) (APG, 1998 ;Hoot et al., 1999;Savolainen
et al., 2000a;Soltis et al., 2000, 2003;APG II, 2003;Kim
et al., 2004a).
The Ranunculales sensu APG II (2003) is a strongly
supported monophyletic group within basal eudicots,
which are sister to all other eudicots (Chase et al., 1993;
Savolainen et al., 2000b;Soltis et al., 1997, 2000;Hilu
et al., 2003). The consensus is that the order comprises
three main constituent clades: the two earlier-diverging
ranunculids Eupteleaceae and Papaveraceae on the one
hand, and the well-supported clade of core Ranuncu-
lales, consisting of the other five families, which is more
derived, on the other (see Kim et al., 2004a). The
relationship among these three clades, however, remains
uncertain, because previous analyses, employing
different taxon and character sampling schemes and
phylogenetic methods, have produced conflicting topo-
logies that are in part only weakly supported (e.g., Hoot
and Crane, 1995;Hoot et al., 1999;Qiu et al., 2005,
2006;Savolainen et al., 2000a, b;Soltis et al., 2000, 2003;
Nickrent et al., 2002;Hilu et al., 2003;Zanis et al., 2003;
Kim et al., 2004a;Worberg et al., 2007). Furthermore,
the weakly supported sister relationship identified
between Circaeasteraceae and Lardizabalaceae (Hoot
and Crane, 1995;Hoot et al., 1999;Savolainen et al.,
2000a;Soltis et al., 2003;Kim et al., 2004a) needs
reevaluation.
Several monotypic genera within the order, such as
Glaucidium,Hydrastis,Kingdonia,Nandina,Pteridophyl-
lum, and Sargentodoxa, differ in their position and
taxonomic rank among the different classification
systems. For example, Glaucidium has been placed in
Ranunculaceae (e.g., Hutchinson, 1973;Dahlgren, 1980;
Cronquist, 1988), in Hydrastidaceae (Tobe, 2002), or
given familial rank and positioned close to Paeoniaceae
or Paeoniales (e.g., Takhtajan, 1997;Wu et al., 2002;
Thorne, 2007). The taxonomic contention involving
these genera is the basis of the dispute over the
delimitation of the order Ranunculales as well as that
of some of its constituent families. Recent broad-based
phylogenetic analyses in angiosperms or eudicots have
placed each of these monotypic genera into a family;
however, their positions have not been sufficiently
clarified due to the relatively limited taxon sampling in
each family. Analyses focusing on selected families in
the Ranunculales, such as Berberidaceae (Kim and
Jansen, 1996, 1998;Kim et al., 2004b;Wang et al.,
2007a), Lardizabalaceae (Hoot et al., 1995;Wang et al.,
2002), Papaveraceae (Hoot et al., 1997), and Ranuncu-
laceae (Johansson and Jansen, 1993;Hoot, 1995;
Johansson, 1995;Kosuge et al., 1995;Ro et al., 1997),
resolved the positions of some of these monotypic
genera, however, in most cases, did not follow through
with the taxonomic consequences.
There exists a striking disproportion in species
number among the families in the order. At one end
of the spectrum are Circaeasteraceae and Eupteleaceae,
each with only two species; Lardizabalaceae contains 50,
Menispermaceae 450, Berberidaceae ca. 650 and Papa-
veraceae 750 species. At the other end of the spectrum
are Ranunculaceae, with some 2000 species. Of the ca.
200 genera in Ranunculales, less than 20 genera have
been sampled in previous studies (e.g., Chase et al.,
1993;Hoot and Crane, 1995;Soltis et al., 1997, 2000,
2003;Hoot et al., 1999;Savolainen et al., 2000a, b;Hilu
et al., 2003;Kim et al., 2004a;Worberg et al., 2007).
Thus to date the patterns of morphological character
evolution in Ranunculales have not been sufficiently
well elucidated. In particular, the morphological syna-
pomorphies that define Ranunculales are still unclear.
The objectives of this study are (1) to investigate
the phylogenetic relationships at generic level within
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W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–11082
Ranunculales using four molecular loci from the
chloroplast and nuclear genomes and morphological
data with a more extensive sampling than in any
previous study, (2) to examine the diagnostic characters
at different taxonomic levels, and (3) to update the
classification system for Ranunculales so that it best
reflects the evolutionary history of the group.
Materials and methods
Taxon sampling
A total of 105 taxa of Ranunculales were sampled in
the present study, including 18 species and all 16 genera
of Berberidaceae, one species each of the two monotypic
genera of Circaeasteraceae, both species of the mono-
generic Eupteleaceae, one species each of the 10 genera
of Lardizabalaceae, 46 species from 43 of the 60 genera
of Ranunculaceae, one species each from 17 of the 71
currently recognized genera of Menispermaceae
(Kessler, 1993), including representatives of all major
lineages based on the study that sampled most of the
genera of this family (Ortiz et al., 2007), and one species
each from 10 of the 41 genera of Papaveraceae, spanning
all major lineages according to the analysis of Hoot
et al. (1997). Our outgroups included nine species of 8
genera representing all lineages of other basal eudicots,
one species each of 8 genera of core eudicots and 15
species of 10 genera representating all major lineages of
basal angiosperms, following the results of Savolainen
et al. (2000a, b),Soltis et al. (2000, 2003),Hilu et al.
(2003), and Qiu et al. (2005, 2006).Acorus was used to
represent monocots because it has been shown to be the
earliest-diverging branch in monocots (see Chase, 2004;
Li and Zhou, 2007). Voucher information and GenBank
accession numbers are listed in Table 1.
Morphological data
Sixty-five morphological characters were mainly
obtained from several published morphological matrices
(e.g., Loconte and Estes, 1989;Kadereit et al., 1994;
Loconte et al., 1995;Nandi et al., 1998;Doyle and
Endress, 2000;Wang and Li, 2002) or from the
literature. Morphological characters and their states as
well as the pertinent literature sources are given in
Appendix A.
We analyzed the morphological data using the genus
as a unit. In cases where different species of a genus have
different characters states, the character was coded as
polymorphic. Our morphological matrix contained 129
terminals (genera), 99 of them from Ranunculales. In
cases in which a character is poorly known or for which
information is currently unavailable, these were scored
as missing or inapplicable. The complete character
matrix is provided in Appendix B.
Molecular data
Forty-two new sequences of rbcL,49ofmatK,42of
trnL-F, and 48 of 26S rDNA were generated in this
study, including sequences for 3 genera and 6 species
reported here for the first time, and have been deposited
at GenBank. Most of the sequences used in this study
were generated in our previous studies (Wang et al.,
2007a, b, 2005;Wang and Chen, 2007); others were
taken from GenBank. Taxa analyzed, voucher informa-
tion and GenBank accession numbers are given in
Table 1. We were unable to obtain material of a few taxa
and for these sequences are missing as follows: Achlys
triphylla DC. (lacking trnL-F and 26S), Bongardia
chrysogonum Spach. (lacking trnL-F), Helleborus foetidus
L. (lacking rbcL), Hypecoum imberbe Sm. (lacking matK
and trnL-F), Megaleranthis saniculiflia Ohwi (lacking
rbcL and trnL-F), Ranzania japonica Ito (lacking trnL-F
and 26S), and Sanguinaria canadensis L. (lacking
trnL-F). In addition, we encountered difficulties in
obtaining some 26S rDNA sequences, and thus were
unable to obtain sequences of the following taxa:
Albertisia laurifolia Yamamoto, Anamirta cocculus (L.)
Wight & Arn., Archakebia apetala (Xia et al.) Wu, Chen
& Qin, Boquila trifoliata (DC.) Decaisne, Hypserpa
nitida Miers, Leptopyrum fumarioides (L.) Reichb.,
Macleaya microcarpa (Maxim.) Fedde, Naravelia
zeylanica (L.) DC., Paropyrum anemonoides (Kar. &
Kir.) Ulbr., Parvatia brunoniana spp. elliptica
Griffith, Pycnarrhena lucida (Teijsm. & Binn.) Miq.
and Stauntonia hexaphylla (Thunb.) Decne. Conse-
quently, the combined molecular data matrix (rbcL,
matK, trnL-F, and 26S), including outgroups, comprised
137 exemplars (Table 1); lacking sequences were scored
as missing.
Total genomic DNA was extracted from fresh silica
gel-dried leaves or seeds or from herbarium specimens
using the modified CTAB procedure of Doyle and
Doyle (1987). The selected DNA regions were amplified
with standard polymerase chain reaction (PCR). All
regions were amplified following the PCR protocols
used in Li et al. (2004) and Wang et al. (2005). The rbcL
gene was amplified and sequenced using the 1F and
1494R primers as well as the 991R internal primer (Chen
et al., 1998). The matK gene was amplified and
sequenced using the matK-AF (Li et al., 2004)or
matK-AF2 and matK-8R2 primers and the internal
primers matK-mF2 and matK-mR2 (Wang et al., 2007a).
The trnL-F region was sequenced using the trnF and
trnR primers (Taberlet et al., 1991). The 26S rDNA was
amplified and sequenced using F1 and R4 primers and
R5 internal primer (Wang et al., 2005). The PCR
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Table 1. GenBank accession numbers and vouchers/references for the sequences used in this study.
Taxon GenBank accession numbers Vouchers/References
rbcL matK trnL-F 26S
Ranunculaceae
Aconitum racemulosum Franch. *AY954488 FJ626484 FJ626533 *AY954473 Chongqing: Wang Wei 081 (PE)
Actaea asiatica Hara FJ626575 FJ626485 FJ626436 Chongqing: Chen et al. 960060 (PE)
1
AJ222985
1
Compton et al. (1998)
Adonis amurensis Regel & Radde *AY954487 FJ626486 FJ626534 *AY954472 Jilin: Chen Z-D 003 (PE)
Anemoclema glaucifolium (Franch.) W. T.
Wang FJ626576 FJ626487 FJ626535 FJ626437 Yunnan: Wang Wei YN070 (PE)
Anemone hupehensis Lem. FJ626577 FJ626488 FJ626536 FJ626438 Guizhou: Wang Wei 002 (PE)
Anemonopsis macrophylla Sieb. & Zucc. FJ626578 FJ626489 provided by Hoot: Reznicek 9977 (MICH)
1
AJ222984
2
AF131289
1
Compton et al. (1998),
2
Ro et al. (2003)
Aquilegia ecalcarata Maxim. *AY954495 *EF437127 *EF437096 *AY954481 Chongqing: Wang Wei 117 (PE)
Aquilegia oxysepala Trautv. & Kir. *EF437140 *EF437128 *EF437097 FJ626439 Jilin: Chen Z-D 001 (PE)
Asteropyrum cavaleriei (Le´vl. et Vant.)
Drumm. & Hutch.
1
AF079453
1
Wang and Chen, unpubl.
FJ626490 FJ626537 *AY954466 Chongqing: Wang Wei 110 (PE)
Batrachium bungei (Steud.) L. Liou. FJ626579 FJ626491 FJ626538 FJ626440 Yunnan: Wang Wei YN092 (PE)
Beesia calthifolia (Maxim.) Ulbr.
1
AF079452
2
AJ496612
1
Wang and Chen, unpubl.;
2
Compton and
Culham (2002)
FJ626492 *AY954468 Hubei: Chen 200010661 (PE)
Callianthemum taipaicum W. T. Wang FJ626580 FJ626493 FJ626539 FJ626441 Shanxi: Wang Wei SX004 (PE)
Caltha palustris L.
1
L02431
2
AB069845
3
AJ496610
4
U52632
1
Albert et al. (1992),
2
Adachi et al., unpubl.;
3
Compton and Culham (2002),
4
Ro et al.
(1997)
Caltha palustris var. membranacea Turcz. FJ626581 FJ626494 FJ626540 FJ626442 Jilin: Chen 2072 (PE)
Cimicifuga simplex Wormsk. *AY954483 *AY954469 Jilin: Chen 2079 (PE)
1
AB044754
2
AJ223008 Li et al., unpubl.;
2
Compton et al. (1998)
Clematis ganpiniana (Le´vl. & Vant.) Tamura *AY954491 FJ626495 FJ626541 *AY954476 Chongqing: Wang Wei 119 (PE)
Consolida ajacis (L.) Schur FJ626582 FJ626496 FJ626443 Xi’an (cult.): Wang Wei Seed2 (PE)
1
AY150259
1
Luo et al., unpubl.
Coptis chinensis Franch. *AY954497 *DQ478614 *EF437110 *AY954482 Sichuan: Chen et al. 960105 (PE)
Delphinium bonvalotii Franch. FJ626583 FJ626497 FJ626542 FJ626444 Guizhou: Wang Wei 030 (PE)
Dichocarpum sutchuenense W. T. Wang &
Hsiao *AY954493 *EF437130 *EF437099 *AY954479 Guizhou: Wang Wei 069 (PE)
Enemion raddeanum Regel *AY954494 *EF437131 *EF437100 *AY954478 Jilin: Chen Z-D 2090 (PE)
Eranthis stellata Maxim. *AY954484 FJ626498 FJ626543 *AY954467 Jilin: Chen Z-D 001 (PE)
Glaucidium palmatum Sieb. & Zucc.
1
AF093723
2
AB069850
3
AF389267
1
Hoot et al. (1999),
2
Adachi et al., unpubl.;
3
Kim et al. (2004a)
*EF437113 Japan: Zhou J 001 (PE)
Halerpestes cymbalaria (Pursh) Green *AY954490 FJ626499 FJ626544 *AY954475 Guizhou: Wang Wei 003 (PE)
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–11084
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Helleborus foetidus L.
1
AJ414322
1
AJ413283
2
U52634
1
Sun et al. (2001),
2
Ro et al. (1997)
Helleborus thibetanus Franch. *AY954485 FJ626500 *AY954470 Shanxi: Wang Wei SX 032 (PE)
1
AJ413296
1
Sun et al. (2001)
Hepatica henryi (Oliv.) Steward FJ626584 FJ626501 FJ626545 FJ626445 Hubei: Chen 200010660 (PE)
Hydrastis canadensis L.
1
AF093725
2
AB069849
3
AF389268
1
Hoot et al. (1999),
2
Adachi et al., unpubl.;
3
Kim et al. (2004a)
*EF437112 USA: Chen Z-D 2002016 (PE)
Isopyrum manshuricum Kom. *EF437143 *EF437133 *EF437102 FJ626446 Liaoning: Wang Wei LN004 (PE)
Leptopyrum fumarioides (L.) Reichb. *EF437145 *EF437135 *EF437104 Xinjiang: Man Y-G T101 (PE)
Megaleranthis saniculiflia Ohwi
1
AY515243
2
AF131285
1
Lee and Heo, unpubl.;
2
Ro et al. (2003)
Myosurus minimus L.
1
DQ099441
2
AJ413305
1
Anderson et al. (2005),
2
Sun et al. (2001)
FJ626502 FJ626447 provided by Hoot: Hoot 98-2 (UWM)
Naravelia zeylanica (L.) DC. FJ626585 FJ626503 FJ626546 Yunnan: Wang Wei YN126 (PE)
Nigella damascena L. FJ626586 FJ626504 FJ626448 Yunnan: Wang Wei Seed1 (PE)
1
AY150260
1
Luo et al., unpubl.
Oxygraphis glacialis (Fisch.) Bunge. FJ626587 FJ626505 FJ626547 FJ626449 Shanxi: Wang Wei SX006 (PE)
Paraquilegia microphylla (Royle) Drumm. &
Hutch. *EF437146 *EF437136 *EF437105 FJ626450 Chongqing: Li C-Y 001 (PE)
Paropyrum anemonoides (Kar. & Kir.) Ulbr. *EF437142 *EF437132 *EF437101 Xinjiang: Wundish U. 177 (PE)
Pulsatilla cernua (Thunb.) Bercht. & Opiz. *AY954492 FJ626548 *AY954477 Jilin: Chen 2048 (PE)
1
AB110531 Miikeda et al. (2006)
Ranunculus cantoniensis DC. *AY954489 FJ626506 FJ626549 *AY954474 Guizhou: Wang Wei 031 (PE)
Semiaquilegia adoxoides (DC.) Makino *EF437147 *EF437137 *EF437106 FJ626451 Hunan: Shao Q 001 (PE)
Souliea vaginata (Maxim.) Franch. FJ626588 FJ626507 FJ626452 Shanxi: Wang Wei SX015 (PE)
1
AJ222983
1
Compton et al. (1998)
Thalictrum javanicum Bl. *AY954496 *DQ478615 *EF437107 *AY954480 Guizhou: Wang Wei 067 (PE)
Trautvetteria carolinensis (Walt.) Vail. FJ626589 FJ626508 FJ626550 provided by Hoot: Hoot 92-18 (UWM)
1
U52630
1
Ro et al. (1997)
Trollius laxus Salisb. *AY954486 FJ626509 *AY954471 USA: CL 108 (PE)
1
AH012578
1
Despres et al. (2003)
Urophysa henryi (Oliv.) Ulbr. *EF437149 *EF437139 *EF437109 FJ626453 Guizhou: Wang Wei 096 (PE)
Xanthorhiza simplicissima Marshall
1
L12669
2
AB069848
3
AF389270
1
Qiu et al. (1993),
2
Adachi et al., unpubl.;
3
Kim
et al. (2004a)
*EF437111 Provided by Qiu: Qiu Y-L 91030 (UCN)
Berberidaceae
Achlys triphylla DC.
1
L75868
2
AB069825
1
Kim and Jansen (1996),
2
Adachi et al.,
unpubl.
Berberis thunbergii DC.
1
AF139878
2
AB069827
1
Feng et al., unpubl.;
2
Adachi et al., unpubl.
FJ626551 FJ626454 Beijing (cult.): Hong Y-P H464 (PE)
Bongardia chrysogonum Spach.
1
L75870
2
AB069840
3
X83830
1
Kim and Jansen (1996),
2
Adachi et al.,
unpubl.;
3
Oxelman and Lide´ n (1995)
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Table 1. (continued )
Taxon GenBank accession numbers Vouchers/References
rbcL matK trnL-F 26S
Caulophyllum robustum Maxim.
1
AF190441
2
AB069832
3
AF325911
1
Xiang et al. (2000),
2
Adachi et al., unpubl.;
3
Liu et al. (2002)
FJ626455 Chongqing: Chen et al. 960601 (PE)
Caulophyllum thalictroides (L.) Michx.
1
AF190442
2
AB069831
3
AF389240
1
Xiang et al. (2000),
2
Adachi et al., unpubl.;
3
Kim et al. (2004a)
FJ626552 provided by Qiu: Qiu Y-L 05023 (PE)
Diphylleia cymosa Michx.
1
L75866
1
Kim and Jansen (1996)
*DQ478620 FJ626553 FJ626456 USA: Chen Z-D 2002019 (PE)
Diphylleia sinensis H. L. Li *DQ478618 *DQ478618 FJ626554 FJ626457 Shanxi: Wang Wei SX034 (PE)
Dysosma versipellis (Hance) M. Cheng ex
Ying *EF173669 *DQ478619 FJ626458 Sichuan: Chen et al. 960583 (PE)
1
AF325908
1
Liu et al. (2002)
Epimedium koreanum Nakai
1
L75869
2
AB069837
1
Kim and Jansen (1996),
2
Adachi et al.,
unpubl.;
FJ626555 FJ626459 Jilin: Zhou Y (PE)
Gymnospermium microrrhynchum Takht. *EF173671 FJ626556 FJ626460 Jilin: Chen Z-D 2047 (PE)
1
AB069833
1
Adachi et al., unpubl.
Jeffersonia diphylla (L.) Pers.
1
L75867
2
AB069836
3
U52604
1
Kim and Jansen (1996),
2
Adachi et al.,
unpubl.;
3
Ro et al. (1997)
FJ626557 USA: Chen 2002010 (PE)
Mahonia bealei (Fort.) Carr.
1
L12657
1
Qiu et al. (1993)
*DQ478617 FJ626558 FJ626461 Sichuan: Chen et al. 960046 (PE)
Nandina domestica Thunb.
1
L75843
2
AB069830
3
AF325912
4
AF389241
1
Kim and Jansen (1996),
2
Adachi et al.,
unpubl.;
3
Liu et al. (2002),
4
Kim et al. (2004a)
Plagiorhegma dubia Maxim. *EF173673 FJ626559 FJ626462 Jilin: Feng M. (PE)
1
AB069836
1
Adachi et al., unpubl.
Podophyllum peltatum L.
1
AF093716
2
AB069843
3
AF325904
4
DQ008614
1
Hoot et al. (1999),
2
Adachi et al., unpubl.;
3
Liu et al. (2004),
4
Qiu et al. (2005)
Ranzania japonica Ito
1
L75853
2
AB069829
1
Kim and Jansen (1996),
2
Adachi et al.,
unpubl.;
Sinopodophyllum hexandrum (Royle) Ying
1
AF079455
2
AF325905
1
Feng and Wang, unpubl.;
2
Liu et al. (2002)
*DQ478616 FJ626463 Qinghai (PE)
Vancouveria hexandra C. Morren & Decne. *EF173674 FJ626560 Germany (cult.): Zhang M-L
1
AB069839
2
U52602
1
Adachi et al., unpubl.;
2
Ro et al. (1997)
Menispermaceae
Albertisia laurifolia Yamamoto FJ626590 *EF143849 EF143880 Hainan: Hong Y-P 99371 (PE)
Anamirta cocculus (L.) Wight & Arn. FJ626591 *EF143856 EF143887 Thailand: Wang H-C 103 (HIB)
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–11086
ARTICLE IN PRESS
Arcangelisia gusanlung Lo FJ626592 *EF143852 EF143883 FJ626464 Hainan: Hong Y-P 99406 (PE)
Aspidocarya uvifera Hook. f. & Thoms. FJ626593 *EF143853 EF143884 FJ626465 Yunnan: Hong Y-P 99190 (PE)
Cissampelos paraira Linn.
1
AF197590*
2
DQ008616
1
Qiu et al. (1999),
2
Qiu et al. (2005)
*EF143858 EF143889 Yunnan: Wang H-C BN-001 (HIB)
Cocculus trilobus (Thunb.) DC.
1
L12642*
1
Qiu et al. (1993)
*DQ478611 EF143892 FJ626466 Guizhou: Hong Y-P H310 (PE)
Cyclea hypoglauca (Schauer) Diels FJ626594 *EF143862 EF143894 FJ626467 Guangdong: Chen et al. 9812108 (PE)
Diploclisia glaucescens (Bl.) Diels FJ626595 *EF143867 EF143899 FJ626468 South China Bot Gard (cult.): Hong Y-P 99403
Hypserpa nitida Miers FJ626596 *EF143868 EF143900 Hainan: Hong Y-P 99378 (PE)
Menispermum dauricum DC.
1
AF190436
2
AF335293
1
Xiang et al. (2000),
2
Wang et al. (2002)
*DQ478613 FJ626469 Beijing (cult.): Hong Y-P 99095 (PE)
Parabaena sagittata Miers FJ626597 *EF143854 EF143885 FJ626470 Guizhou: Hong Y-P H346 (PE)
Pericampylus glaucus (Lam.) Merr. FJ626598 *EF143869 EF143901 FJ626471 Guangdong: Chen et al. 9812095 (PE)
Pycnarrhena lucida (Teijsm. & Binn.) Miq. FJ626599 *EF143851 EF143882 Hainan: Hong Y-P HN167 (PE)
Sinomenium acutum (Thunb.) Redd. & Wils. FJ626600 *EF143870 EF143902 FJ626472 Henan: Hong Y-P H006 (PE)
Stephania longa Lour. FJ626601 *EF143875 EF143907 FJ626473 Guangxi: Hong Y-P H101 (PE)
Tinomiscium petiolare Hook. f . & Thoms. *EF173675 *DQ478612 EF143888 FJ626474 Yunnan: Hong Y-P H142 (PE)
Tinospora sinensis (Lour.) Merr. FJ626602 *EF143855 EF143886 FJ626475 Thailand: Wang H-C 109 (HIB)
Lardizabalaceae
Akebia quinata (Houtt.) Decne.
1
L12627
2
AF542587
3
AM397152
4
AF389253
1
Qiu et al. (1993),
2
Hilu et al. (2003),
3
Worberg
et al. (2007),
4
Kim et al. (2004a)
Archakebia apetala (Xia et al.) Wu, Chen &
Qin
1
AF335306
1
AF335288
1
Wang et al. (2002)
FJ626510 Gansu: Hong Y-P 99109 (PE)
Boquila trifoliata (DC.) Decaisne FJ626603 FJ626511 Chile: Qin H-N 643 (PE)
1
AF335291
1
Wang et al. (2002)
Decaisnea insignis (Griff.) Hook. f. et
Thoms. FJ626604 FJ626512 Hubei: Chen Z-D 961051 (PE)
1
AF335303
2
AF389254
1
Wang et al. (2002),
2
Kim et al. (2004a)
Holboellia grandiflora Reaub.
1
AF398181
1
AF335290
1
Wang et al. (2002)
FJ626513 FJ626476 Guizhou: Wang Wei 011 (PE)
Lardizabala biternata (Molina) Looser
1
L37919
2
AY437809
3
AF335282
2
DQ008618
1
Hoot et al. (1995),
2
Qiu et al. (2005),
3
Wang et
al. (2002)
Parvatia brunoniana spp. elliptica Griffith
1
AF335307
1
AF335283
1
Wang et al. (2002)
FJ626514 Guangxi: Wang Wei GH0425-3 (PE)
Sargentodoxa cuneata (Oliv.) Rehd. & Wils. FJ626605 FJ626515 Yunnan: Hong Y-P 99238 (PE)
1
AF335301
2
DQ008620
1
Wang et al. (2002),
2
Qiu et al. (2005)
Sinofranchetia chinensis (Franch.) Hemsl. *EF173676 FJ626516 Shanxi: Wang Wei SX040 (PE)
1
AF335284
2
AF389255
1
Wang et al. (2002),
2
Kim et al. (2004a)
Stauntonia hexaphylla (Thunb.) Decne. FJ626606 FJ626517 Guangdong: Chen Z-D H29905 (PE)
1
AF335286
1
Wang et al. (2002)
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–110 87
ARTICLE IN PRESS
Table 1. (continued )
Taxon GenBank accession numbers Vouchers/References
rbcL matK trnL-F 26S
Circaeasteraceae
Circaeaster agrestis Maxim. FJ626607 FJ626518 Shanxi: Ren Y 001 (PE)
1
AF335298
2
AF389246
1
Wang et al. (2002),Kim et al. (2004a)
Kingdonia uniflora Balf. f. & W. W. Sm. FJ626608 FJ626519 Shanxi: Ren Y 002 (PE)
1
DQ185598
2
AF389245
1
Zha and Sun, unpubl.;
2
Kim et al. (2004a)
Papaveraceae
Corydalis sp. FJ626609 FJ626520 FJ626561 FJ626477 Gansu: Hong Y-P 99105 (PE)
Dicentra eximia (Ker) Torrey
1
L37917
2
DQ182345
3
AY145361
4
AF389262
1
Hoot et al. (1995),
2
Mu¨ ller et al. (2006),
3
Borsch et al. (2003),
4
Kim et al. (2004a)
Eomecon chionantha Hance FJ626610 FJ626521 FJ626562 FJ626478 Guizhou: Wang Wei 059 (PE)
Hylomecon japonica (Thunb.) Prantl. &
Kundig. FJ626611 FJ626522 FJ626563 FJ626479 Jilin: Chen 2046 (PE)
Hypecoum imberbe Sm.
1
U86628
2
AF389263 1Hoot et al. (1997),
2
Kim et al. (2004a)
Macleaya microcarpa (Maxim.) Fedde FJ626612 FJ626523 FJ626564 Beijing (cult.): Hong Y-P H542 (PE)
Meconopsis quintuplinervia Regel. FJ626613 FJ626524 FJ626565 FJ626480 Shanxi: Wang Wei SX009 (PE)
Papaver rhoeas L. FJ626614 FJ626525 FJ626566 FJ626481 Kunming (cult.): Wang Wei YN100 (PE)
Pteridophyllum racemosum Sieb. & Zucc.
1
U86631
2
AF389264
1
Hoot et al. (1997),
2
Kim et al. (2004a)
FJ626526 FJ626567 provided by Hoot: Chase 531 (K)
Sanguinaria canadensis L.
1
L01951*
2
DQ401350
3
DQ008621
1
Albert et al. (1992),
2
Qiu et al. (2006),
3
Qiu
et al. (2005)
Eupteleaceae
Euptelea pleiospermum Hook. f. & Thoms.
1
AY048174
2
AM39715
1
Wang et al. (2002),
2
Worberg et al. (2007)
FJ626527 FJ626482 Yunnan: Feng M. 967011 (PE)
Euptelea polyandra Sieb. & Zucc.
1
L12645*
2
AF389249 Qiu et al. (1993),
2
Kim et al. (2004a)
FJ626528 FJ626568 Japan: Ren B-Q (PE)
Other basal eudicots
Buxus sempervirens L.
1
AF093717
2
AF543728
3
AY145357
4
AF389243
1
Hoot et al. (1999),
2
Mu¨ ller et al. (2006),
3
Borsch et al. (2003),
4
Kim et al. (2004a)
Grevillea robusta Cunn. & R. Br.
1
AF197589
2
DQ008612
1
Qiu et al. (1999),
2
Qiu et al. (2005)
FJ626529 FJ626569 Hainan: Hong Y-P 99354 (PE)
Meliosma veitchiorum Hemsl. View.
1
AF206793
2
AF389271
1
Soltis et al. (1999),
2
Kim et al. (2004a)
FJ626530 FJ626570 Henan: Hong Y-P 99061 (PE)
Nelumbo lutea (Willd.) Pers.
1
DQ182337
1
AF543740
1
AY145359
2
AF389259
1
Mu¨ ller et al. (2006),
2
Kim et al. (2004a)
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–11088
ARTICLE IN PRESS
Nelumbo nucifera Gaertn. FJ626615 FJ626531 FJ626571 FJ626483 Beijing (cult.): Wang W 08010 (PE)
Platanus occidentalis L.
1
L01943
2
AF543747
3
AY145358
4
AF274662
1
Albert et al. (1992),
2
Mu¨ ller et al. (2006),
3
Borsch et al. (2003),
4
Fishbein et al. (2001)
Sabia swinhoei Hemsl. FJ626616 FJ626532 FJ626572 no voucher
1
AF389272
1
Kim et al. (2004a)
Tetracentron sinense Oliv.
1
L12668
2
AM396504
2
AM397165
3
AF274670
1
Qiu et al. (1993),
2
Worberg et al. (2007),
3
Fishbein et al. (2001)
Trochodendron aralioides Sieb. & Zucc.
1
L01958
2
AF543751
3
AY145360
4
AF274671
1
Albert et al. (1992),
2
Mu¨ ller et al. (2006),
3
Borsch et al. (2003),
4
Fishbein et al. (2001)
Core eudicots
Altingia excelsa Noron.
1
DQ352374
2
AF013037
1
DQ352226
3
AF479208
1
Ickert-Bond and Wen (2006),
2
Li et al. (1997),
3
Soltis et al. (2003)
Paeonia suffruticosa Andr.
1
AJ402982
2
AF033593
3
AF274659
1
Savolainen et al. (2000a, b),
2
Sang et al.
(1997),
3
Fishbein et al. (2001)
FJ626573 Beijing (cult.): Wang W 08012 (PE)
Hamamelis virginiana L.
1
DQ352368
2
AF013046
1
DQ352196
3
AF036495
1
Ickert-Bond and Wen (2006),
2
Li et al. (1997),
3
Kuzoff et al. (1998)
Coriaria myrtifolia L.
1
L01897
2
AB016459
3
AY091824
4
AY968406
1
Albert et al. (1992),
2
Yokoyama et al. (2000),
3
Yang, unpubl.;
4
Zhang et al. (2006)
Cornus mas L.
1
L11216
2
AJ429275
2
AJ430866
3
AF297535
1
Xiang et al. (1993),
2
Bremer et al. (2002),
3
Fan
and Xiang (2001)
Aextoxicon punctatum Ruiz & Pav.
1
X83986
2
AF543720
3
AY145362
4
AF389239
1
Savolainen et al. (1997),
2
Mu¨ ller et al. (2006),
3
Borsch et al. (2003),
4
Kim et al. (2004a)
Vitis aestivalis Michx.
1
L01960
2
AF274635
3
AB235073
4
AF479207
1
Albert et al. (1992),
2
Fishbein et al. (2001),
3
Soejima and Wen (2006),
4
Soltis et al. (2003)
Tetracera asiatica (Lour.) Hoogl.
1
AJ235796
2
AY042665
3
AF479097
1
Savolainen et al. (2000a, b),
2
Cuenoud et al.
(2002),
3
Soltis et al. (2003)
FJ626574 Guangdong: Xu K-X 012 (PE)
Basal angiosperms
Acorus gramineus L.
1
D28866
2
DQ182341
3
AY145336
4
AF036490
1
Kawano, unpubl.;
2
Mu¨ ller et al. (2006),
3
Borsch et al. (2003),
4
Kuzoff et al. (2003)
Amborella trichopoda Baill.
1
L12628
2
AF543721
3
AY145324
4
AF479238
1
Qiu et al. (1993),
2
Mu¨ ller et al. (2006),
3
Borsch
et al. (2003),
4
Soltis et al. (2003)
Annona muricata L.
1
L12629
2
AF543722
3
AY145352
4
DQ008634
1
Qiu et al. (1993),
2
Mu¨ ller et al. (2006),
3
Borsch
et al. (2003),
4
Qiu et al. (2005)
Austrobaileya scandens C. T. White
1
L12632
2
DQ182344
3
AY145326
4
AY095452
1
Qiu et al. (1993),
2
Mu¨ ller et al. (2006),
3
Borsch
et al. (2003),
4
Zanis et al. (2003)
Brasenia schreberi J. Gmelin
1
M77031
2
AF092973
3
AY145329
4
DQ008661
1
Les et al. (1991),
2
Les et al. (1999),
3
Borsch
et al. (2003),
4
Qiu et al. (2005)
Calycanthus floridus L. (occidentalis Hook. &
Arn. for 26S)
1
L14291
2
AF543730
3
AY145349
4
AY095454
1
Olmstead et al. (1993),
2
Mu¨ ller et al. (2006),
3
Borsch et al. (2003),
4
Zanis et al. (2003)
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–110 89
ARTICLE IN PRESS
Table 1. (continued )
Taxon GenBank accession numbers Vouchers/References
rbcL matK trnL-F 26S
Canella winterana (L.) Gaertn.
1
AJ131928
2
AF543731
3
AY004152
4
AY095455
1
Qiu et al. (1993),
2
Mu¨ ller et al. (2006),
3
Karol
et al. (2000),
4
Zanis et al. (2003)
Ceratophyllum demersum L.
1
D89473
2
AF543732
3
AY145335
4
AY095456
1
Ueda et al. (1997),
2
Mu¨ ller et al. (2006),
3
Borsch et al. (2003),
4
Zanis et al. (2003)
Hedyosmum arborescens Sw.
1
L12649
2
DQ401339
3
AY236750
4
AF479226
1
Qiu et al. (1993),
2
Mu¨ ller et al. (2006),
3
Zhang
and Renner (2003),
4
Soltis et al. (2003)
Illicium floridanum Ellis
1
DQ182334
1
AF543738
2
AY145325
3
DQ008659
1
Mu¨ ller et al. (2006),
2
Borsch et al. (2003),
3
Qiu
et al. (20050
Magnolia denudata Desr.
1
AY008913
2
AF123465
3
AY009052
4
AF479244
1
Kim et al., unpubl.;
2
Jin et al. (1999),
3
Su
et al., unpubl.;
4
Soltis et al. (2003)
Orontium aquaticum L.
1
AJ005632
2
AF543744
3
AY145338
4
DQ008652
1
Cho and Palmer (1999),
2
Mu¨ ller et al. (2006),
3
Borsch et al. (2003),
4
Qiu et al. (2005)
Saruma henryi Oliv.
1
L12664
2
AF543748
3
AY145340
4
DQ008644
1
Qiu et al. (1993),
2
Mu¨ ller et al. (2006),
3
Borsch
et al. (2003),
4
Qiu et al. (2005)
Saururus cernuus L.
1
L14294
2
AF543749
3
AF332970
4
AY095468
1
Olmstead et al. (1993),
2
Mu¨ ller et al. (2006),
3
Meng et al., unpubl.;
4
Zanis et al. (2003)
Takhtajania perrieri M. Baranova & J. Leroy
1
AF206824
2
DQ401371
3
AY004146
4
DQ008645
1
Soltis et al. (1999),
2
Qiu et al. (2006),
3
Karol
et al. (2000),
4
Qiu et al. (2005)
The accession numbers published in our previous studies (Wang et al., 2005, 2007a, b;Wang and Chen, 2007) are marked with an asterisk.
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–11090
products were purified using a GFX
TM
PCR DNA and
Gel Band Purification Kit (Amersham Pharmacia
Biotech, Piscataway, NJ, USA) then directly sequenced.
Sequencing reactions were conducted using the
DYEnamic
TM
ET Dye Terminator Cycle Sequencing
Kit (Amersham Pharmacia Biotech) and analyzed using
MegaBACE
TM
1000 DNA Analysis Systems, following
the manufacturer’s protocols.
Alignment and phylogenetic analysis
Three of the four DNA regions (all except trnL-F)
were aligned using the default alignment parameters in
CLUSTAL X (Thompson et al., 1997) with manual
adjustment. Because of the great length variation in
trnL-F regions, these were manually aligned in BioEdit
(Hall, 1999) according to the rules outlined by Borsch
et al. (2003). Following Borsch et al. (2003) and
Worberg et al. (2007), we identified eight mutational
hotspots in the trnL-F matrix, encompassing 1168
positions, which were excluded from the analyses. Two
difficult-to-align regions in 26S rDNA, representing 45
sites, were also excluded from the analyses. Gaps were
coded as missing data. All aligned matrices are available
upon request from the corresponding author.
We used maximum parsimony (MP) and Bayesian
inference (BI) to analyze each of the individual
molecular data sets as well as the morphological data
set. Because simultaneous analysis of combined data has
been proposed as the best approach to phylogenetic
inference (Kluge, 1989;Chippindale and Wiens, 1994;
Nixon and Carpenter, 1996;Cognato and Vogler, 2001),
we visually inspected the individual bootstrap consensus
trees on a node to node basis to test the congruence
among the individual DNA data sets by identifying
contradictory nodes with 470% BS support as de-
scribed in Mason-Gamer and Kellogg (1996). Since no
strongly supported (470% BS) nodes were found, data
sets were combined as follows: first all molecular data
were combined to obtain a combined molecular data set,
then the combined molecular data set was combined
with the morphological data set yielding a total evidence
data set. The combined molecular data set included 137
taxa, in which the amount of missing data was:
rbcL ¼0.014% (2/137), matK ¼0.007% (1/137),
trnL-F ¼0.036% (5/137), and 26S ¼0.117% (16/137).
The total evidence data set included 129 taxa, in which
the amount of missing data was: rbcL ¼0.008%
(1/129), matK ¼0.008% (1/129), trnL-F ¼0.039%
(5/129), and 26S ¼0.124% (16/129).
Parsimony analyses were performed using PAUP*
version 4.0b10 (Swofford, 2003). Heuristic searches were
conducted with 1000 replicates of random addition,
one tree held at each step during stepwise addition,
tree-bisection-reconnection (TBR) branch swapping,
MulTrees in effect, and steepest descent off. Boot-
strapping was conducted with 1000 replicates, using a
heuristic search strategy (five random addition repli-
cates, saving five trees per replicate). The BI analyses
were carried out using MrBayes version 3.1.2 (Ronquist
and Huelsenbeck, 2003). Each of the four regions (rbcL,
matK, trnL-F and 26S rDNA) was assigned its own
model of nucleotide substitution, as determined by the
Akaike information criterion (AIC) in Modeltest
version 3.06 (Posada and Crandall, 1998). The morpho-
logical data were run under the datatype ¼standard
option, and only variable sites had the possibility of
being sampled (coding ¼variable). For both the com-
bined molecular data set and the total evidence data set,
the prior assumption of rate heterogeneity across the
data partitions was set at variable (ratepr ¼variable).
For more information on the settings used, contact the
first author or see the MrBayes manual on the Internet
at http://mrbayes.csit.fsu.edu/manual.php. Searches
were based on 1,000,000 generations with four chains
of the Markov Chain Monte Carlo (MCMC). Runs
were started from a random tree and allowed to proceed
in parallel while sampling and recording the topology
every 100 generations of the MCMC chain. Performance
of individual runs was assessed and phylogenies
compared between runs. Majority rule (450%) con-
sensus trees were constructed after removing the
‘‘burn-in period’’ samples (the first 15% of sampled
trees).
In order to identify the diagnostic morphological
characters at different taxonomic levels, we optimized
selected characters onto the strict consensus tree from
the MP analysis based on the total evidence data set
using MacClade 4.06 (Maddison and Maddison, 2003).
Results
Molecular data
The aligned trnL-F matrix included 2777 positions,
the aligned 26S rDNA sequences were 1345 nucleotides
in length, and the aligned rbcL and matK data sets had
1395 and 1440 positions, respectively. Table 2 sum-
marizes the number of variable and parsimony-
informative sites and tree statistics for the molecular
data sets.
The topologies based on individual DNA data sets
were nearly identical except in some of the terminal
branches (trees not shown, but available upon request
from the corresponding author). The aligned matrix of
the combined DNA data comprised 6957 characters, of
which 2984 were variable and 2207 were potentially
parsimony-informative. The MP analysis generated
eight most parsimonious trees (MPTs) of 15,799 steps,
with a CI of 0.33, RI of 0.66 and an RC of 0.22.
ARTICLE IN PRESS
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–110 91
Bayesian analysis yielded trees with topologies that
are consistent with those retrieved with MP analysis
except that Hypecoum is sister to the clade containing
Pteridophyllum,Dicentra and Corydalis (PP 0.61),
Helleborus is sister to the clade E (PP 0.92), Asteropyrum
is sister to the clade D (PP 0.74), Nigella is sister to the
clades B and C (PPo0.50), and clades B-E, Caltha,
Asteropyrum,Helleborus, and Nigella in Ranunculaceae
form a poorly supported clade F (Fig. 1).
Morphological data
The morphological data set generated 15 MPTs of 561
steps, with a CI of 0.22, RI of 0.76 and an RC of 0.16.
Cladograms from BI and MP analyses were poorly
resolved (Fig. 2). Both Menispermaceae and Papaver-
aceae are resolved and each is supported as a mono-
phyletic group. Within Papaveraceae, Pteridophyllum,
Dicentra,Hypecoum, and Corydalis formed a clade
(BS 81%, PP 1.00). Circaeaster and Kingdonia formed a
clade (BS 80%, PP 0.93).
Total evidence data
The total evidence data set generated eight MPTs of
16,639 steps, with a CI of 0.35, RI of 0.65 and an RC of
0.23. Patterns of relationship are almost identical with
those obtained from the combined molecular data
except that support for Eupteleaceae as sister to other
Ranunculales increased notably (BS 87%), Pteridophyllum
and Hypecoum form a strongly supported (BS 96%)
clade, and in Ranunculaceae, clade F is recovered, which
unites clades B-E, Caltha,Asteropyrum,Helleborus, and
Nigella, but is with poor support (Fig. 3).
The trees generated by BI were highly congruent with
those of MP analysis except for the presence of several
nodes with weak support (PP o0.50) in Ranunculaceae:
Helleborus is sister to the clade containing Asteropyrum,
Caltha and Clade D, and Nigella is sister to the clades
B-C (Fig. 3). All further discussion will be based on the
total evidence trees (Fig. 3).
Ranunculales and its seven families are all strongly
supported as monophyletic. Three major clades were
identified within Ranunculales: Eupteleaceae, Papaver-
aceae and core Ranunculales. Papaveraceae and core
Ranunculales form a clade (BS 87%, PP 0.98), sister to
Eupteleaceae. Within core Ranunculales, Lardizabala-
ceae and Circaeasteraceae form a subclade with strong
support (BS 87%, PP 1.00), which is sister to a subclade
(BS 76%, PP 1.00) comprised of the other three families.
Papaveraceae is strongly supported (BS 100%, PP
1.00) as monophyletic and is divided into two subclades
(each with 100% BS and 1.00 PP support), the first
containing Pteridophyllum,Hypecoum,Dicentra and
Corydalis; the second containing Eomecon, Hylomecon,
Macleaya, Meconopsis and Papaver.
Within Lardizabalaceae, Sargentodoxa is sister to the
remaining taxa of the family (BS 100%, PP 1.00),
followed by Decaisnea (BS 100%, PP 1.00), then
Sinofranchetia (BS 96%, PP 1.00).
Within Menispermaceae, two major clades are recog-
nized. The first clade consists of tribe Pachygoneae
Miers (represented by Albertisia and Pycnarrhena) and
tribe Menispermeae DC., with the former embedded
within the latter. The second clade is composed of tribes
Tinosporeae Hook. f. & Thoms. and Fibraureae Diels,
which are not supported as monophyletic. Anamirta of
tribe Fibraureae and Arcangelisia of tribe Tinosporeae
form a strongly supported (BS 100%, PP 1.00) subclade
that is sister to a subclade (BS 95%, PP 1.00) comprised
of Tinomiscium of tribe Fibraureae and Tinospora of
tribe Tinosporeae, which in turn is sister to the
AspidocaryaParabaena subclade (BS 100%, PP 1.00).
Within Berberidaceae, Nandinoideae Heintz is em-
bedded within Berberidoideae Kostel and forms a
clade with Gymnospermium and Caulophyllum (BS
99%, PP 1.00).
Ranunculaceae contains three major clades: Glaucidium
(BS 100%, PP 1.00), Hydrastis (BS 76%, PP 1.00)
and Ranunculaceae s. str. (sensu Takhtajan, 1997,
excluding Kingdonia) (BS 100%, PP 1.00), with
Glaucidium sister to the other two clades. Except for
two monogeneric subfamilies (Thalictroideae and
Hydrastidoideae), Isopyroideae, Helleboroideae and
ARTICLE IN PRESS
Table 2. Statistics from analyses of the molecular data sets. Tree length, consistency index (CI), retention index (RI), and rescaled
consistency index (RC) were calculated based on parsimony-informative characters only.
Data set No.
taxa
Total
length
No. variable
character
No. informative
character
No. of
trees
Length of
trees
CI RI RC Model
rbcL 135 1395 633 458 105 3099 0.31 0.66 0.20 GTR+I+G
matK 136 1440 1020 843 105 6111 0.32 0.68 0.22 TVM+I+G
trnL-F 131 2777 718 557 11185 3426 0.40 0.72 0.29 TVM+I+G
26S rDNA 121 1345 613 349 15 2993 0.30 0.58 0.18 GTR+I+G
Combined
molecular data
137 6957 2984 2207 8 15799 0.33 0.66 0.22
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–11092
ARTICLE IN PRESS
Ranunculus
Batrachium
1.00
Halerpestes
1.00
Myosurus
1.00/99
Trautvetteria
Oxygraphis
1.00/100
1.00/100
Naravelia
Clematis
1.00
Anemoclema
1.00/86
Pulsatilla
Anemone
1.00
1.00/83
Hepatica
1.00/100
1.00/100
Callianthemum
0.76/53
Trollius
Megaleranthis
1.00
Adonis
1.00/100
Asteropyrum
Caltha palu
Caltha memb
1.00/100
Cimicifuga
Actaea
Souliea
1.00/100
Eranthis
1.00/100
Beesia
Anemonopsis
1.00
1.00/100
Helleborus thib
Helleborus foet
1.00/100
Delphinium
Consolida
1.00
Aconitum
1.00
Nigella
Aquilegia oxys
Aquilegia ecal
1.00/100
Semiaquilegia
Urophysa
1.00/100
Isopyrum
Enemion
1.00/100
Dichocarpum
Paropyrum
Paraquilegia
Leptopyrum
1.00/99
Thalictrum
1.00/95
1.00/100
0.93/96
Xanthorhiza
Coptis
1.00/100
1.00/100
Hydrastis
1.00/82
Glaucidium
1.00/100
Sinopodophyllum
Podophyllum
1.00/80
Diphylleia sine
Diphylleia cymo
1.00/90
Dysosma
1.00/100
Achlys
0.83/62
Bongardia
1.00/90
Vancouveria
Epimedium
1.00/100
1.00/95
Plagiorhegma
Jeffersonia
1.00/100
1.00/55
Caulophyllum thal
Caulophyllum robu
1.00/100
Gymnospermium
1.00
Nandina
1.00
Berberis
Mahonia
1.00/100
Ranzania
1.00/100
0.77/
1.00/100
1.00/100
1.00/100
1.00/81
Holboellia
Parvatia
Stauntonia
1.00/97
Akebia
Archakebia
1.00/100
Lardizabala
Boquila
1.00/99
1.00/100
Sinofranchetia
1.00/99
Decaisnea
1.00/100
Sargentodoxa
1.00/100
Kingdonia Circaeaster
1.00/100
1.00/100
Sanguinaria
Eomecon
1.00/100
Macleaya Hylomecon
1.00
0.94/85
Papaver
Meconopsis
1.00/100
1.00/100
Dicentra Corydalis
1.00/100
Pteridophyllum
1.00
1.00/100
0.63/68
Euptelea poly
Euptelea plei
1.00/100
1.00/100
Thalictroideae
Isopyroideae
Isopyroideae
Helleboroideae
Isopyroideae
Ranunculoideae
Berberidaceae Menispermaceae Lardizabalaceae
Helleboroideae
Ranunculoideae
Outgroups
CIR Papaveraceae EUP
Ranunculaceae
Nandinoideae
Berberidoideae
Berberidoideae
Hydrastidoideae
Hypecoum
0.61/-
A
B
C
E
D
F
1.00
1.00/95
1.00/100
1.00/100
1.00/100
1.00/89
1.00/100
0.89/63
1.00/100
1.00/97
1.00/100
1.00/100
1.00/99
Pycnarrhena
Albertisia
Cocculus Cyclea
Cissampelos
Stephania
Hypserpa
Pericampylus
Menispermum
Diploclisia
Tinospora
Tinomiscium
Aspidocarya
1.00
0.69/-
Sinomenium
Anamirta
Arcangelisia
Parabaena
0.84/-
0.92/57
0.71/-
1.00/96
0.61/
0.74/-
1.00/<50
Helleboroideae
0.05
Altingia
Hamamelis Paeonia
Vitis
Aextoxicon Cornus Coriaria Tetracera
Tetracentron
Trochodendron
Buxus
Meliosma
Sabia Grevillea
Platanus
Nelumbo lute
Nelumbo nuci Annona
Magnolia Canella
TakhtajaniaSaruma Saururus
Calycanthus Acorus Orontium
Ceratophyllum
Hedyosmum
Austrobaileya
Illicium
Amborella Brasenia
1.00/100
1.00/100
1.00/100
0.97/
1.00/100
1.00/99
1.00/99
1.00/99
0.88/-
1.00/-
0.85/
1.00/100
1.00/100
1.00/100
1.00/87
1.00
1.00/100
0.53/- 1.00/100
0.76/-
1.00/91 0.69
0.68/-
0.87/-1.00
0.99/-
1.00/76
0.92/-
0.90/-
93
100
100
100
100
100
100
100
/-
/-
100
100
100
100
1.00
91
1.00
90
98
100
54
60
1.00
89
100
Ranunculus
Batrachium
1.00
Halerpestes
1.00
Myosurus
1.00/99
Trautvetteria
Oxygraphis
1.00/100
1.00/100
Naravelia
Clematis
1.00
Anemoclema
1.00/86
Pulsatilla
Anemone
1.00
1.00/83
Hepatica
1.00/100
1.00/100
Callianthemum
0.76/53
Trollius
Megaleranthis
1.00
Adonis
1.00/100
Asteropyrum
Caltha palu
Caltha memb
1.00/100
Cimicifuga
Actaea
Souliea
1.00/100
Eranthis
1.00/100
Beesia
Anemonopsis
1.00
1.00/100
Helleborus thib
Helleborus foet
1.00/100
Delphinium
Consolida
1.00
Aconitum
1.00
Nigella
Aquilegia oxys
Aquilegia ecal
1.00/100
Semiaquilegia
Urophysa
1.00/100
Isopyrum
Enemion
1.00/100
Dichocarpum
Paropyrum
Paraquilegia
Leptopyrum
1.00/99
Thalictrum
1.00/95
1.00/100
0.93/96
Xanthorhiza
Coptis
1.00/100
1.00/100
Hydrastis
1.00/82
Glaucidium
1.00/100
Sinopodophyllum
Podophyllum
1.00/80
Diphylleia sine
Diphylleia cymo
1.00/90
Dysosma
1.00/100
Achlys
0.83/62
Bongardia
1.00/90
Vancouveria
Epimedium
1.00/100
1.00/95
Plagiorhegma
Jeffersonia
1.00/100
1.00/55
Caulophyllum thal
Caulophyllum robu
1.00/100
Gymnospermium
1.00
Nandina
1.00
Berberis
Mahonia
1.00/100
Ranzania
1.00/100
0.77/
1.00/100
1.00/100
1.00/100
1.00/81
Holboellia
Parvatia
Stauntonia
1.00/97
Akebia
Archakebia
1.00/100
Lardizabala
Boquila
1.00/99
1.00/100
Sinofranchetia
1.00/99
Decaisnea
1.00/100
Sargentodoxa
1.00/100
Kingdonia Circaeaster
1.00/100
1.00/100
Sanguinaria
Eomecon
1.00/100
Macleaya Hylomecon
1.00
0.94/85
Papaver
Meconopsis
1.00/100
1.00/100
Dicentra Corydalis
1.00/100
Pteridophyllum
1.00
1.00/100
0.63/68
Euptelea poly
Euptelea plei
1.00/100
1.00/100
Thalictroideae
Isopyroideae
Isopyroideae
Helleboroideae
Isopyroideae
Ranunculoideae
Berberidaceae Menispermaceae Lardizabalaceae
Helleboroideae
Ranunculoideae
Outgroups
CIR Papaveraceae EUP
Ranunculaceae
Nandinoideae
Berberidoideae
Berberidoideae
Hydrastidoideae
Hypecoum
0.61/-
A
B
C
E
D
F
1.00
1.00/95
1.00/100
1.00/100
1.00/100
1.00/89
1.00/100
0.89/63
1.00/100
1.00/97
1.00/100
1.00/100
1.00/99
Pycnarrhena
Albertisia
Cocculus Cyclea
Cissampelos
Stephania
Hypserpa
Pericampylus
Menispermum
Diploclisia
Tinospora
Tinomiscium
Aspidocarya
1.00
0.69/-
Sinomenium
Anamirta
Arcangelisia
Parabaena
0.84/-
0.92/57
0.71/-
1.00/96
0.61/
0.74/-
1.00/<50
Helleboroideae
0.05
Altingia
Hamamelis Paeonia
Vitis
Aextoxicon Cornus Coriaria Tetracera
Tetracentron
Trochodendron
Buxus
Meliosma
Sabia Grevillea
Platanus
Nelumbo lute
Nelumbo nuci Annona
Magnolia Canella
TakhtajaniaSaruma Saururus
Calycanthus Acorus Orontium
Ceratophyllum
Hedyosmum
Austrobaileya
Illicium
Amborella Brasenia
1.00/100
1.00/100
1.00/100
0.97/
1.00/100
1.00/99
1.00/99
1.00/99
0.88/-
1.00/-
0.85/
1.00/100
1.00/100
1.00/100
1.00/87
1.00
1.00/100
0.53/- 1.00/100
0.76/-
1.00/91 0.69
0.68/-
0.87/-1.00
0.99/-
1.00/76
0.92/-
0.90/-
93
100
100
100
100
100
100
100
/-
/-
100
100
100
100
1.00
91
1.00
90
98
100
54
60
1.00
89
100
Fig. 1. Phylogram obtained from Bayesian inference based on the combined molecular data set. Numbers above the branches are
Bayesian posterior probabilities (40.50) and bootstrap percentages (450%), respectively. ‘‘–’’ indicates the nodes not found in MP
trees. Familial classification is based on APG II (2003), subfamilial classifications based on Tamura (1993) for Ranunculaceae and
Loconte (1993) for Berberidaceae. CIR ¼Circaeasteraceae, EUP ¼Eupteleaceae.
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–110 93
Ranunculoideae of Tamura (1993) are not monophy-
letic. Within Ranunculaceae s. str., the Coptis-Xanthor-
hiza clade (BS 100%, PP 1.00) is sister to the clade
(BS 96%, PP 1.00) containing the remaining genera.
Within the latter clade, five monophyletic groups are
identified: clade A (BS 100%, PP 1.00) contains
Thalictroideae and nine genera of Isopyroideae; clade
B (BS 100%, PP 1.00) contains Adonis of Ranunculoi-
deae and Trollius and Megaleranthis of Helleboroideae;
clade C (BS 100%, PP 1.00) comprises tribe Delphinieae
Warm.; clade D (BS 100%, PP 1.00) contains tribe
Cimicifugeae Torrey & A. Gray together with Beesia
and Eranthis of tribe Helleboreae DC.; clade E
(BS 74%, PP 0.98) contains tribe Anemoneae DC. and
tribe Ranunculeae DC. and Callianthemum of tribe
Adonideae Kunth.
Mapping morphological characters onto the strict
consensus tree from the MP analysis of the total
evidence data set indicates that monophyly of Ranun-
culales and each of its seven constituent families, and
inter-family and intra-family major clades are supported
by morphology (Fig. 3). Specific morphological chara-
cters are discussed in detail below.
Discussion
Monophyly and synapomorphies of Ranunculales
Ranunculales as well as each of its seven constituent
families sensu APG II (2003) (Berberidaceae, Circaeast-
eraceae, Eupteleaceae, Lardizabalaceae, Menisperma-
ceae, Papaveraceae, and Ranunculaceae) is strongly
supported as monophyletic. Within the order, family
inter-relationships are consistent with the results of
previous molecular studies (e.g., Chase et al., 1993;Hoot
and Crane, 1995;Hoot et al., 1999;Savolainen et al.,
2000a, b;Soltis et al., 2000, 2003;Hilu et al., 2003;Kim
et al., 2004a;Worberg et al., 2007), and combined
molecular and morphological studies (Nandi et al.,
1998;Doyle and Endress, 2000), but with our expanded
taxon sampling, are resolved usually with greater
support for clades found therein (Table 3).
Recent analyses (e.g., Chase et al., 1993;Hoot and
Crane, 1995;Hoot et al., 1999;Savolainen et al.,
2000a, b;Soltis et al., 2000, 2003;Hilu et al., 2003;
Kim et al., 2004a;Worberg et al., 2007; this study)
strongly support Eupteleaceae, a monogeneric family
endemic to East Asia, as a member of Ranunculales.
This conflicts with earlier morphological and anatomical
studies (Hutchinson, 1973;Cronquist, 1988;Dahlgren,
1983;Endress, 1986, 1993;Kubitzki et al., 1993;Thorne,
2000;Wu et al., 2002), which considered that the genus
belonged to the ‘‘lower’’ hamamelids and was related
to Trochodendraceae (including Trochodendron and
ARTICLE IN PRESS
Batrachium
Halerpestes
Oxygraphis
Trautvetteria
Circaeaster
Kingdonia
Hepatica
Anemoclema
Anemone
Clematis
Naravelia
Pulsatilla
Ranunculus
Myosurus
Callianthemum
Coptis
Thalictrum
Adonis
Aconitum
Consolida
Delphinium
Nigella
Asteropyrum
Eranthis
Caltha
Helleborus
Megaleranthis
Actaea
Beesia
Souliea
Anemonopsis
Cimicifuga
Trollius
Enemion
Aquilegia
Glaucidium
Hydrastis
Urophysa
Semiaquilegia
Paraquilegia
Isopyrum
Paropyrum
Leptopyrum
Dichocarpum
Corydalis
Hypecoum
Dicentra
Pteridophyllum
Meconopsis
Papaver
Hylomecon
Eomecon
Macleaya
Sanguinaria
Jeffersonia
Epimedium
Vancouveria
Bongardia
Achlys
Plagiorhegma
Diphylleia
Dysosma
Podophyllum
Sinopodophyllum
Ranzania
Caulophyllum
Gymnospermium
Berberis
Mahonia
Nandina
Decaisnea
Sargentodoxa
Sinofranchetia
Parvatia
Holboellia
Stauntonia
Lardizabala
Boquila
Archakebia
Akebia
Albertisia
Hypserpa
Pycnarrhena
Cissampelos
Cyclea
Stephania
Cocculus
Pericampylus
Sinomenium
Diploclisia
Menispermum
Anamirta
Arcangelisia
Aspidocarya
Parabaena
Tinomiscium
Tinospora
Xanthorhiza
Coriaria
Cornus
Aextoxicon
Buxus
Grevillea
Meliosma
Sabia
Altingia
Hamamelis
Platanus
Tetracera
Tetracentron
Trochodendron
Acorus
Saururus
Saruma
Brasenia
Ceratophyllum
Annona
Magnolia
Calycanthus
Austrobaileya
Canella
Takhtajania
Paeonia
Illicium
Euptelea
Nelumbo
Hedyosmum
Amborella
Vitis
0.86/55
0.94/69
1.00/81
0.98/75
0.79/70
0.80/70
0.95/79
80
1.00/97
0.96/75
0.61/
0.93
Batrachium
Halerpestes
Oxygraphis
Trautvetteria
Circaeaster
Kingdonia
Hepatica
Anemoclema
Anemone
Clematis
Naravelia
Pulsatilla
Ranunculus
Myosurus
Callianthemum
Coptis
Thalictrum
Adonis
Aconitum
Consolida
Delphinium
Nigella
Asteropyrum
Eranthis
Caltha
Helleborus
Megaleranthis
Actaea
Beesia
Souliea
Anemonopsis
Cimicifuga
Trollius
Enemion
Aquilegia
Glaucidium
Hydrastis
Urophysa
Semiaquilegia
Paraquilegia
Isopyrum
Paropyrum
Leptopyrum
Dichocarpum
Corydalis
Hypecoum
Dicentra
Pteridophyllum
Meconopsis
Papaver
Hylomecon
Eomecon
Macleaya
Sanguinaria
Jeffersonia
Epimedium
Vancouveria
Bongardia
Achlys
Plagiorhegma
Diphylleia
Dysosma
Podophyllum
Sinopodophyllum
Ranzania
Caulophyllum
Gymnospermium
Berberis
Mahonia
Nandina
Decaisnea
Sargentodoxa
Sinofranchetia
Parvatia
Holboellia
Stauntonia
Lardizabala
Boquila
Archakebia
Akebia
Albertisia
Hypserpa
Pycnarrhena
Cissampelos
Cyclea
Stephania
Cocculus
Pericampylus
Sinomenium
Diploclisia
Menispermum
Anamirta
Arcangelisia
Aspidocarya
Parabaena
Tinomiscium
Tinospora
Xanthorhiza
Coriaria
Cornus
Aextoxicon
Buxus
Grevillea
Meliosma
Sabia
Altingia
Hamamelis
Platanus
Tetracera
Tetracentron
Trochodendron
Acorus
Saururus
Saruma
Brasenia
Ceratophyllum
Annona
Magnolia
Calycanthus
Austrobaileya
Canella
Takhtajania
Paeonia
Illicium
Euptelea
Nelumbo
Hedyosmum
Amborella
Vitis
0.86/55
0.94/69
1.00/81
0.98/75
0.79/70
0.80/70
0.95/79
80
1.00/97
0.96/75
0.61/
0.93
Fig. 2. Strict consensus tree of 15 MPTs of Ranunculales
based on the morphological data set. Numbers above the
branches are Bayesian posterior probabilities (40.50) and
bootstrap percentages (450%), respectively.
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–11094
Tetracentron) or Cercidiphyllaceae. Eupteleaceae differs
from Cercidiphyllaceae in that stipules are absent (vs.
present), seeds lack appendages (vs. with appendages),
and in having strongly peltate carpels (vs. epeltate), and
from Trochodendraceae in having longitudinal anther
dehiscence (vs. valvate), carpels stipitate (vs. sessile),
long anther connective tips (vs. short), ovary superior
(vs. semi-inferior), and the micropyle formed from both
integuments (vs. from inner integument only). Addi-
tionally, the chromosome number for Trochodendron,
Tetracentron and Cercidiphyllum is n¼20, 23/24 and
19, respectively (Ratter and Milne, 1976;Cronquist,
1981), whereas that of Euptelea is n¼14 (Ratter and
Milne, 1976;Pan et al., 1991), which is more similar to
those found in Lardizabalaceae (n¼11, 14–16, 18) (Wu
and Kubitzki, 1993;Shi et al., 1994;Qin, 1997) and
Menispermaceae (n¼11–13) (Kessler, 1993;Wang
et al., 2004). Floral morphology (free carpels,
basifixed anthers, whorled phyllotaxis) also supports a
position within Ranunculales for Eupteleaceae (Ren
et al., 2007).
A micropyle formed from both integuments is a
synapomorphy of Ranunculales (char. 50:0), whereas
one formed from the inner integument (found only in
Lardizabalaceae and some Ranunculaceae) is indepen-
dently derived (Fig. 3). Epicuticular wax tubules
(char. 4:0) (Barthlott and Theisen, 1995) and large and
homogeneous S sieve element plastids (char. 14:0)
(Behnke, 1995) are ubiquitous in Ranunculales. How-
ever, S sieve element plastids are also present in other
eudicots. At present, the epicuticular anatomy of
Eupteleaceae is unknown. Thus, it is unclear whether
ARTICLE IN PRESS
CORE
RANUNCULALES
Corydalis
Dicentra
Hypecoum
Pteridophyllum
Eomecon
Sanguinaria
Hylomecon
Macleaya
Meconopsis
Papaver
Euptelea
Aextoxicon
Cornus
Altingia
Paeonia
Hamamelis
Coriaria
Tetracera
Vitis
Buxus
Tetracentron
Trochodendron
Meliosma
Sabia
Grevillea
Platanus
Nelumbo
Annona
Magnolia
Canella
Takhtajania
Calycanthus
Saruma
Saururus
Acorus
Ceratophyllum
Hedyosmum
Austrobaileya
Illicium
Brasenia
Amborella
Papaveraceae
Eupteleaceae
Outgroups
50:0
52:05:2
12:1
41:1
45:4
26:2
23:1 35:3
35:1
23:0
1.00/100
1.00/100
1.00/100
**
1.00/99
*
1.00/98
1.00/99
*
1.00/
0.74/
1.00/79
1.00/92
1.00/100
1.00/97 1.00/100
1.00/100
0.97/64
1.00/75
*
1.00/100
1.00/64
1.00/100
0.98/87 1.00/100
1.00/100
1.00/100
1.00/97
1.00/100
0.93/96
1.00/100
1.00/100
1.00
100
CORE
RANUNCULALES
Corydalis
Dicentra
Hypecoum
Pteridophyllum
Eomecon
Sanguinaria
Hylomecon
Macleaya
Meconopsis
Papaver
Euptelea
Aextoxicon
Cornus
Altingia
Paeonia
Hamamelis
Coriaria
Tetracera
Vitis
Buxus
Tetracentron
Trochodendron
Meliosma
Sabia
Grevillea
Platanus
Nelumbo
Annona
Magnolia
Canella
Takhtajania
Calycanthus
Saruma
Saururus
Acorus
Ceratophyllum
Hedyosmum
Austrobaileya
Illicium
Brasenia
Amborella
Papaveraceae
Eupteleaceae
Outgroups
50:0
52:05:2
12:1
41:1
45:4
26:2
23:1 35:3
35:1
23:0
1.00/100
1.00/100
1.00/100
**
1.00/99
*
1.00/98
1.00/99
*
1.00/
0.74/
1.00/79
1.00/92
1.00/100
1.00/97 1.00/100
1.00/100
0.97/64
1.00/75
*
1.00/100
1.00/64
1.00/100
0.98/87 1.00/100
1.00/100
1.00/100
1.00/97
1.00/100
0.93/96
1.00/100
1.00/100
1.00
100
Fig. 3. Strict consensus tree of eight MPTs of Ranunculales based on the total evidence data set. Numbers above and below
branches are Bayesian posterior probabilities (40.50) and bootstrap percentages (450%), respectively. ‘‘*’’ indicates the nodes not
found in Bayesian trees. Morphological character state changes within Ranunculales are indicated by boxes on the branches, with
character numbers and states. The accelerated transformation (ACCTRAN) optimization is shown. Filled boxes represent non-
homoplastic synapomorphies and empty boxes indicate homoplastic changes. Crosses indicate reversals and parallel lines indicate
parallelisms.
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–110 95
ARTICLE IN PRESS
Ranunculus
Batrachium
Halerpestes
Myosurus
Trautvetteria
Oxygraphis
Naravelia
Clematis
Anemoclema
Pulsatilla
Anemone
Hepatica
Callianthemum
Asteropyrum
Caltha
Actaea
Cimicifuga
Souliea
Eranthis
Anemonopsis
Beesia
Helleborus
Nigella
Delphinium
Consolida
Aconitum
Adonis
Megaleranthis
Trollius
Enemion
Isopyrum
Dichocarpum
Aquilegia
Semiaquilegia
Urophysa
Paraquilegia
Paropyrum
Leptopyrum
Thalictrum
Coptis
Xanthorhiza
Hydrastis
Glaucidium
Achlys
Diphylleia
Dysosma
Podophyllum
Sinopodophyllum
Bongardia
Epimedium
Vancouveria
Jeffersonia
Plagiorhegma
Berberis
Mahonia
Ranzania
Caulophyllum
Gymnospermium
Nandina
Albertisia
Pycnarrhena
Cissampelos
Cyclea
Stephania
Cocculus
Diploclisia
Hypserpa
Pericampylus
Anamirta
Arcangelisia
Aspidocarya
Parabaena
Tinomiscium
Tinospora
Menispermum
Sinomenium
Akebia
Archakebia
Holboellia
Parvatia
Stauntonia
Boquila
Lardizabala
Sinofranchetia
Decaisnea
Sargentodoxa
Circaeaster
Kingdonia
Berberidaceae
Menispermaceae
Lardizabalaceae
Circaeasteraceae
A
B
C
D
E
F
59:0
60:0 35:0 46:0
58:2
19:2
Ranunculaceae
35:2, 47:1, 51:1
57:3
45:1
22:2 55:1
54:1
29:1
26:1
59:1
60:1
3:1
48:2
1:1
39:1 40:0
35:2 64:3
37:1
46:2
36:0
64:1
46:0
29:0
39:0
64:2
64:1
9:0
63:1
60:1
62:1
60:0
56:1
27:0
47:1
64:0
65:1
54:0
44:0
44:2
44:1
37:0
61:1
60:0
1.00/100
1.00/87
1.00/100
1.00/100
1.00/100
1.00/96
1.00/100
1.00/100
1.00/99
1.00/98
1.00/97
1.00/100
0.96/65
1.00/99 1.00/100
1.00/100
1.00/100
1.00/95
1.00
99
1.00/90
0.99/95
1.00/100
1.00/100
1.00/100
*
1.00/100
1.00/100
1.00/76
1.00/100
1.00/100
0.97/79
1.00
99
1.00/100
1.00/100
1.00/100
1.00/90
1.00/73
1.00/93
0.93/70
1.00/100
1.00/82
*
1.00/100 1.00/100
1.00/76
1.00/100
1.00/96
0.53/
1.00/100
1.00/87
1.00/98
1.00/99
1.00/1000.57/90
1.00/51 1.00/96
1.00/100
1.00/100 1.00/100
*
1.00/100
1.00/99
1.00/100
1.00/100
1.00/96
0.98/78
1.00/97
1.00/90 1.00/100
1.00/88
1.00/100
1.00
99
1.00/100
1.00/100
1.00/100
1.00/100
1.00/91
1.00/
*
1.00/100
Ranunculus
Batrachium
Halerpestes
Myosurus
Trautvetteria
Oxygraphis
Naravelia
Clematis
Anemoclema
Pulsatilla
Anemone
Hepatica
Callianthemum
Asteropyrum
Caltha
Actaea
Cimicifuga
Souliea
Eranthis
Anemonopsis
Beesia
Helleborus
Nigella
Delphinium
Consolida
Aconitum
Adonis
Megaleranthis
Trollius
Enemion
Isopyrum
Dichocarpum
Aquilegia
Semiaquilegia
Urophysa
Paraquilegia
Paropyrum
Leptopyrum
Thalictrum
Coptis
Xanthorhiza
Hydrastis
Glaucidium
Achlys
Diphylleia
Dysosma
Podophyllum
Sinopodophyllum
Bongardia
Epimedium
Vancouveria
Jeffersonia
Plagiorhegma
Berberis
Mahonia
Ranzania
Caulophyllum
Gymnospermium
Nandina
Albertisia
Pycnarrhena
Cissampelos
Cyclea
Stephania
Cocculus
Diploclisia
Hypserpa
Pericampylus
Anamirta
Arcangelisia
Aspidocarya
Parabaena
Tinomiscium
Tinospora
Menispermum
Sinomenium
Akebia
Archakebia
Holboellia
Parvatia
Stauntonia
Boquila
Lardizabala
Sinofranchetia
Decaisnea
Sargentodoxa
Circaeaster
Kingdonia
Berberidaceae
Menispermaceae
Lardizabalaceae
Circaeasteraceae
A
B
C
D
E
F
59:0
60:0 35:0 46:0
58:2
19:2
Ranunculaceae
35:2, 47:1, 51:1
57:3
45:1
22:2 55:1
54:1
29:1
26:1
59:1
60:1
3:1
48:2
1:1
39:1 40:0
35:2 64:3
37:1
46:2
36:0
64:1
46:0
29:0
39:0
64:2
64:1
9:0
63:1
60:1
62:1
60:0
56:1
27:0
47:1
64:0
65:1
54:0
44:0
44:2
44:1
37:0
61:1
60:0
1.00/100
1.00/87
1.00/100
1.00/100
1.00/100
1.00/96
1.00/100
1.00/100
1.00/99
1.00/98
1.00/97
1.00/100
0.96/65
1.00/99 1.00/100
1.00/100
1.00/100
1.00/95
1.00
99
1.00/90
0.99/95
1.00/100
1.00/100
1.00/100
*
1.00/100
1.00/100
1.00/76
1.00/100
1.00/100
0.97/79
1.00
99
1.00/100
1.00/100
1.00/100
1.00/90
1.00/73
1.00/93
0.93/70
1.00/100
1.00/82
*
1.00/100 1.00/100
1.00/76
1.00/100
1.00/96
0.53/
1.00/100
1.00/87
1.00/98
1.00/99
1.00/1000.57/90
1.00/51 1.00/96
1.00/100
1.00/100 1.00/100
*
1.00/100
1.00/99
1.00/100
1.00/100
1.00/96
0.98/78
1.00/97
1.00/90 1.00/100
1.00/88
1.00/100
1.00
99
1.00/100
1.00/100
1.00/100
1.00/100
1.00/91
1.00/
*
1.00/100
Fig. 3. (Continued)
W. Wang et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 81–11096