Worberg & al. • Phylogenetic position of Huerteales
TAXON 58 (2) • May 2009: 468–478
Huerteales sister to Brassicales plus Malvales, and newly circumscribed
to include Dipentodon, Gerrardina, Huertea, Perrottetia, and Tapiscia
Andreas Worberg1, Mac H. Alford2, Dietmar Quandt1,3 & Thomas Borsch1,4
1
Nees-Institut für Biodiversität der Pflanzen, Rheinische Friedrich-Wilhelms-Universität Bonn,
Meckenheimer Allee 170, 53115 Bonn, Germany. andreas@worberg.de (author for correspondence)
2
Department of Biological Sciences, University of Southern Mississippi, 118 College Drive 5018,
Hattiesburg, Mississippi 39406, U.S.A.
3
Institut für Botanik, Technische Universität Dresden, Zellescher Weg 20b, 01217 Dresden, Germany
4
Current address: Botanischer Garten und Botanisches Museum Berlin-Dahlem und Institut für Biologie,
Freie Universität Berlin, Königin Luise-Str. 6–8, 14195 Berlin, Germany
Sequence data from the matK gene, the trnK group II intron, the trnL group I intron and the trnL-F spacer
were analysed for a broad sampling of the rosids and other eudicots. For the first time all putative genera of
Dipentodontaceae and Tapisciaceae (Dipentodon, Huertea, Perrottetia, Tapiscia), as well as the recently described Gerrardinaceae were included in a molecular phylogenetic dataset. All genera were found in a well
supported Huerteales clade. Moreover, with the rapidly evolving and non-coding plastid sequence data we
were able to resolve the Huerteales clade to branch after Sapindales, and to be sister to a Brassicales-Malvales
clade. Increased resolution and support among the malvids underscore the potential of plastid introns and
spacers as well as the matK gene as phylogenetic markers in rosids.
KEYWORDS: Huerteales, matK, rosids, trnK, trnL-F
INTRODUCTION
The large scale phylogenetic analysis of angiosperms
based on rbcL, atpB, and 18S sequence data of Soltis &
al. (2000) included one genus that was unresolved in either Brassicales, Malvales, or Sapindales in the eurosid II
(malvid) clade. This genus, Tapiscia, was later inferred to
be sister to Dipentodon (Dipentodontaceae), a relationship
strongly supported (94% JK) based on combined analyses
of matR, rbcL, and 18S DNA data (Peng & al., 2003) and
matR alone (Zhu & al., 2007). The order Huerteales was
thus described by Doweld (2001) to include the genus
Dipentodon (Dipentodontaceae) and the two small genera Tapiscia and Huertea (Tapisciaceae), formerly placed
together as subfamily Tapiscioideae of Staphyleaceae. Wu
& al. (2002) later recognized the Dipentodontales as a
new order in their vascular plant classification system,
consisting of Dipentodon sinicus alone. All three genera
are poorly known, but toothed leaf margins, 1–2 ovules
per carpel, and relatively small embryos seem to be shared
morphological characters (Stevens, 2001 onwards). Recently, an analysis based on several plastid and nuclear
genes by Zhang & Simmons (2006) also placed Perrottetia (formerly Celastraceae) with Tapiscia and Dipentodon.
Later, an analysis based on rbcL and atpB by Alford
(2006) indicated that the genus Gerrardina (formerly Flacourtiaceae) was unplaced in the eurosids II (malvids) but
potentially related to the Huerteales based on having two
468
ovules per carpel. However, analyses of rbcL and atpB
datasets remained inconclusive, and Gerrardina could not
be placed unambiguously in Huerteales. In the latest update of their angiosperm classification Thorne and Reveal
(Thorne, 2007) grouped all three families into Huerteales
within the newly described superorder named Huerteanae.
No study, though, has included all of the genera potentially
placed in Huerteales, thus underscoring the need for an
analysis with complete taxon sampling.
Pre-phylogenetic placements of genera that are now
candidates as members of a monophyletic order Huerteales
varied considerably among diverse angiosperm orders. Dipentodon sinicus Dunn is a small tree native to southern
China, Burma and northern India and can be recognized
by stipulate, serrate leaves, umbelliform inflorescences,
and essentially free-central placentation (Takhtajan,
1997). This taxon was formerly included in Santalales
(Cronquist, 1981) because of its placentation or in Violales (now Malpighiales) because of perianth morphology,
wood anatomy, and pollen (Dahlgren, 1980; Thorne, 1992;
Takhtajan, 1997). Dunn (1911) originally placed Dipentodon in Celastraceae, whereas a series of subsequent authors hypothesized it to be a member of Flacourtiaceae
(including Samydaceae) mostly based on floral features
(Sprague, 1925; Fischer, 1941; Loesener, 1942; Metcalfe
& Chalk, 1950; Lobreau, 1969). Tapisciaceae consist of
the two genera Tapiscia Ruiz & Pav. and Huertea Oliv.
(Takhtajan, 1987). Tapiscia sinensis is native to China,
TAXON 58 (2) • May 2009: 468–478
and the four known Huertea species are distributed from
the West Indies and Central America through the Andes
to Peru. They comprise small trees with spirally arranged
compound leaves that are odd-pinnate or trifoliolate and
have serrate margins (Krause, 1942; Stevens, 2001 onwards). Whereas Tapiscia was originally described in
Sapindaceae by Oliver (1890), Huertea and Tapiscia were
then mostly included together in Staphyleaceae (Bean,
1909; Diels, 1909; Schneider, 1912: p. 1026 and Fig. 607;
Dickison, 1986) and placed in Sapindales (Cronquist,
1981). Takhtajan (1987) later described a separate family Tapisciaceae that remained classified in Sapindales.
Perrottetia comprises about 15 species and is distributed
in Asia from China to Malesia and southwards to northeastern Australia but also occurs in the Neotropics from
Mexico to Peru. The floral structure of Perrottetia was
studied in some detail by Matthews & Endress (2005),
whereas that of the other genera is poorly known. All species of Perrottetia share two carpels per ovule and spirally
arranged leaves (Stevens, 2001 onwards), characters also
present in Dipentodon and Tapiscia. Gerrardina Oliv.
consists of two species, Gerrardina eylesiana and Gerrardina foliosa, both of which are small trees or shrubs
native to southern Africa (Malawi and Tanzania south to
the Eastern Cape Province of South Africa). Species of
Gerrardina have simple, alternate, stipulate leaves with
serrate margins, stamens opposite the petals, and two apical ovules per carpel. Formerly Gerrardina was placed
in Flacourtiaceae (including Samydaceae; Oliver, 1870;
Warburg, 1894; Gilg, 1925).
Sequences of rapidly evolving plastid spacers and
introns have been very successfully used in phylogenetic analyses to infer deep relationships among families
and orders of angiosperms (Borsch & al., 2003, 2005;
Löhne & Borsch, 2005; Müller & al., 2006; Wanke &
al., 2007; Worberg & al., 2007; Korotkova & al., 2009),
and they were expected to provide some resolution to the
above-mentioned phylogenetic ambiguity. Well known
examples of such rapidly evolving genomic regions that
also can be amplified using universal primers are the
so-called trnT-trnF-region (composed of the trnT-L and
trnL-F spacers and the group I intron in trnL; Taberlet
& al., 1991; Borsch & al., 2003; Quandt & al., 2004), and
the group II introns within trnK (Johnson & Soltis, 1994;
Müller & Borsch, 2005a, b), rpl16 (Kelchner, 2002) and
petD (Löhne & Borsch, 2005; Korotkova & al., 2009).
In addition to high phylogenetic structure per nucleotide
sequenced (Müller & al., 2006) these non-coding datasets yield additional indel matrices that usually further
improve resolution and support of the reconstructed trees
(Worberg & al., 2007; Borsch & al., 2007). Combining
such intron and spacer sequence data with sequences of
the rapidly evolving matK gene (Hilu & al., 2003) has
been shown to lead to further improved resolution and
Worberg & al. • Phylogenetic position of Huerteales
support of phylogenetic hypotheses, for example, in early
branching angiosperms (Borsch & al., 2005; Müller &
al., 2006) or early branching eudicots (Worberg & al.,
2007).
Our approach was therefore to first combine matK sequence data with trnK group II intron sequence data. The
matK gene is located within domain V of the trnK intron,
and therefore both markers can be co-amplified easily. In
the context of work aimed at understanding the mutational
dynamics and phylogenetic signal in non-coding plastid regions in eudicots, a large dataset is currently being
assembled for the rosids, a clade which comprises more
than 70,000 species in ca. 140 families. One of the most
prominent results is that we found strong support for the
Huerteales clade in a well resolved eurosid II ( = malvid)
clade, based on a combined matK/trnK dataset. Moreover,
trnL-intron and trnL-F spacer data confirmed the monophyly of Huerteales and provided molecular evidence for
the inclusion of Huertea, for which any kind of sequence
data were previously unavailable.
Considering the still unclear phylogenetic position
of Gerrardinaceae (Alford, 2006) and the hitherto unresolved position of Dipentodontaceae and Tapisciaceae
(see APG II, 2003), our study aims at contributing to the
ongoing discussion by first clarifying the composition of
the Huerteales clade, and by second resolving the phylogenetic relationships of Gerrardinaceae and Huerteales,
respectively.
MATERIAL AND METHODS
Taxon sampling. — The dataset comprises 54 taxa
from core eudicots with three representatives of Gunnerales as outgroup. Representatives for all rosid orders
recognized by APG II (2003) were sampled with a focus
on the eurosids II (malvid) clade to establish the framework for positioning Huerteales. Moreover, the sampling
of this study represents major lineages also found in a
more extensive combined trnK/matK plus trnL-F rosid
analyses by the authors ( > 180 taxa, not shown here). All
genera with putative affinities to Huerteales are included
in this study. Taxa with complete matK sequences already
available (Worberg & al., 2007) were completed by adding
the trnK intron, what could easily be achieved through
further sequencing reactions on already existing amplicons. In addition, some species with published completely
sequenced plastid genomes were added. Material for further taxa came from the living collections at the Botanical Gardens Bonn, Dresden, and Berlin (FU-BGBM). A
DNA extraction of Gerrardina foliosa was provided from
the DNA Bank of the Royal Botanic Gardens, Kew, U.K.
A list of all sampled taxa, their origin and voucher information is given in Appendix 1.
469
TAXON 58 (2) • May 2009: 468–478
Worberg & al. • Phylogenetic position of Huerteales
DNA isolation, amplification and sequencing.
— DNA was isolated from fresh or silica gel-dried plant
material, using a CTAB method with three extractions
(Borsch & al., 2003), designed to yield high amounts of genomic DNA. Fresh leaves were generally dried in silica gel
before extraction. Dry tissue was ground to a fine powder
using a mechanical homogenizer (Retsch MM200) with
5 mm beads at 30 Hz for 2 min. To identify mutational
hotspots complete sequences of spacers and introns are
necessary. We amplified with primers that were located
sufficiently far away from the actual region under study.
Sequencing was performed with either the universal
primers already used for amplification or with additional
internal primers, some of which were newly designed using SeqState v1.36 (Müller, 2005b). The matK gene was
amplified within the trnK intron, either entirely or in two
overlapping halves. Primers annealing to the trnK exons
were trnKFbryo forward (Wicke & Quandt, in press) and
trnK2R (Johnson & Soltis, 1994). To amplify two overlapping fragments, internal primers with higher taxon specificity primers were placed roughly 600 nt (reverse) and
450 nt (forward) downstream from the matK-start codon.
Internal primers for respective families were either automatically selected from the eudicot matK primer database
(www.eudicots.de) or designed newly with SeqState v1.36.
The trnL intron and the trnL-F spacer (in the following
called trnL-F region) were co-amplified using primers c
and f of Taberlet & al. (1991) and sequenced with primers
d of Taberlet & al. (1991) and trnL460F (Worberg & al.,
2007) that is a forward primer universal for angiosperms
annealing within the trnL intron. All primers used in this
study are listed in Appendix 2 (in the online version of
this article).
Amplification and sequencing reactions were performed in a T3 Thermocycler (Biometra, Göttingen,
Germany). Amplicons were purified with an Avegene gel
extraction kit (Avegene) after running them out on a 1.2%
agarose gel and excising the bands. The BeckmannCoulter
DTCS QuickStart Reaction kit was used for direct sequencing. Temperature profiles and PCR reaction conditions for
trnK/matK followed Müller & Borsch (2005a) and Borsch
& al. (2003) for trnL-F. Extension products were run on
BeckmannCoulter CEQ 8000 automated sequencers in
Bonn or Dresden. Alternatively, sequences were generated
on ABI377 autosequencers. Sequences were edited manually with PhyDE v0.995 (Müller & al., 2005).
Alignment, indel coding and phylogenetic analysis. — The presence of microstructural changes, such as
deletions, single sequence repeats, other insertions, and inversions, necessitates special attention to the alignment of
sequences. Alignment was carried out by eye using PhyDE
v0.995, applying the rules outlined in Borsch & al. (2003)
and Löhne & Borsch (2005), as there are still no automated
alignment algorithms that can handle length-variable DNA
470
regions adequately (Simmons & al., 2007). These alignment rules are based on recognizing sequence motifs that
result from microstructural changes (Kelchner & Clark,
1997; Kelchner, 2000, 2002) rather than globally applying
fixed gap costs. Inclusion of ambiguously alignable regions
into character matrices (Lutzoni & al., 2000; Aagesen,
2004) were not pursued here. Mutational hotspots with
unclear primary homology (Borsch & al., 2003) were excluded from tree inference in order to achieve maximum
accuracy. To utilize indel characters we applied the simple
indel coding method of Simmons & Ochoterena (2000) via
SeqState v1.36 (Müller, 2005b). The resulting indel matrix
was directly combined with the nucleotide-sequence matrix for parsimony and Bayesian Inference, as an increase
of resolution and support by adding indel information from
matK/trnK could be expected based on previous evidence
(Müller & Borsch, 2005b; Worberg & al., 2007).
To infer most parsimonious trees we used the Parsimony Ratchet (Nixon, 1999) as implemented in the
program PRAP (Müller, 2004). Ratchet settings were 20
random addition cycles of 200 ratchet replicates and upweighting 25% of the characters. If more than one shortest
tree was found, strict consensus trees were created. Nodes
were evaluated by jackknifing in PAUP* 4b10 (Swofford,
2001) with 36.79% deletion of characters and 10,000 replicates saving only one tree per replicate. This approach
follows studies on the reliability of jackknife percentages (Freudenstein & Simmons, 2004; Müller, 2005a).
Bayesian inference (BI) was performed using the program
MrBayes v3.1 (Ronquist & Huelsenbeck, 2003) applying
the GTR + Γ + I model for sequence data and the adaption
of the restriction site model (“F81”) for presence/absence
for the indel matrix. Four runs (106 generations each) with
four chains each were run simultaneously, starting from
random trees. Chains were sampled every 10th generation. Stationarity of the runs was reached within the first
250,000 generations (burn-in). Calculations of the consensus tree and the posterior probability (PP) of clades
were done based upon the trees sampled after the burn-in.
Only PP’s of 90 and higher were considered significant
(alpha = 0.1). Trees were drawn using TreeGraph (Müller
& Müller, 2004).
RESULTS
The combined trnK and matK dataset, excluding
mutational hotspots, comprises 3,334 substitution characters, of which 1,402 were parsimony-informative, 757
variable but not parsimony-informative, and 1,175 constant,
and an additional 348 indel characters. The parsimony
ratchet analysis yielded four most parsimonious trees with
a length of 8,697 steps (consistency index, CI = 0.420;
retention index, RI = 0.410; rescaled consistency index,
TAXON 58 (2) • May 2009: 468–478
Worberg & al. • Phylogenetic position of Huerteales
RC = 0.172; homoplasy index, HI = 0.580). The strict
consensus tree is depicted in Fig. 1 and displays a topology with Gerrardina (Gerrardinaceae) well supported as
part of the Huerteales clade (100% JK, 100 PP). Gerrardina is then sister to Tapisciaceae plus Dipentodontaceae
(100% JK, 100 PP), Tapisciaceae, including Huertea and
Tapiscia (100% JK, 100 PP) are sister to Dipentodontaceae (99% JK, 100 PP), and Dipentodon is sister to the
100
100
53
51
100
100
100
100
50
-
94
94
68
54
100
100
100
100
94
93
100
100
malvids 99
98
100
100
100
100
99
99
100
100
54
-
100
99
100
100
66
100
100
97
96
99
99
77
76
100
100
rosids 97
89
100
100
50
51
fabids 69
57
54
-
69
69
100
100
93
77
100
100
67
64
90
75
100
99
56
57
100
100
55
61
58
65
100
100
51
51
72
70
100
100
100
98
70
70
100
100
two species of Perrottetia sampled here (100% JK, 100
PP). The Huerteales are a highly supported clade (100%
JK, 100 PP). They are well positioned within the eurosids II ( = malvids), which are overall highly supported
(99% JK, 100 PP). Within malvids, Sapindales are sister
to Huerteales + Brassicales + Malvales (94% JK, 100 PP),
and Huerteales are sister to Brassicales + Malvales (94%
JK, 100 PP). Separate analyses of trnK and matK (trees not
BIX Bixa
CIST Cistus
MUNT Muntingia
MALV Gossypium
BOMB Durio
THYM Daphne
TROP Tropaeolum
CARIC Vasconcellea
RESED Reseda
BRASS Lobularia
DIPEN Perrottetia long.
DIPEN Perrottetia ovat.
DIPEN Dipentodon
TAP Tapiscia
TAP Huertea
GERR Gerrardina
RUTA Citrus
MELI Melia
SIMA Ailanthus
SAPIN Acer
ANAC Schinus
PARN Parnassia
CELAS Salacia
OXAL Oxalis
SALIC Populus
PASS Passiflora
EUPH Euphorbia
RHIZO Kandelia
CORI Coriaria
CUCUR Cucumis
JUG Juglans
ROSAC Malus
ROSAC Spiraea
URTI Urtica
FAB Glycine
POLY Polygala
GERAN Erodium
ZYGO Larrea
MELAS Melastoma
ONAG Oenothera
COMBR Combretum
MYRT Eucalyptus
STACH Stachyurus
VIT Vitis
LEEA Leea
AQUI Ilex
BALS Impatiens
API Daucus
ARALI Panax
SAX Chrysosplenium
AEX Aextoxicon
MYRO Myrothamnus flab.
MYRO Myrothamnus mosc.
GUNN Gunnera
Malvales
Brassicales
Huerteales
Sapindales
Celastrales
Oxalidales
Malpighiales
Cucurbitales
Fagales
Rosales
Fabales
Geraniales
Zygophyllales
Myrtales
Crossosomatales
Vitales
Asterids
Gunnerales
Outgroup
Fig. 1. Combined strict consensus tree based on substitutions and indels of trnK and matK, inferred with maximum parsimony. Bold values above branches are Jackknife percentages with indels coded, plain ones below are Jackknife values
without simple indel coding applied.
471
TAXON 58 (2) • May 2009: 468–478
Worberg & al. • Phylogenetic position of Huerteales
shown) found identical topologies as depicted here but with
generally lower node support. The Bayesian Analysis (Fig.
2) yielded nearly the same topology as Maximum Parsimony. Minor differences concern the unsupported position
of the Geraniales and Zygophyllales, i.e., the monophyly
of the fabids (compare Figs. 1 and 2). Whereas Geraniales
and Zygophyllales are resolved within the fabid clade and
share a moderate supported sister group relation under
parsimony, the position is unresolved under a Bayesian
framework. However, the remaining fabid clade reaches
significance in the Bayesian analyses if information obtained from indels was included in the analyses. Moreover, two strong supported clades appear: (1) Malpighiales, Oxalidales and Celastrales, and (2) Rosales, Fabales,
Cucurbitales, and Juglans (Fagales), with the former being
resolved with high support under parsimony as well. To
further clarify the composition of Huerteales we sampled
trnL-F data for a smaller dataset (26 taxa). Considering
the high statistical confidence of the matK/trnK trees with
larger sampling, we restricted the analysis of trnL-F data
BIX Bixa
CIST Cistus
THYM Daphne
100
MALV Gossypium
100
BOMB Durio
100
MUNT Muntingia
TROP Tropaeolum
CARIC Vasconcellea
100
RESED Reseda
100
100
BRASS Lobularia
DIPEN Perrottetia long.
100
100
DIPEN Perrottetia ovat.
100
DIPEN Dipentodon
TAP Tapisci
malvids 100
100
TAP Huertea
100
GERR Gerrardina
RUTA Citrus
100
SIMA Ailanthus
100
MELI Melia
SAPIN Acer
100*
ANAC Schinus
PARN Parnassia
100
CELAS
Salacia
100
OXAL Oxalis
SALIC Populus
100
98
PASS Passiflora
100
EUPH Euphorbia
rosids 100 92*
RHIZO Kandelia
CORI Coriaria
100
CUCUR Cucumis
ROSAC Malus
100
100
ROSAC Spirea
URTI Urtica
100
FAB Glycine
100
POLY Polygala
JUG Juglans
ZYGO Larrea
GERAN Erodium
MELAS Melastoma
99
MYRT Eucalyptus
100
COMBR Combretum
ONAG Oenothera
100
STACH Stachyurus
VIT Vitis
100
LEEA Leea
API Daucus
100
100
ARALI Panax
100
AQUI Ilex
100
BALS Impatiens
AEX Aextoxicon
SAX Chrysosplenium
MYRO Myrothamnus flab.
100
MYRO Myrothamnus mosc.
GUNN Gunnera
0.1
100
472
Malvales
Brassicales
Huerteales
Sapindales
Celastrales
Oxalidales
Malpighiales
Cucurbitales
Rosales
Fabales
Fagales
Zygophyllales
Geraniales
Myrtales
Crossosomatales
Vitales
Asterids
Gunnerales
Fig. 2. Bayesian phylogram based on the combined trnK + matK matrix.
Posterior probabilities
are depicted above
branches. * indicates that
the respective node did
not reach significance
( > 90) in the analysis after
exclusion of the indel
matrix.
TAXON 58 (2) • May 2009: 468–478
Worberg & al. • Phylogenetic position of Huerteales
to a malvid subset that included all six putative Huerteales
genera (Fig. 3). The matrix comprised 1,231 characters of
which 274 were parsimony-informative, and an additional
183 indel characters. Parsimony analysis yielded six shortest trees of 1,168 steps (CI = 0.622, RI = 0.440, RC = 0.273).
The Huerteales clade was resolved with 88% JK, again
rendering Gerrardina as sister to the remaining genera.
Huertea was found as sister to Tapiscia (Fig. 3). Notably,
the rather small trnL-F dataset also yielded good support
for the support within all other malvid orders, Brassicales,
Malvales, and Sapindales.
BOMB Bombax
BIX Bixa
89
90
MUNT Muntingia
58
76
82
80
97
94
CARIC Vasconcellea
BRASS Lobularia
TROP Tropaeolum
99
96
75
77
malvids 87
78
TAP Huertea
DIPEN Perrottetia long.
99
100
88
85
TAP Tapiscia
DIPEN Dipentodon
81
76
Brassicales
CIST Cistus
53
-
Huerteales
64
76
MALV Gossypium
Malvales
100
100
DIPEN Perrottetia ovat.
GERR Gerrardina
100
99
100
100
RUTA Citrus
MELI Melia
ANAC Schinus
SIMA Brucea
Sapindales
100
100
SAPIN Acer
ZYGO Larrea
GERAN Erodium
MYRT Eucalyptus
CORI Coriaria
CELAS Salacia
Outgroup
62
77
ROSAC Malus
STACH Stachyurus
Fig. 3. Combined strict consensus tree based on substitutions and indels of trnL-intron and trnL-F intergenic spacer
region for a smaller sampling, inferred with MP. Bold values above branches are Jackknife percentages with indels
coded, plain ones below are Jackknife values without simple indel coding applied.
DISCUSSION
Although morphological features and previous
analyses of DNA sequence data could not confidently
place Gerrardina (Alford, 2006), this study using matK/
trnK sequence data provides unambiguous support for a
placement of Gerrardinaceae sister to a Dipentodontacae + Tapisciaceae clade (100% JK, 100 PP). We have thus
enlarged the Huerteales to include Gerrardina. Moreover,
phylogenetic analysis of trnK/matK and trnL-F sequence
data shows for the first time with molecular data that
Huertea is the sister group of Tapiscia. Furthermore, our
results provide strong evidence for the interrelationships
of the orders of the eurosids II (malvids). Brassicales and
Malvales are sister, which are then sister to Huerteales,
which are then sister to Sapindales. This branching pattern
has strong support, regardless of the analytical approach.
Our results are a further example of the effectiveness
of matK/trnK and trnL-F as phylogenetic markers at deeper
levels in eudicots. High performance of partial matK was
earlier shown by Hilu & al. (2003) for angiosperms. In
addition to larger quantities of informative sites in such
rapidly evolving and non-coding genomic regions, Müller
& al. (2006) found increased phylogenetic signal per informative character of rapidly evolving or non-coding
markers like matK or trnT-F in contrast to slow evolving
regions like rbcL. Complete matK sequences alone (tree
not shown) inferred a topology similar to the complete
angiosperm analysis made by Hilu & al. (2003; Huerteales
not included) with high support for most nodes. This
study is the first to apply trnK group II intron sequences
at deeper level in angiosperms. Similar to the addition of
petD group II intron sequence data to a matK + trnL-F matrix in eudicots (Worberg & al., 2007), the addition of trnK
drastically improved the signal, especially for deeper nodes
(e.g., raising the JK support for rosids from 88% to 97%).
The results presented here were based on analyses of
all putative genera of the Huerteales clade. Gerrardina is
consistently inferred as sister to all other genera (Figs. 1–3)
whereas the detailed relationships within the clade comprised of Perrottetia, Dipentodon, Huertea and Tapiscia
await additional sequence data. Both Huertea and Tapiscia
share a large number of indels in trnL-F and trnK/matK
that further underscores their close relationship. Whereas
trnK/matK depict Dipentodon and Perrotetia as sisters
with 99% JK (100 PP) support, trnL-F signal is inconsistent (75% JK) and therefore not conclusive to this question.
Perrottetia, which previously had been classified
within Celastraceae, does not belong in this family anymore. Matthews & Endress (2005) found substantial floral
structural differences between Perrottetia and Celastrales.
While reconstructing Celastrales phylogeny, Zhang &
Simmons (2006) found Perrottetia among the outgroup
taxa of their taxon set. Zhang & Simmons (2006) further
473
Worberg & al. • Phylogenetic position of Huerteales
investigated the position of Perrottetia by including newly
generated 18S, atpB and rbcL sequences into the 567-taxon
dataset of Soltis & al. (2000) and found Perrottetia as sister
to the monotypic Tapiscia with 100% JK support. Our study
supports these results, placing the two Perrottetia species
with high statistical confidence into the Dipentodontaceae
clade (JK 98%, 100 PP) sister to Dipentodon sinicus. Alford
(2006) recovered Gerrardinaceae in the malvid clade using
rbcL+atpB sequences from Soltis & al. (2000) with the
addition of Gerrardina foliosa and Dipentodon sinicus to
the sampling. Although Gerrardinaceae were recovered in
a clade of Huerteales + Malvales + Brassicales (with 70%
JK), its relationships among those orders were unresolved.
While ndhF-sequences (3 end only), for which less comparative data are available, showed an affinity to Tapiscia,
Dipentodon, Brassicales, and Malvales, analyses of atpB
and rbcL sequences did not unambiguously reveal the relationship of Gerrardinaceae within the eurosids II.
Phylogenetic relationships in the malvids ( = eurosids
II) are depicted in our study as Brassicales being sister
to Malvales, with Huerteales being sister to the Brassicales + Malvales clade. Finally, Sapindales are shown to
be sister of all remaining malvids. The latter topology
was already hypothesised by partial matK sequences
(Hilu & al., 2003), although no member of the Huerteales
was represented. The three gene analysis of Soltis & al.
(2000) could not resolve relationships among Malvales,
Sapindales, Brassicales and Tapiscia using maximum parsimony. Huerteales were represented by Tapiscia alone in
Soltis & al. (2000). However, in their subsequent Bayesian analysis of the same dataset, Soltis & al. (2007) arrived at a different malvid topology, with Tapiscia sister
to Brassicales (unsupported), and this clade being sister to
a well-supported Malvales + Sapindales clade. Similarly,
the recent addition of mitochondrial matR sequences to a
rbcL + atpB + 18SrDNA dataset (Zhu & al., 2007) indicated
a position of Tapiscia sister to Brassicales, albeit without
support. The presented analyses, however, clearly resolve
the Huerteales sister to Malvales and Brassicales irrespective of the optimization criterion with strong support.
As a taxonomic order, Huerteales is a recent creation
based almost entirely on analyses of DNA sequence data
(Soltis & al., 2000; Doweld, 2001; Peng & al., 2003). The
taxa of Huerteales, in general, are poorly known and have
been considered as peripheral members of various families, often shuffled among notoriously diverse families
such as Celastraceae and Flacourtiaceae. Much of the detailed morphological and anatomical work for adequate
comparisons, however, remains to be done. In particular,
Gerrardina is lacking study of wood anatomy, and data
on seed anatomy and secondary chemistry for all of the
genera are scarce. Thus, one would not be surprised to
learn that morphological synapomorphies for Huerteales
are not yet clear. Furthermore, since the composition of
474
TAXON 58 (2) • May 2009: 468–478
Huerteales has been modified drastically in its short lifespan, phylogenetic studies utilizing morphological data
or mapping morphological features onto molecular trees
have not incorporated all of the relevant genera (cf. Soltis
& al., 2005; Ronse de Craene & Haston, 2006).
The highly supported resolution resulting from this
study now permits the formation of preliminary hypotheses about the evolution of the order and highlights types
of data that may be useful in further clarifying morphological evolution within the eurosids II. Table 1 summarizes the current state of knowledge on morphology and
anatomy of Huerteales taxa and is the basis for our discussion. Within Huerteales, Dipentodon and Perrottetia are
sister, a relationship suspected even in morphological and
pre-phylogenetic works (e.g., Dunn, 1911; Fischer, 1941;
Liu & Cheng, 1991). They both have stipulate, toothed
leaves, poorly differentiated perianth (sepals and petals
similar), a nectary disk, and similar wood anatomy. Zhang
& Simmons (2006) recognize them together in the family
Dipentodontaceae. Huertea and Tapiscia are not obvious relatives of Dipentodon and Perrottetia morphologically, but they do share scalariform perforation plates in
the vessels (Carlquist & Hoekman, 1985), septate fibers
(lacking in Perrottetia), stamen number equal to the petal
number, and stamens alternating with the petals. Unlike
Dipentodontaceae, Huertea and Tapiscia have compound
leaves, a calyx tube, and clearly differentiated calyx and
corolla. Tapiscia also lacks a nectary disk. Previously recognized as a subfamily of the Staphyleaceae, Huertea and
Tapiscia are now recognized as the family Tapisciaceae.
Rarely, the two genera have been placed in monogeneric
families, Tapisciaceae and Huerteaceae (Doweld, 2001).
The final member of Huerteales is the monogeneric family Gerrardinaceae. Like other Huerteales, Gerrardina
has toothed leaves, cymose inflorescences, a hypanthium
(short in Tapiscia and Huertea), a unilocular ovary with
1–2 ovules per carpel, and small embryos. Although not
present in all members of Huerteales, Gerrardina also
has stipules (lacking in Huertea), mucilage cells (lacking
in Dipentodon), and a nectary disk (lacking in Tapiscia).
Gerrardina differs from the other members of Huerteales
in having stamens opposite the petals.
Comparison of the features of Huerteales and the other
orders of the eurosids II (Sapindales, Malvales, Brassicales)
may provide clues for uncovering potential synapomorphies
for Huerteales. Wood anatomy seems a good place to start.
Sapindales, Malvales, and Brassicales usually have simple
perforation plates and axial parenchyma in the wood, but
Huerteales have scalariform plates (rarely simple in Perrottetia; unknown for Gerrardina) and lack axial parenchyma (except Perrottetia, where it is paratracheal; again,
unknown for Gerrardina). Although nectary disks are common in Sapindales, they are not usually associated with a
hypanthium (albeit short in Tapisciaceae), as in Huerteales.
TAXON 58 (2) • May 2009: 468–478
Worberg & al. • Phylogenetic position of Huerteales
Table 1. Representative anatomical and morphological features of the taxa included in Huerteales, as well as predominant
states for the related orders Sapindales, Malvales, and Brassicales.
Feature
Gerrardina
Tapiscia
Huertea Dipentodon Perrottetia Sapindales Malvales
Vessel perforation plates
?
Scalariform
Scalariform
Scalariform
Scalariform (rarely
simple)
Simple
Simple
Simple
Septate fibers
?
Present
Present
Present
Absent
Often
present
Absent
Absent or
present (then
plesiomorphic)
Axial parenchyma in wood
Brassicales
?
Absent
Absent
Absent
Present
Present
Present
Present
Mucilage cells
Present
Present
Present
Absent
Present
Sometimes
present
Present
(except
Cistaceaee)
Absent
Leaf complexity
Simple
Simple
Simple
Simple or Simple or
compound compound
Stipules
Present
Present
(modified)/
absent
Present
Present
Absent
Present
Usually
absent
Imbricate +/– valvate
Valvate
Open to
valvate
Imbricate
Valvate
Mostly
imbricate
Sepal aestivation
Imbricate
Compound Compound
Present
Simple or
compound
Hypanthium (floral cup)
Present
Present
Present
(but short)
Present
Present
Usually
absent
Absent
Usually
absent
Nectary disk
Present
Absent
Present
Present
Present
Present
Absent
Absent
Multilocular
Usually
multilocular
Usually
multilocular
Ovary
Unilocular Unilocular Unilocular Unilocular Unilocular
(with basal
(with apical
septum)
septum)
Carpels
2
2
2
3
2
2–5(–6)
2–5(–many)
2–6(–12)
Ovules/carpel
2
1
1
2
2
1–2
1–∞
1–2(–∞)
Placentation
Apical
Basal
Axile
Axile or
parietal
Embryo
Straight
Straight
Folded
Straight or
curved
Axile/ Free-central Basal and
Axile,
basal-axile
lateral basal-axile,
apical-axile
Straight
Straight
?
Straight or
curved
General information compiled from Metcalfe & Chalk (1950), Cronquist (1981), Takhtajan (1997), Stevens (2001 onwards),
and Ronse de Craene & Haston (2006). Specific data for Gerrardina from Alford (2006); for Tapiscia and Huertea from Krause
(1942), Carlquist & Hoekman (1985), and Dickison (1986); for Dipentodon from Dunn (1911), Merrill (1941), Liu & Cheng
(1991), and Zhang & Gao (1995); and for Perrottetia from Matthews & Endress (2005).
The nectary disks in Huerteales are non-vascularized or
only supplied by phloem (Dickison, 1986; Matthews &
Endress, 2005). Huerteales also share unilocular ovaries,
although this feature may occasionally occur in the other
orders. Some characters show remarkable diversity within
Huerteales. Leaf complexity, presence/absence of stipules,
and position of stamens have already been mentioned, but
the order also includes taxa with imbricate, valvate, and
open sepal aestivation, uniseriate and homocellular to multiseriate and heterocellular wood rays, and apical, basal,
partially axile, and free-central placentation.
APG (2003) treat Tapisciaceae as an unplaced family in malvids. In addition to insufficient tree resolution,
earlier analyses were also incomplete in taxon sampling,
thereby hindering the development of a strongly supported ordinal classification. We therefore recognise
the order Huerteales undoubtedly as a monophyletic
group of families with ordinal level that has a distinct
position within the malvid grade with high support.
Thus we support an angiosperm classification system
with the order Huerteales included into the rosids.
ACKNOWLEDGEMENTS
We acknowledge support by the Deutsche Forschungsgemeinschaft (DFG) for the project “Mutational dynamics of
non-coding genomic regions and their potential for reconstructing eudicot evolution” (grants BO1815/2 to T.B. and QU153/2
to D.Q.). Most of the material was provided by the living collections of the Botanical Gardens of Bonn University and
Dresden Technical University. The authors like to thank Peter
475
Worberg & al. • Phylogenetic position of Huerteales
Stevens for helpful comments on the manuscript. For various kinds of support and helpful discussions we are grateful
to Wilhelm Barthlott (Bonn), Mark W. Chase (K), Jay Horn
(FTG), Nadja Korotkova (Bonn), Xia Lei (Cornell University),
Anna-Magdalena Barniske (Dresden), Kai Müller (Bonn). We
would like to express our sincere thanks to Armando Urquiloa
(Jardín Botánico de Pinar del Río), Ramona Oviedo (HAC)
and Werner Greuter (B) in organizing to collect fresh material
of Huertea cubensis, and to Angela T. Leiva Sánchez, Jardín
Botánico Nacional, Havana, for arranging the permission to
export material. Helpful comments of three anonymous reviews
are gratefully acknowledged. T.B. acknowledges a Heisenberg
scholarship by the Deutsche Forschungsgemeinschaft (DFG)
that greatly facilitated his work on angiosperm phylogenetics.
This study is in partial fulfilment for the requirements to obtain
a Ph.D. from Bonn University by the first author.
LITERATURE CITED
Aagesen, L. 2004. The information content of an ambiguously
alignable region, a case study of the trnL intron from the
Rhamnaceae. Org. Divers. Evol. 4: 35–49.
Alford, M.H. 2006. Gerrardinaceae: a new family of African flowering plants unresolved among Brassicales,
Huerteales, Malvales, and Sapindales. Taxon 55: 959–964.
APG II. 2003. An update of APG classification for the orders and
families of flowering plants. Bot. J. Linn. Soc. 141: 399–436.
Bean, W.J. 1909. Garden notes on new trees and shrubs. Bull.
Misc. Inform. Kew 1909: 355–356.
Borsch, T. 2000. Phylogeny and Evolution of the Genus Nymphaea. Ph.D. thesis, University of Bonn, Bonn.
Borsch, T., Hilu, K.W., Quandt, D., Wilde, V., Neinhuis,
C. & Barthlott, W. 2003. Non-coding plastid trnT-trnF
sequences reveal a well supported phylogeny of basal angiosperms. J. Evol. Biol. 16: 558–576
Borsch, T., Hilu, K.W., Wiersema, J.H., Löhne, C., Barthlott, W. & Wilde, V. 2007. Phylogeny of Nymphaea (Nymphaeaceae): evidence from substitutions and microstructural changes in the chloroplast trnT-trnF region. Int. J.
Pl. Sci. 168: 639–671.
Borsch, T., Löhne, C., Müller, K., Hilu, K.W., Wanke, S.,
Worberg, A., Barthlott, W., Neinhuis, C. & Quandt,
D. 2005. Towards understanding basal angiosperm diversification: recent insights using rapidly evolving genomic
regions. Nova Acta Leop. 92: 85–110.
Carlquist, S. & Hoekman, D.A. 1985. Wood anatomy of Staphyleaceae: ecology, statistical correlations, and systematics.
Flora 177: 195–216.
Cronquist, A. 1981. An Integrated System of Classification of
Flowering Plants. Columbia University Press, New York.
Dahlgren, R. 1980. A revised system of classification of the
angiosperms. Bot. J. Linn. Soc. 80: 91–124.
Dickison, W.C. 1986. Floral morphology and anatomy of Staphyleaceae. Bot. Gaz. 147: 312–326.
Diels, L. 1909. Staphyleaceae. Bot. Jahrb. Syst. 29: 448.
Doweld, A.B. 2001. Prosyllabus Tracheophytorum. Tentamen
systematis plantarum vascularium (Tracheophyta). Geos,
Moscow.
476
TAXON 58 (2) • May 2009: 468–478
Dunn, A. 1911. Dipentodon, a new genus of uncertain systematic position. Bull. Misc. Inform. Kew 1911: 310–313.
Fischer, C.E.C. 1941. Contributions to the flora of Burma:
XVIII. Kew Bull. 1940: 282–294.
Freudenstein, J.V. & Simmons, M.P. 2004. Relative effects of
increasing genetic distance on alignment and phylogenetic
analysis. Cladistics 20: 83–83.
Gilg, E. 1925. Flacourtiaceae. Pp. 377–457 in: Engler, A. &
Prantl, K. (eds.), Die natürlichen Pflanzenfamilien, ed. 2,
vol. 21. Duncker & Humblot, Berlin.
Hilu, K.W., Borsch, T., Müller, K., Soltis, D.E., Soltis, P.S.,
Savolainen, V., Chase, M., Powell, M., Alice, L.A.,
Evans, R., Sauquet, H., Neinhuis, C., Slotta, T.A., Rohwer, J.G., Campbell, C.S. & Chatrou, L. 2003. Angiosperm phylogeny based on matK sequence information.
Amer. J. Bot. 90: 1758–1776.
Johnson, L.A. & Soltis, D.E. 1994. MatK DNA sequences and
phylogenetic reconstruction in Saxifragaceae sensu stricto.
Syst. Bot. 19: 143–156.
Johnson, L.A. & Soltis, D.E. 1995. Phylogenetic inference in
Saxifragaceae sensu stricto and Gilia (Polemoniaceae) using matK sequences. Ann. Missouri Bot. Gard. 82: 149–175.
Kelchner, S.A. 2000. The evolution of noncoding chloroplast
DNA and its application in plant systematics. Ann. Missouri Bot. Gard. 87: 482–498.
Kelchner, S.A. 2002. Group II introns as phylogenetic tools:
structure, function, and evolutionary constraints. Amer. J.
Bot. 89: 1651–1669.
Kelchner, S.A. & Clark, L.G. 1997. Molecular evolution and
phylogenetic utility of the rpl16 intron in Chusquea and the
Bambusoideae (Poaceae). Molec. Phylog. Evol. 8: 385–397.
Korotkova, N., Schneider, J.V., Quandt, D., Worberg, A.,
Zizka, G. & Borsch, T. 2009. Phylogeny of the eudicot
order Malpighiales — analysis of a recalcitrant clade with
sequences of the petD group II intron. Pl. Syst. Evol. doi
10.1007/s00606-008-0099-7. [Special issue “Molecular
evolution and phylogenetic utility of non-coding DNA –
applications from species to deep level questions.”]
Krause, J. 1942. Staphyleaceae. Pp. 255–321 in: Engler, A.
(ed.), Die natürlichen Pflanzenfamilien, ed. 2, vol. 20b.
Duncker & Humblot, Berlin.
Liang H. & Hilu K.W. 1996. Application of the matK gene
sequences to grass systematics. Canad. J. Bot. 74: 125–134.
Liu, J. & Cheng, C. 1991. On the systematic position of genus
Dipentodon Dunn. J. Wuhan Bot. Res. 9: 29–39.
Lobreau, D. 1969. Les limites de l’ “ordre” des Celastrales
d’apres le pollen. Pollen & Spores 11: 499–555.
Loesener, T. 1942. Celastraceae. Pp. 87–197 in: Engler, A. (ed.),
Die natürlichen Pflanzenfamilien, ed. 2, vol. 20b. Duncker
& Humblot, Berlin.
Löhne, C. & Borsch, T. 2005. Molecular evolution and phylogenetic utility of the petD group II intron: a case study in
basal angiosperms. Molec. Biol. Evol. 22: 317–332.
Lutzoni, F., Wagner, P., Reeb, V. & Zoller, S. 2000. Integrating ambiguously aligned regions of DNA sequences in
phylogenetic analyses without violating positional homology. Syst. Biol. 49: 628–651.
Matthews, M.L. & Endress, P.K. 2005. Floral structure and
systematics in Celastrales (Celastraceae, Parnassiaceae,
Lepidobotryaceae). Bot. J. Linn. Soc. 149: 129–194.
Matthews, M.L. & Endress, P.K. 2006. Floral structure and
systematics in four orders of rosids, including a broad
TAXON 58 (2) • May 2009: 468–478
survey of floral mucilage cells. Pl. Syst. Evol. 260: 199–
221.
Merrill, E.D. 1941. The Upper Burma plants collected by Captain F. Kingdon Ward on the Vernay-Cutting Expedition,
1938–1939. Brittonia 4: 20–188.
Metcalfe, C.R. & Chalk, L. 1950. Anatomy of the Dicotyledons: Leaves, Stem, and Wood in Relation to Taxonomy
with Notes on Economic Uses, 1st ed., vols. 1–2. Clarendon
Press, Oxford, U.K.
Müller, K. 2004. PRAP-computation of Bremer support for
large data sets. Molec. Phylog. Evol. 31: 780–782.
Müller, K. 2005a. The efficiency of different search strategies
in estimating parsimony jackknife, bootstrap, and Bremer
support. BMC Evol. Biol. 5: 58.
Müller, K. 2005b. SeqState: primer design and sequence statistics for phylogenetic DNA datasets. Appl. Bioinformatics
4: 65–69.
Müller, K. & Borsch, T 2005a. Phylogenetics of Amaranthaceae based on matK/trnK sequence data — evidence from
parsimony, likelihood, and Bayesian analyses. Ann. Missouri Bot. Gard. 92: 66–102.
Müller, K. & Borsch, T. 2005b. Phylogenetics of Utricularia
(Lentibulariaceae) and molecular evolution of the trnK
intron in a lineage with high substitutional rates. Pl. Syst.
Evol. 250: 39–67.
Müller, K., Borsch, T. & Hilu, K.W. 2006. Phylogenetic utility of rapidly evolving DNA at high taxonomical levels:
contrasting matK, trnT-F, and rbcL in basal angiosperms.
Molec. Phylog. Evol. 41: 99–117.
Müller, J. & Müller, K. 2004. TREEGRAPH: automated
drawing of complex tree figures using an extensible tree
description format. Molec. Ecol. Notes 4: 786–788.
Müller, J., Müller, K., Quandt, D. & Neinhuis, C. 2005.
PhyDE — www.phyde.de
Nandi, O. I., Chase, M.W. & Endress, P.K. 1998. A combined
cladistic analysis of angiosperms using rbcL and non-molecular data sets. Ann. Missouri Bot. Gard. 85: 137–212.
Nixon, K.C. 1999. The parsimony ratchet, a new method for
rapid parsimony analysis. Cladistics 15: 407–414.
Oliver, D. 1870. Gerrardina foliosa Oliv. T. 1075 in: Hooker,
J.D. (ed.), Hooker’s Icones Plantarum, vol. 11. Longman,
Rees, Orme, Brown, Green, & Longman, London.
Oliver, D. 1890. Tapiscia sinensis. T. 1928 in: Hooker, J.D.
(ed.), Hooker’s Icones Plantarum, vol. 20. Longman, Rees,
Orme, Brown, Green, & Longman, London.
Peng, Y., Chen, Z., Gong, X., Zhong, Y. & Shi, S. 2003. Phylogenetic position of Dipentodon sinicus: evidence from
DNA sequences of chloroplast rbcL, nuclear ribosomal
18S, and mitochondrial matR. Bot. Bull. Acad. Sin. 44:
217–222.
Quandt, D., Müller, K., Stech, M., Frahm, J.-P., Frey, W.,
Hilu, K.W. & Borsch, T. 2004. Molecular evolution of
the chloroplast trnL-F region in land plants. Pp. 13–37 in:
Goffinet, B., Hollowell, V. & Magill, R. (eds.), Molecular Systematics of Bryophytes. Monographs in Systematic
Botany from the Missouri Botanical Garden 98. Missouri
Botanical Garden, St. Louis.
Ronquist, F. & Huelsenbeck, J.P. 2003. MrBayes 3: Bayesian
phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.
Ronse de Craene, L.P. & Haston, E. 2006. The systematic
relationships of glucosinolate-producing plants and related
Worberg & al. • Phylogenetic position of Huerteales
families: a cladistic investigation based on morphological
and molecular characters. Bot. J. Linn. Soc. 151: 453–494.
Schneider, C.K. 1912. Illustriertes Handbuch der Laubholzkunde, vol. 2. Gustav Fischer, Jena.
Simmons, M.P., Müller, K.F. & Norton, A.P. 2007. The relative performance of indel-coding methods in simulations.
Molec. Phylog. Evol. 44: 724–740.
Simmons, M.P. & Ochoterena, H. 2000. Gaps as characters
in sequence-based phylogenetic analyses. Syst. Biol. 49:
369–381.
Solereder, H. 1932. Über die Staphyleaceengattung Tapiscia
Oliv. Ber. Deutsch. Bot. Ges. 10, 545-551.
Soltis, D.E., Gitzendanner, M.A. & Soltis, P.S. 2007. A 567taxon data set for angiosperms: the challenges posed by
Bayesian analyses of large data sets. Int. J. Pl. Sci. 168:
137–157.
Soltis, D.E., Soltis, P.S., Chase, M.W., Mort, M.E., Albach,
D.C., Zanis, M., Savolainen, V., Hahn, W.H., Hoot,
S.B., Fay, M.F., Axtell, M., Swensen, S.M., Price, L.M.,
Kress, W.J., Nixon, K.C. & Farris, J.S. 2000. Angiosperm phylogeny inferred from 18S rDNA, rbcL, and atpB
sequences. Bot. J. Linn. Soc. 133: 381–461.
Soltis, D.E., Soltis, P.S., Endress, P.K. & Chase, M.W. 2005.
Phylogeny and Evolution of Angiosperms. Sinauer Associates, Sunderland.
Sprague, T.A. 1925. Samydaceae. J. Bot. 63: 9–13.
Steele, K.P. & Vilgalys, R. 1994. Phylogenetic analyses of
Polemoniaceae using nucleotide sequences of the plastid
gene matK. Syst. Bot. 19: 126–142.
Stevens, P.F. 2001 onwards. Angiosperm Phylogeny Website, vers.
9, June 2008. www.mobot.org/MOBOT/research/ APweb/
Swofford, D.L. 2001. PAUP*: Phylogenetic Analysis Using
Parsimony (*and Other Methods). Sinauer, Sunderland.
Taberlet, P., Gielly, L., Pautou, G. & Bouvet, J. 1991. Universal primers for amplification of three non-coding regions
of chloroplast DNA. Pl. Molec. Biol. 17: 1105–1109.
Takhtajan A.L. 1987. System of Magnoliophyta. Academy of
Sciences U.S.S.R., Leningrad.
Takhtajan, A.L. 1997. Diversity and Classification of Flowering Plants. Columbia University Press, New York.
Thorne, R.F. 1992. An updated phylogenetic classification of
the flowering plants. Aliso 13: 365–390.
Thorne, R.F. 2007. An updated classification of the class Magnoliopsida (“Angiospermae”). Bot. Rev. 73: 67–182. [“with
many nomenclatural additions by James L. Reveal.”]
Wanke, S., Gonzalez, F. & Neinhuis, C. 2006a. Systematics
of pipevines: combining morphological and fast-evolving
molecular characters to investigate the relationships within
subfamily Aristolochioideae (Aristolochiaceae). Int. J. Pl.
Sci. 167: 1215–1227.
Wanke, S., Jaramillo, M.A., Borsch, T., Samain, M.-S.,
Quandt, D. & Neinhuis, C. 2007. Evolution of Piperales — combined matK gene and trnK intron sequence data
reveal lineage specific resolution contrast. Molec. Phylog.
Evol. 42: 477–497.
Wanke, S., Samain, M.S., Vanderschaeve, L., Mathieu, G.,
Goetghebeur, P. & Neinhuis, C. 2006b. Phylogeny of
the genus Peperomia (Piperaceae) inferred from the trnK/
matK region (cpDNA). Pl. Biol. 8: 93–102.
Warburg, O. 1894. Flacourtiaceae. Pp. 1–56 in: Engler, A. &
Prantl, K. (eds.), Die natürlichen Pflanzenfamilien, ed. 1,
vol. 3(6a). Engelmann, Leipzig.
477
Worberg & al. • Phylogenetic position of Huerteales
Wicke, S. & Quandt, D. In press. Universal primers for the amplification of the plastid trnK/matK region in land plants.
Anales Jard. Bot. Madrid 66.
Worberg, A., Quandt, D., Barniske, A.-M., Löhne, C., Hilu,
K.W. & Borsch, T. 2007. Phylogeny of basal eudicots:
insights from non-coding and rapidly evolving DNA. Org.
Divers. Evol. 7: 55–77.
Wu, Z.-Y., Lu, A.-M., Tang, Y.-C., Chen, Z.-D. & Li, D.Z. 2002. Synopsis of a new “polyphyletic-polychronicpolytopic” system of the angiosperms. Acta Phytotax. Sin.
40: 298–322.
TAXON 58 (2) • May 2009: 468–478
Zhang, L.-B. & Simmons, M.P. 2006. Phylogeny and delimitation of the Celastrales inferred from nuclear and plastid
genes. Syst. Bot. 31: 122–137.
Zhang, X.-Y. & Gao, X.-Z. 1995. Anatomical studies on secondary xylem and secondary phloem of Dipentodon sinicus. Acta Bot. Sin. 37: 534–538.
Zhu, X.-Y., Chase, M.W., Qiu, Y.-L., Kong, H.-Z., Dilcher,
D.L., Li, J.-H. & Chen, Z.-D. 2007. Mitochondrial matR
sequences help to resolve deep phylogenetic relationships
in rosids. BMC Evol. Biol. 7: 217
Appendix 1. Taxa and GenBank/EMBL/DDBJ accession numbers of the sequences used in this study.
Species in alphabetical order and their corresponding family following APG II (2003), voucher information, garden or field origin and GenBank/
EMBL/DDBJ accession numbers of matK, trnL-F and trnK with corresponding citations. New sequences generated in this study are labeled with
“this study”. Dashes indicate missing data.
Acer campestre L., Sapindaceae, EMBL/GenBank, –, AJ438793 Bittkau & Mueller-Starck (2002), AF401189 Tian & al. (2001), AJ438793 Bittkau & MuellerStarck (2002); Aextoxicon punctatum Ruiz & Pav., Aextoxicaceae, T. Borsch 3459 (BONN), BG Bonn, DQ182342 Müller & al. (2006), – , DQ182342 this
study; Ailanthus altissima Swingle, Simaroubaceae, A. Worberg 025 (BONN), BG Bonn, FM179922 this study, – , FM179922 this study; Bixa orellana L.,
Bixaceae, A. Worberg 040 (BONN), BG Bonn, FM179929 this study, FM179540 this study, FM179929 this study; Bombax malabaricum DC., Bombacaceae,
EMBL/GenBank, – , – , AY328149 Yuan & al. (2003), – ; Brucea javanica (L.) Merr., Simaroubaceae, EMBL/GenBank, – , – , AB365015, AB365024 Tanaka
& al. (2007), – ; Chrysosplenium alternifolium L., Saxifragaceae, T. Borsch s.n. (BONN), Germany, AM396496 Worberg & al. (2007), –, AM396496 this
study; Cistus ladanifer L., Cistaceae, A. Worberg s.n. (BONN), BG Bonn, FM179939 this study, FM179538 this study, FM179939 this study; Citrus sinensis
Osbeck, Rutaceae, EMBL/GenBank, – , NC_008334 Bausher & al. (2006), NC_008334 Bausher & al. (2006), NC_008334 Bausher & al. (2006); Combretum
molle R. Br. ex G. Don, Bombacaceae, IPEN-xx-0-B-1560480, BG Berlin, FM179938 this study, – , FM179938 this study; Coriaria myrtifolia L., Coriariaceae, T. Borsch 3415 (BONN), BG Bonn, AF542600 Worberg & al. (2007), AM397179 Worberg & al. (2007), AF542600 this study; Cucumis sativus L.,
Cucurbitaceae, EMBL/GenBank, – , DQ119058 Kim & al. (2006), – , DQ119058 Kim & al. (2006); Daphne bholua Buch.-Ham. ex D. Don, Thymeleaeceae,
A. Worberg 026 (BONN), BG Bonn, FM179927 this study, – , FM179927 this study; Daucus carota L., Apiaceae, EMBL/GenBank, – , DQ898156 Ruhlman & al. (2006), – , DQ898156 Ruhlman & al. (2006); Dipentodon sinicus Dunn, Dipentodontaceae, EMBL/GenBank, – , AJ429397 Bremer & al. (2002),
AJ430865 Bremer & al. (2002), – ; Durio zibethinus Murr., Bombacaceae, EMBL/GenBank, – , AY321188 Nyffeler & al. (2005), – , AY321188 Nyffeler &
al. (2005); Erodium cicutarium (L.) L’Hér, Geraniaceae, T. Borsch 3483 (BONN), Germany, Eifel, AM396500 Worberg & al. (2007), AM397178 Worberg
& al. (2007), AM396500 this study; Eucalyptus globulus Labill., Myrtaceae, EMBL/GenBank, – , NC_008115 Steane (2005), NC_008115 Steane (2005),
NC_008115 Steane (2005); Euphorbia milii Desmoul., Euphorbiaceae, A. Worberg 002 (BONN), BG Bonn, FM179936 this study, – , FM179936 this study;
Gerrardina foliosa Oliv., Gerrardinaceae, Balkwill & al. 11983, KEW, FM179924 this study, FM179535 this study, FM179924 this study; Glycine max Merr.,
Fabaceae, EMBL/GenBank, – , DQ317523 Saski & al. (2005), – , DQ317523 Saski & al. (2005); Gossypium hirsutum L., Malvaceae, EMBL/GenBank, – ,
NC_007944 Lee & al. (2006), NC_007944 Lee & al. (2006), NC_007944 Lee & al. (2006); Gunnera tinctoria (Molina) Mirb., Gunneraceae, N. Korotkova 50
(BONN), BG Bonn, AM396506 Worberg & al. (2007), –, AM396506 this study; Huertea cubensis Griseb., Tapisciaceae, R. Oviedo & al. s.n. (B, HAC), Cuba,
FM179926 this study, FM179539 this study, FM179926 this study; Ilex aquifolium L., Aquifoliaceae, T. Borsch 3419 (BONN), BG Bonn, AF542607 Worberg
& al. (2007), – , AF542607 this study; Impatiens noli-tangere L., Balsaminaceae, T. Borsch 3485 (BONN), BG Bonn, AF542608 Worberg & al. (2007), – ,
AF542608 this study; Juglans microcarpa Berland., Juglandaceae, EMBL/GenBank, – , AF118034 Stanford & al. (2000), – , AF118034 Stanford & al. (2000);
Kandelia candel Druce, Rhizophoraceae, EMBL/GenBank, – , AF105090 Shi & al. (1999), – , – ; Larrea tridentata Coult., Zygophyllaceae, A. Worberg 012
(BONN), BG Bonn, AM396502 Worberg & al. (2007), AM397180 Worberg & al. (2007), AM396502 this study; Leea coccinea Planch., Leeaceae, T. Borsch
3418 (BONN), BG Bonn, AM396497 Worberg & al. (2007), – , AM396497 this study; Lobularia maritime L. (Desv.), Brassicaceae, EMBL/GenBank, – ,
AP009375 Hosouchi & al. (2007), AP009375 Hosouchi & al. (2007), AP009375 Hosouchi & al. (2007); Malus trilobata C.K. Schneid., Rosaceae, EMBL/
GenBank, – , DQ860463 Campbell & al. (2006), DQ863235 Campbell & al. (2006), DQ860463 Campbell & al. (2006); Melastoma septemnervium Jacq.,
Melastomataceae, T. Borsch s.n. (BONN), BG Bonn, FM179940 this study, –, FM179940 this study; Melia azedarach L., Meliaceae, A. Worberg 035 (BONN),
BG Bonn, FM179921 this study, FM179536 this study, FM179921 this study; Muntingia calabura L., Muntingiaceae, EMBL/GenBank, –, –, AY328166 Yuan
& al. (2003), – ; Muntingia calabura L., Muntingiaceae, D. Quandt s.n. (BONN), BG Bonn, FM179930 this study, – , FM179930 this study; Myrothamnus
flabellifolia Welw., Myrothamnaceae, A. Worberg 011 (BONN), BG Bonn, AM396507 Worberg & al. (2007), – , AM396507 this study; Myrothamnus moschata Baill., Myrothamnaceae, E. Fischer & W. Höller s.n. (BONN), BG Bonn, AF542591 Worberg & al. (2007), – , AF542591 this study; Oenothera elata
Kunth, Onagraceae, EMBL/GenBank, – , NC_002693 Hupfer & al. (2000), – , NC_002693 Hupfer & al. (2000); Oxalis hedysaroides Kunth, Oxalidaceae, N.
Korotkova 55 (BONN), BG Bonn, FM179935 this study, – , FM179935 this study; Panax ginseng C.A. Mey., Araliaceae, EMBL/GenBank, – , NC_006290
Kim & Lee (2004), – , NC_006290 Kim & Lee (2004); Parnassia palustris L., Parnassiaceae, EMBL/GenBank, – , AY935911 Zhang & Simmons (2006), – , – ;
Passiflora quadrangularis L., Passifloraceae, N. Korotkova 56 (BONN), BG Bonn, FM179937 this study, – , FM179937 this study; Perrottetia ovata Hemsl.,
Dipentodontaceae, EMBL/GenBank, – , AY935916 Zhang & Simmons (2006), AY935771 Zhang & Simmons (2006), – ; Perrottetia longistylis Rose, Dipentodontaceae, EMBL/GenBank, – , AY935915 Zhang & Simmons (2006), AY935770 Zhang & Simmons (2006), – ; Polygala californica Nutt. ex Torr. & A.
Gray, Polygalaceae, EMBL/GenBank, – , AY386842 Wojciechowski & al. (2004), – , – ; Populus alba L., Salicaceae, EMBL/GenBank, – , AP008956 Okumura
& al. (2005), – , AP008956 Okumura & al. (2005); Reseda lutea L., Resedaceae, A. Worberg 027 (BONN), BG Bonn, FM179932 this study, – , FM179932 this
study; Salacia lehmbachii Loes., Celastraceae, T. Borsch 3549 (BONN), BG Bonn, AF542599 this study update, FM179534 this study, AF542599 this study;
Schinus molle L., Anacardiaceae, EMBL/GenBank, – , – , AY640463 Yi & al. (2004), – ; Schinus molle L., Anacardiaceae, A. Worberg s.n. (BONN), BG Bonn,
FM179923 this study, – , FM179923 this study; Stachyurus chinensis Franch., Stachyuraceae, A. Worberg s.n. (BONN), BG Bonn, AM396501 Worberg & al.
(2007), – , AM396501 this study; Stachyurus chinensis Franch., Stachyuraceae, EMBL/GenBank, – , – , AB066335, Ohi & al. (2003), – ; Spiraea thunbergii
Siebold ex Blume, Rosaceae, A. Worberg s.n. (BONN), BG Bonn, FM179934 this study, – , FM179934 this study; Tapiscia sinensis Oliver, Tapisciaceae,
T. Borsch s.n. (BONN), BG Bonn, FM179925 this study, FM179541 this study, FM179925 this study; Tropaeolum majus L., Tropaeolaceae, A. Worberg
s.n. (BONN), BG Bonn, FM179931 this study, – , FM179931 this study; Urtica cannabina L., Urticaceae, A. Worberg s.n. (BONN), BG Bonn, FM179933
this study, – , FM179933 this study; Vasconcellea parviflora A. DC., Caricaceae, A. Worberg 038 (BONN), BG Bonn, FM179928 this study, FM179537 this
study, FM179928 this study; Vitis riparia A. Gray, Vitaceae, T. Borsch 3458 (BONN), BG Bonn, AF542593 Worberg & al. (2007), – , AF542593 this study.
478
TAXON 58 (2) • May 2009: E1
Worberg & al. • Phylogenetic position of Huerteales
Appendix 2. List of primers used in this study.
Primers used for the amplification of matK along with their sequences and the taxa for which they were designed. References are
given for primers that were not designed for this study.
Primer name
MG15
MG1
trnKFbryo
trnK3914Fdi
trnK2R
psbA-R
NYmatK480F
ARmatK660R
ARmatK1200F
ARmatK2400R
ASmatK460F
ACmatK873R
ACmatK100F
ACmatK1400R
ACmatK634R
ACmatK1746F
ACmatK2231R
ACmatK499F
PImatK1060F
PImatK1480F
ROSmatK655R
ROSmatK530F
DIDYmatK1035R
DIDYmatK1107F
DIDYmatK570F
EUPTmatK1006F
NYmatK980R
Sequence
ATC TGG GTT GCT AAC TCA ATG
AAC TAG TCG GAT GGA GTA GAT
GGG TTG CTA ACT CAA TGG TAG AG
GGG GTT GCT AAC TCA ACG G
AAC TAG TCG GAT GGA GTA G
CGC GTC TCT CTA AAA TTG CAG TCA T
CAT CTG GAA ATC TTG STT C
AYG GAT TCG CAT TCA TA
TTC CAA AGT CAA AAG AGC G
ATT TTC TAG CAT TTG ACT CC
TAC TTC CCT TTT T(ACT)G AGG
ATA TAC TCC TGA AAG AGA AGT GG
CTC GAC TGT ATC AAC AGA ATC
GGA TTC GTA TTC ACA TAC A
ATA GGA ACA AGA ATA ATC T
CTT CAA AAG GGA CAT CTC TTC TG
TCT TCC AAA AAT TCT GAA CCT
CCT CKT CTT TGC ATT TAT TAC G
ACT T(AG)T GGT CTC AAC (CT)G
TCG TAA ACA (CT)AA AAG TAC
GGA TTC GTA TTC ACA TAC AT
AGA TGC CTC TTC TTT GC
CGA ACT CGT AAA GAC TCG A
CAA TTA CTC CTC TGA TTG GAT C
CGA GTA TCA GAA CTG GAG
GGC TAT CTT TCA AGT GTA CG
GGT TAG AAT CAT TAG CRG
Taxa
Angiosperms
Angiosperms
Land plants
Angiosperms
Angiosperms
Angiosperms
Angiosperms
Angiosperms
Angiosperms
Angiosperms
Angiosperms
Eudicots
Proteaceae
Aquifoliaceae
Basal eudicots
Aextoxicaceae
Cercidiphyllaceae
Stachyuraceae
Trochodendraceae
Papaveraceae
Rosids
Rosids
Didymelaceae
Didymelaceae
Didymelaceae
Eupteleaceae
Rosids
Reference
Liang & Hilu (1996)
Liang & Hilu (1996)
Wicke & Quandt (in press)
Johnson & Soltis (1995)
Johnson & Soltis (1995)
Steele & Vilgalys (1994)
Borsch (2000)
Wanke & al. (2006a)
Wanke & al. (2006a)
Wanke & al. (2006a)
Wanke & al. (2006a)
Müller & Borsch (2005)
Müller & Borsch (2005)
Müller & Borsch (2005)
Müller & Borsch (2005)
Müller & Borsch (2005)
Müller & Borsch (2005)
Müller & Borsch (2005)
Wanke & al. (2006b)
Wanke & al. (2006b)
Worberg & al. (2007)
Worberg & al. (2007)
Worberg & al. (2007)
Worberg & al. (2007)
Worberg & al. (2007)
Worberg & al. (2007)
Worberg & al. (2007)
Primers used for the amplification of trnK along with their sequences and the taxa for which they were designed. References are
given for primers that were not designed for this study.
Primer name
Sequence
trnK-f bryo
trnK-2R
MG15
psbAR
GGG
AAC
ATC
CGC
TTG
TAG
TGG
GTC
CTA
TCG
GTT
TCT
ACT
GAT
GCT
CTA
CAA
GGA
AAC
AAA
TGG
GTA
TCA
TTG
TAG AG
G
ATG
CAG TCA T
Taxa
Reference
Angiosperms
Angiosperms
Angiosperms
Angiosperms
Wicke & Quandt (in press)
Johnson & Soltis (1995)
Liang & Hilu (1996)
Steele & Vilgalys (1994)
Primers used for the amplification of trnL-F region along with their sequences and the taxa for which they were designed. References
are given for primers that were not designed for this study.
Primer name
Sequence
trnTc
trnTf
trnTd
trnL110R
trnL460F
CGA
ATT
GGG
GAT
GAG
AAT
TGA
GAT
TTG
AAT
CGG
ACT
AGA
GCT
AAA
TAG
GGT
GGG
CAG
GAT
ACG
GAC
ACT
GAT
AGA
CTA
ACG
TGA
TGC
GTC
CG
AG
AC
CC
C
Taxa
Reference
Angiosperms
Angiosperms
Angiosperms
Angiosperms
Angiosperms
Taberlet & al. (1991)
Taberlet & al. (1991)
Taberlet & al. (1991)
Borsch & al. (2003)
Worberg & al. (2007)
E1