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Towards resolving Lamiales relationships:
Insights from rapidly evolving chloroplast
sequences
Article in BMC Evolutionary Biology · November 2010
DOI: 10.1186/1471-2148-10-352 · Source: PubMed
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Schäferhoff et al. BMC Evolutionary Biology 2010, 10:352
http://www.biomedcentral.com/1471-2148/10/352
RESEARCH ARTICLE
Open Access
Towards resolving Lamiales relationships: insights
from rapidly evolving chloroplast sequences
Bastian Schäferhoff1*, Andreas Fleischmann2, Eberhard Fischer3, Dirk C Albach4, Thomas Borsch5,
Günther Heubl2, Kai F Müller1
Abstract
Background: In the large angiosperm order Lamiales, a diverse array of highly specialized life strategies such as
carnivory, parasitism, epiphytism, and desiccation tolerance occur, and some lineages possess drastically accelerated
DNA substitutional rates or miniaturized genomes. However, understanding the evolution of these phenomena in
the order, and clarifying borders of and relationships among lamialean families, has been hindered by largely
unresolved trees in the past.
Results: Our analysis of the rapidly evolving trnK/matK, trnL-F and rps16 chloroplast regions enabled us to infer
more precise phylogenetic hypotheses for the Lamiales. Relationships among the nine first-branching families in
the Lamiales tree are now resolved with very strong support. Subsequent to Plocospermataceae, a clade consisting
of Carlemanniaceae plus Oleaceae branches, followed by Tetrachondraceae and a newly inferred clade composed
of Gesneriaceae plus Calceolariaceae, which is also supported by morphological characters. Plantaginaceae (incl.
Gratioleae) and Scrophulariaceae are well separated in the backbone grade; Lamiaceae and Verbenaceae appear in
distant clades, while the recently described Linderniaceae are confirmed to be monophyletic and in an isolated
position.
Conclusions: Confidence about deep nodes of the Lamiales tree is an important step towards understanding the
evolutionary diversification of a major clade of flowering plants. The degree of resolution obtained here now
provides a first opportunity to discuss the evolution of morphological and biochemical traits in Lamiales. The
multiple independent evolution of the carnivorous syndrome, once in Lentibulariaceae and a second time in
Byblidaceae, is strongly supported by all analyses and topological tests. The evolution of selected morphological
characters such as flower symmetry is discussed. The addition of further sequence data from introns and spacers
holds promise to eventually obtain a fully resolved plastid tree of Lamiales.
Background
With more than 23,000 species in at least 23 families
[1], Lamiales (eudicots/asterids) are one of the largest
orders of flowering plants, with representatives found all
over the world. The highest diversity is contributed by
herbaceous plants with mono-symmetric flowers. Some
members are economically important, such as Lamiaceae (pot-herbs like mint, sage, oregano or basil), Oleaceae (olives), Pedaliaceae (sesame), Verbenaceae (timber,
medicinal) Plantaginaceae (drugs like digitalis, ornamentals) and Scrophulariaceae (ornamentals). The order
* Correspondence: schaeferhoff@uni-muenster.de
1
Institute for Evolution and Biodiversity, University of Muenster, Hüfferstraße
1, 48149 Münster, Germany
Full list of author information is available at the end of the article
contains lineages with highly specialized life forms and
traits of particular scientific interest. So far, their comparative study has been limited by the lack of a robust
phylogenetic framework for Lamiales. Desiccationtolerant members (so-called “resurrection plants”, see
Figure 1a) of the recently described family Linderniaceae
[2] are a focus of molecular and evolutionary studies
[3,2]. Extreme metabolic and genomic shifts are exhibited by parasitic plants. With Orobanchaceae, Lamiales
harbor the largest number of parasitic angiosperms
(Figure 1b). The family comprises both hemi- and holoparasites [4], with some species causing serious damage
in agriculture [5]. Chloroplast genomes of members of
Orobanchaceae show gene order rearrangements, high
evolutionary rates and gene losses, potentially as a
© 2010 Schäferhoff et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Schäferhoff et al. BMC Evolutionary Biology 2010, 10:352
http://www.biomedcentral.com/1471-2148/10/352
Page 2 of 22
Figure 1 Example taxa from Lamiales, showing representatives of desiccation-tolerant, parasitic, and carnivorous lineages, as well as
members from families frequently referred to in the text. a: the desiccation-tolerant Craterostigma pumilum from Linderniaceae; b: the
holoparasitic Orobanche gracilis from Orobanchaceae, a family that contains all hemi- and holoparasites from Lamiales; c: Pinguicula leptoceras from
Lentibulariaceae, the largest family of carnivorous plants in angiosperms; d: Pinguicula filifolia, with a habit resembling Byblis; e: Byblis gigantea from
Byblidaceae, another carnivorous lineage previously suspected to be the closest relative of Lentibulariaceae; f: Rhynchoglossum gardneri from
Gesneriaceae and g Calceolaria andina from Calceolariaceae, two families inferred here as sister groups based on molecular data, alveolated seeds
and pair-flowered cymes; h Prunella grandiflora (Lamiaceae), i: Verbena bonariensis (Verbenaceae); both families were long regarded as close
relatives but are inferred as only distantly related (Figure 2). Photos: a: E.F.; c, d, e: A.F.; f: Nadja Korotkova; g: D.C.A.; b, h, i: K.F.M.
Schäferhoff et al. BMC Evolutionary Biology 2010, 10:352
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consequence of parasitism in this family. One line of
current research in the family concentrates on gradual
plastid evolution under increasingly relaxed functional
constraints [Wicke et al., in prep].
Carnivory in Lamiales
Lentibulariaceae, the most species-rich family of carnivorous plants (ca. 350 spp.) belongs to Lamiales
(Figure 1c, d). This family is unique for a variety of
reasons: traps of Utricularia (bladderworts) are
regarded as a complex modification of leaves [6,7], and
the typical angiosperm body plan is strongly relaxed in
members of this genus [8-10]. Utricularia and its sister
genus, Genlisea (the corkscrew plants), are the only
carnivorous angiosperms known to feed on protozoa
[11]. They have the smallest holoploid genome sizes
among angiosperms, with some nuclear genomes as
small as 63 Mbp or less [12], and exhibit the highest
relative DNA substitution rates for some of the investigated chloroplast genome regions [13,14]. Pinguicula
(butterworts), the third genus of Lentibulariaceae, is
far less extreme in genome size, substitution rate and
morphology, and exhibits glandular leaves that function as adhesive ("flypaper”) traps (Figure 1c, d).
Apart from Lentibulariaceae, the monogeneric Australian family Byblidaceae (Figure 1e) also attracts and
catches insects with simple flypaper traps comparable in
function to those of Pinguicula. The carnivorous syndrome of Byblis was questioned by some authors, as the
plants were considered to lack their own digestive
enzymes and have not been demonstrated to be able to
take up released nutrients, thus being ranked as merely
“protocarnivorous” [15]. However, a recent study [16]
detected phosphatase activity, thereby restoring the rank
of carnivory to Byblis. Morphological links - flypaper
trap leaves that are densely covered with multicellular,
non-vascularized epidermal glands, as well as embryology [17,18] - and early phylogenetic studies suggested a
sister relationship of Byblidaceae and Lentibulariaceae
[19], thus hypothesizing a single origin of carnivory in
the order, which was questioned later [14]. With the
recently described genus Philcoxia [20], a further supposedly “protocarnivorous” lineage emerged and was
placed in Lamiales [21]. Although a first test of enzymatic activity was negative [21], this might have been an
artifact caused by the minuteness of the leaves, and
further experiments to test its status as potentially fully
carnivorous are underway.
Understanding the evolution of the morphological,
ecological, and genomic peculiarities in the order heavily
relies on having robust hypotheses on organismal relationships. For example, knowledge of the closest relatives of resurrection plants, parasites, and carnivores,
respectively, would enable us to infer (pre-) adaptations
Page 3 of 22
and genomic changes on the evolutionary path leading
to each of these specialized groups.
Phylogeny and systematics of Lamiales: current state of
knowledge
While the monophyly of many of the currently accepted
families has been inferred with confidence by a number
of molecular phylogenetic studies [22,23], there has
been only little progress on understanding the relationships among families. Nearly all phylogenetic trees
produced so far lacked resolution and support for interfamiliar relationships of Lamiales [24-26]. This has
earned Lamiales the reputation of being among the
most difficult angiosperm clades to resolve [27].
Circumscription of Lamiales and the inclusion of
Hydrostachys
The current concept of Lamiales [28] expands the earlier order Lamiales from pre-cladistic classification systems [29,30] to also include former Scrophulariales and
Oleales. While there is overwhelming evidence for the
monophyly of Lamiales circumscribed like this [28], the
surprising inclusion of Hydrostachys as an early branch
in Lamiales was recently proposed [31]. Hydrostachys is
a rheophyte from Africa and Madagascar suggested to
be related to Cornales in most previous analyses of
DNA sequence data, albeit without consistent placement
in this order [32-34].
Most studies converged on a set of most likely candidates for the first branches of the Lamiales tree. Oleaceae have been consistently identified as being among
the first branches [2,14,24,35]. Whenever the monotypic
Plocospermataceae from Central America had been
included in the sampling [26,35], they were found to be
sister to the remaining Lamiales. In contrast, the Carlemanniaceae-suspected to have affinities of some kind to
early branching Lamiales - have never been analyzed in
the context of a broad Lamiales sampling. Tetrachondraceae have been resolved as a branch following Oleaceae [36,26].
No clear picture in more derived parts of tree
In contrast, there has not been any consistent hypothesis on the “backbone” of the remainder of the Lamiales
tree [37,31]. Conflicting hypotheses have been put forward with regard to the relationships of Gesneriaceae
and Calceolariaceae (Figure 1f, g) to each other and to
remaining Lamiales. A successive branching order of
Oleaceae, Calceolariaceae, Gesneriaceae, and remaining
Lamiales was originally suggested [38,39], but support
for the placement of Gesneriaceae and for the monophyly of the more derived remaining Lamiales was
always negligible. On the other hand, a clade including
Gesneriaceae and Calceolariaceae was hypothesized
[2,40,41]. Consequently, relationships of Calceolariaceae
remained indistinct, and until now there has been no
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study sampling all families from early branching
Lamiales with a sufficient amount of sequence data to
provide a clear picture.
The situation is even worse for the more derived, remaining lineages of the Lamiales tree - as far as the backbone
and relationship among families is concerned, almost no
resolution could be obtained by previous studies [42,31,43].
The new circumscription of many traditional families
Lamiales are also known for the decomposition of previously widely accepted families due to phylogenetic
insights.
Scrophulariaceae and Plantaginaceae
The most prominent case for a family that turned out to
be polyphyletic are the Scrophulariaceae. In their traditional circumscription they used to be the largest family
(more than 5000 spp. [44]) among Lamiales. In the first
report on the polyphyly of Scrophulariaceae [45], members of the “old” Scrophulariaceae sensu lato were found
in two different clades, named “scroph I” (including
Scrophularia) and “scroph II” (containing Plantago,
Antirrhinum, Digitalis, Veronica, Hippuris and Callitriche). The first clade was later [38] referred to as Scrophulariaceae sensu stricto (s. str.), while the “scroph II”
clade was called Veronicaceae. However, since Plantago
is contained in that clade, Plantaginaceae as the older
name should be given priority and meanwhile became
accepted for this clade [46,28]. Plantaginaceae experienced an enormous inflation since these early studies,
when more and more genera from former Scrophulariaceae s. l. were included in phylogenetic studies and
identified as members of this newly circumscribed
family [22,37-39]. Some genera from tribe Gratioleae,
including Gratiola itself, have been found in a well supported clade. Based on the unknown relationships to the
the other lamialean families, it has been suggested to
separate this part of the inflated Plantaginaceae by
restoring family rank to former tribe Gratioleae from
Scrophulariaceae as traditionally circumscribed [2].
Orobanchaceae
Initial molecular phylogenetic studies [47,48] showed
that all hemi-parasitic members of the former Scrophulariaceae s. l. should be included in a newly circumscribed Orobanchaceae while the non-parasitic genus
Lindenbergia was found sister to all hemi- and holoparasites and also included in Orobanchaceae. In this
expanded circumscription [4,49], the monophyly of Orobanchaceae is strongly supported by all studies, and the
family now comprises 89 genera with about 2000 species
[49] and unites phototrophic, hemi- and holoparasitic
plants. As next relatives to Orobanchaceae, a clade consisting of the East Asian genera Rehmannia (six species)
and Triaenophora (one or two species) was identified
recently [43,50].
Page 4 of 22
Phrymaceae
Shortly after the first reports on the polyphyly of Scrophulariaceae [45], it was noticed that Mimulus (tribe
Mimuleae) neither clustered with the “scroph I” nor the
“scroph II” clade, but instead was found in a group
together with Lamiaceae, Paulownia and Orobanchaceae
[38]. Sampling the taxonomically isolated Phryma (Phrymaceae), but not Mimulus, Phryma appeared as sister to
Orobanchaceae plus Paulownia [26]. In an attempt to
redefine the Phrymaceae, their circumscription was
expanded to include Mimulus, Hemichaena, Berendtiella, Leucocarpus, Glossostigma, Peplidium, Elacholomia, Lancea, and Mazus [51]. However, relationships to
other families of Lamiales remained unclear. Sampling
six genera from Phrymaceae [39], two clades emerged:
one comprising Mimulus, Phryma, Hemichaena and
Berendita, the other including Mazus and Lancea being
sister to Rehmannia. Thus, the monophyly of Phrymaceae was put into question.
Linderniaceae
Linderniaceae were described as a new family independent from Scrophulariaceae, comprising genera formerly
classified in the tribe Lindernieae of Scrophulariaceae
s. l. and are characterized by stamens in which the abaxial filaments are conspicuously geniculate, zigzag shaped
or spurred [2,52,53]. The original recognition as a distinct clade was based upon a taxon set including the
genera Artanema, Craterostigma, Crepidorhopalon, Torenia and Lindernia. The existence of a Linderniaceae
clade was confirmed by other studies comprising Craterostigma, Lindernia, Torenia and Micranthemum [22] or
Stemodiopsis, Micranthemum, Torenia and Picria [39].
Calceolariaceae
Jovellana and Calceolaria (formerly Calceolarieae/Scrophulariaceae) were identified as another lineage separate
from Scrophulariaceae, which led to recognizing them at
family level (Calceolariaceae) [38]. The authors of this
study initially also listed Porodittia as genus of this new
family, but a subsequent study [41] showed Porodittia to
be nested in Calceolaria.
Schlegeliaceae, Paulowniaceae, and Stilbaceae
The genera Paulownia and Schlegelia, which had been
traditionally included either in Bignoniaceae or Scrophulariaceae, were not found to be related to any of these
families based on molecular data [54] and therefore
treated as families of their own [55,56]. In addition, Halleria was transferred from Scrophulariaceae to Stilbaceae
[38]. Molecular phylogenetic studies later expanded the
circumscription of Stilbaceae to a total of 11 genera
[37,39].
Aims of this study
Using a dataset representing all major lineages from
Lamiales, the goal of the present study was to investigate
Schäferhoff et al. BMC Evolutionary Biology 2010, 10:352
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inter-familial relationships within Lamiales, in the hope
to come up with a better resolved tree that provides the
basis for an interpretation of the evolution of the abovementioned morphological, ecological, and molecular
peculiarities observed in the order.
Since the protein-coding genes usually applied to the
inference problem in Lamiales have not provided satisfactory resolution in the past, the approach in the current study was to employ non-coding and rapidly
evolving chloroplast DNA. Introns and spacers have
been demonstrated to be a valuable source of phylogenetic signal even on deeper taxonomic levels than they
used to be applied to [57-59]. Mutational dynamics of
non-coding regions also include microstructural changes
in addition to substitutions, and generally are less constrained than coding genes [60]. Non-coding markers
have been shown to be significantly more informative
than coding regions [57]. Even more, non-coding markers have been successfully applied to disentangle deep
nodes in angiosperm evolution [58].
Methods
Taxon sampling and plant material
Sequences from the plastid markers trnK/matK, trnL-F
and rps16 were newly generated or downloaded from
GenBank for 98 taxa from Lamiales, two outgroup taxa
from Solanaceae, and one from Rubiaceae. All 23
families currently accepted for Lamiales [28] were
sampled. Since one of the specific questions in our
study was the relationship between Lentibulariaceae and
Byblidaceae, which might have been blurred by long
branch attraction (LBA) problems in previous studies,
we slightly enhanced sampling for both families in one
set of analyses and included two to three species for
each genus. The complete material sampled is shown in
Table 1. Using fewer representatives for either family
did not change results. We also used a somewhat denser
taxon sampling for Gratioleae (Plantaginaceae) in order
to (i) examine whether the distinctness of this tribe [2]
can be confirmed after taxan sampling enhancement
and (ii) doublecheck the position of the apparently “protocarnivorous” genus Philcoxia.
Amplification and sequencing
Total genomic DNA was isolated using the AVE Gene
Plant Genomics DNA Mini Kit (AVE Gene, Korea),
according to the manufacturer’s protocol. As phylogenetic markers, the trnK intron including the coding
matK, the trnL-F region, and the rps16 intron were
amplified using standard PCR protocols. Primers used
for amplification and sequencing are given in Table 2.
Reactions were performed in 50 μl volumes containing
2 μl template DNA (10 ng/μl), 10 μl dNTP mix
(1.25 mM each), 2 μl of each forward and reverse
Page 5 of 22
primer (20 pm/μl), and 0.25 μl Taq polymerase (5 U/μl,
Peqlab). Thermal cycling was performed on an Biometra
T3 thermocycler using the following PCR profiles:
1:30 min at 96°C, 1 min at 50°C, 1:30 min at 72°C, 35
cycles of 30 sec at 96°C, 1 min at 50°C, 1:30 min at
72°C, and a final extension time of 10 min at 72°C for
the trnK intron; 35 cycles of 1 min at 94°C, 1 min at
52°C and 2 min at 72°C, followed by a final extension
time of 15 min at 72°C for the trnL-F region; 1:30 min
at 94°C, 30 cycles of 30 sec at 94°C, 30 sec at 56°C and
1 min at 72°C, and a final extension time of 15 min at
72°C for the rps16 intron. Fragments were gel-purified
on a 1.2% agarose gel (Neeo-agarose, Roth), extracted
with the Gel/PCR DNA Fragments Extraction Kit (AVE
Gene, Korea) and sequenced on an ABI3730XL automated sequencer using the Macrogen sequencing service
(Macrogen Inc., Seoul, Korea). Pherogram editing and
contig assembly was done manually.
Addition and analysis of GenBank sequence data
We additionally took rbcL and ndhF sequences (see
Additional file 1, Table S1) for relevant taxa from GenBank, and in a separate set of analyses combined them
with our three marker dataset. Taxon sampling of these
four- and five-region datasets was adapted to include
only taxa with all regions present.
Because the position of Hydrostachys remained inconsistent in previous studies, all sequences from that genus
existing in GenBank were blasted against the entire data
of GenBank via blastn [61]. Additionally, trnK/matK,
rps16 and trnL-F sequences for Hydrostachys from a collection independent from those previously used
[31,33,62,63] were generated; all sequences used, including voucher information, are given in Table 1. The newly
generated Hydrostachys matK sequence was aligned to
an existing angiosperm matK alignment [35] and subjected to parsimony analysis.
Alignment and indel coding
DNA sequences were manually aligned in PhyDE [64],
taking microstructural changes into account as outlined
elsewhere [58,65]. Regions of uncertain homology were
excluded from phylogenetic analyses. For maximum
parsimony (MP) analyses and Bayesian Inference of
Phylogeny (BI), indels were coded according to simple
indel coding (SIC) [66] using the program SeqState [67].
Parsimony analyses
Searches for the shortest tree were performed using the
parsimony ratchet approach implemented in PRAP2
[68] using the following settings: 10 random addition
cycles with 200 ratchet replicates, setting the weight for
25% of the characters to 2. The files generated were executed in PAUP* v4.0b10 [69]. Bootstrapping was
Genus
Family
trnK/matK
trnLF
Acanthus
Acanthaceae
Acanthus longifolius Poir.; [GenBank:AJ429326.1]
Acanthus sennii Chiov.; [GenBank:DQ054856.1]
rps16
Acanthus sennii Chiov.; [GenBank:DQ059148.1]
Anastrabe
Stilbaceae
Anastrabe integerrima E. Mey. Ex Benth.; H. Joffe
171; (M); [EMBL:FN773529]
Anastrabe integerrima E. Mey. Ex Benth.; H. Joffe
171; (M); [EMBL:FN794042 ]
Anastrabe integerrima E. Mey. Ex Benth.; [GenBank:
AJ609216]
Angelonia
Plantaginaceae
Angelonia sp.; Löhne; BG Bonn; [EMBL:FN773530]
Angelonia sp.; Löhne; BG Bonn; [EMBL:FN794043]
Angelonia sp.; Löhne; BG Bonn; [EMBL:FN794079]
Antirrhinum
Plantaginaceae
Antirrhinum majus L.; [GenBank:AF051978]
Antirrhinum majus L.; [GenBank:AY316707]
Antirrhinum majus L.; [GenBank:AJ431054]
Avicennia
Acanthaceae
Avicennia germinans L.; [GenBank:AF531771]
Avicennia germinans L.; [GenBank:AY008819]
Avicennia marina (Forssk.) Vierh.; [GenBank:
AJ431038]
Bacopa monnieri (L.) Pennell; [GenBank:AY492170]
Bacopa monnieri (L.) Pennell; [GenBank:AY492196]
Bacopa
Plantaginaceae
Bacopa monnieri (L.) Pennell; [GenBank:AY667458]
Barthlottia
Scrophulariaceae
Barthlottia madagascariensis Eb.Fisch.; A. Erpenbach Barthlottia madagascariensis Eb.Fisch.; A. Erpenbach Barthlottia madagascariensis Eb.Fisch.; A. Erpenbach
s.n. (BONN); [EMBL:FN773531]
s.n. (BONN); [EMBL:FN794044]
s.n. (BONN); [EMBL:FN794080]
Bryodes
Linderniaceae
Bryodes micrantha Benth.; E. Fischer 10258; (BONN);
[EMBL:FN773532]
Bryodes micrantha Benth.; E. Fischer 10258;
Madagascar; (BONN); [EMBL:FN794045]
Bryodes micrantha Benth.; E. Fischer 10258;
Madagascar; (BONN); [EMBL:FN794081]
Buchnera
Orobanchaceae
Buchnera hispida D. Don; E. Fischer 10230; (BONN);
[EMBL:FN773533]
Buchnera hispida D. Don; E. Fischer 10230; (BONN);
[EMBL:FN79046]
Buchnera hispida D. Don; E. Fischer 10230; (BONN);
[EMBL:FN794082]
Buddleja
Scrophulariaceae
Buddleja alternifolia Maxim.; [GenBank:AF531772]
Buddleja alternifolia Maxim.; [GenBank:AF380857]
Buddleja asiatica Lour.; [GenBank:AJ431058]
Byblis
Byblidaceae
Byblis gigantea Lindl.; [GenBank:AF531774]
Byblis gigantea Lindl.; Kai Müller KM 733; (BONN);
[EMBL:FN794047]
Byblis gigantea Lindl.; Kai Müller KM 733; (BONN);
[EMBL:FN794083]
Byblis
Byblidaceae
Byblis lamellata Conran & Lowrie; Schäferhoff 49;
(BONN); [EMBL:FN773534]
Byblis lamellata Conran & Lowrie; Schäferhoff 49;
(BONN); [EMBL:FN794048]
Byblis lamellata Conrad & Lowrie; Schäferhoff 49;
(BONN); [EMBL:FN794084]
Byblis
Byblidaceae
Byblis liniflora Salisb.; Schäferhoff 44; (BONN); [EMBL: Byblis liniflora Salisb.; Schäferhoff 44; (BONN); [EMBL: Byblis liniflora Salisb.; [GenBank:AJ431070]
FN773535]
FN794049]
Calceolaria
Calceolariaceae
Calceolaria falklandica Kraenzl.; [GenBank:
AY667457.1]
Calceolaria arachnoidea Graham; [GenBank:
AY423126]
Calceolaria mexicana Benth.; [GenBank:AJ609202]
Callicarpa
Lamiaceae
Callicarpa bodinieri H.Lév.; Schäferhoff 57; (BONN)
Callicarpa japonica Thunb.; [GenBank:AJ505536.1]
Callicarpa japonica Thunb.; [GenBank:AJ505413.1]
Campsis
Bignoniaceae
Campsis radicans Seem.; [GenBank:AF531775]
Campsis radicans Seem.; Kai Müller KM701; (BONN);
[EMBL:FN794050]
Campsis radicans Seem.; Kai Müller KM701; (BONN);
[EMBL:FN794085]
Carlemannia
Carlemanniaceae
Carlemannia griffithii Benth.; Grierson, A.J.C. & Long, Carlemannia griffithii Benth.; Grierson, A.J.C. & Long, Carlemannia griffithii Benth.; Grierson, A.J.C. & Long,
D.D. 3027; (K); [EMBL:FN773536]
D.D. 3027; (K); [EMBL:FN794051]
D.D. 3027; (K); [EMBL:FN794086]
Castilleja linariifolia Benth.; [GenBank:EF103866.1]
Castilleja
Orobanchaceae
Castilleja linariifolia Benth.; [GenBank:AF051981.1]
Clerodendrum
Lamiaceae
Clerodendrum thomsoniae Balf.; [GenBank:AY840129] Clerodendrum thomsoniae Balf.; Schäferhoff 39;
(BONN); [EMBL:FN794052]
Clerodendrum thomsoniae Balf.; Schäferhoff 39;
(BONN); [EMBL:FN794087]
Conobea
Plantaginaceae
Conobea multifida (Michx.) Benth.; V. Mühlenbach
278; (M); [EMBL:FN773563]
Conobea multifida (Michx.) Benth.; V. Mühlenbach
278; (M); [EMBL:FN794053]
Conobea multifida (Michx.) Benth.; V. Mühlenbach
278; (M); [EMBL:FN794088]
Craterostigma
Linderniaceae
Craterostigma hirsutum S.Moore; [GenBank:
AF531776]
Craterostigma hirsutum S.Moore; N. Peine 2;
(BONN); [EMBL:FN794054]
Craterostigma hirsutum S.Moore; N. Peine 2;
(BONN); [EMBL:FN794089]
Dermatobotrys
Scrophulariaceae
Dermatobotrys saundersii Bolus; B. Schäferhoff 64
(BONN); [EMBL:FN773537]
Dermatobotrys saundersii Bolus; [GenBank:AJ608596] Dermatobotrys saundersii Bolus; [GenBank:AJ609191]
Castilleja integrifolia L.f.; [GenBank:EF103789.1]
Diascia capsularis Benth.; [GenBank:AJ608595]
Scrophulariaceae
Diascia barbarae Hook.f.; [GenBank:AY667464]
Scrophulariaceae
Diclis ovata Benth.; E. Fischer 10255; (BONN); [EMBL: Diclis ovata Benth.; E. Fischer 10255; (BONN); [EMBL: Diclis reptans Benth.; [GenBank:AJ609188]
FN773538]
FN794055]
Diascia capsularis Benth.; [GenBank:AJ609190]
Dipteracanthus
Acanthaceae
Dipteracanthus portellae (Hook.f.) Boom; [GenBank:
AF531773 ]
Dipteracanthus portellae (Hook.f.) Boom; Kai Müller
KM734; (BONN); [EMBL:FN794090]
Page 6 of 22
Diascia
Diclis
Dipteracanthus portellae (Hook.f.) Boom; Kai Müller
KM734; (BONN); [EMBL:FN794056]
Schäferhoff et al. BMC Evolutionary Biology 2010, 10:352
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Table 1 Taxa, specimens and GenBank acession numbers for sequences used in the present study
Dodartia
Phrymaceae
Dodartia orientalis L.; N. Hölzl M34434; (M); [EMBL:
FN773539]
Dodartia orientalis L.; N. Hölzl M34434; (M); [EMBL:
FN794057]
Dodartia orientalis L.; N. Hölzl M34434; (M); [EMBL:
FN794091]
Elytraria
Acanthaceae
Elytraria imbricata (Vahl) Persoon; J. Calónico S.&A.
Domínguez M. 4883; (M); [EMBL:FN773540]
Elytraria imbricata (Vahl) Persoon; [GenBank:
AF061819.1]
Elytraria imbricata (Vahl) Persoon; P. Döbbeler 4189;
(M); [EMBL:FN794092]
Euphrasia
Orobanchaceae
Euphrasia stricta D. Wolff ex J.F. Lehmann; Borsch
3785; (BONN); [EMBL:FN773541]
Euphrasia stricta D. Wolff ex J.F. Lehmann; Borsch
3785; (BONN); [EMBL:FN794058]
Euphrasia stricta D. Wolff ex J.F. Lehmann; Borsch
3785; (BONN); [EMBL:FN794093]
Forsythia
Oleaceae
Forsythia suspensa Vahl; [GenBank:EU281175.1]
Forsythia suspensa Vahl; [GenBank:EU281157.1]
Forsythia suspensa Vahl; [GenBank:AF225231.1]
Genlisea
Lentibulariaceae
Genlisea aurea A.St.-Hil.; [GenBank:AF531814.1]
Genlisea aurea A.St.-Hil.; [GenBank:AF482614]
Genlisea aurea A.St.-Hil.; [GenBank:AF482540]
Genlisea
Lentibulariaceae
Genlisea hispidula Stapf; [GenBank:AF531815]
Genlisea hispidula Stapf; [GenBank:AF488528.1]
Genlisea hispidula Stapf; [GenBank:AF488523.1]
Globularia
Plantaginaceae
Globularia nudicaulis L.; [GenBank:AY667473]
Globularia trichosantha Fisch. & C.A.Mey.; [GenBank:
AY591321]
Globularia repens Lam.; [GenBank:AY492206]
Gratiola
Plantaginaceae
Gratiola officinalis L.; [GenBank:AF531777]
Gratiola brevifolia Raf.; [GenBank:AY727201 and
AY727237]
Gratiola pilosa Michx.; [GenBank:AJ609182]
Halleria tetragona Baker; [GenBank:AY667476.1]
Harpagophytum grandidieri Baill.; [GenBank:
AF531813]
Harveya alba Hepper; E. Fischer 11547; (BONN);
[EMBL:FN773564]
Hydrotriche hottoniaeflora Zucc.; E. Fischer 10264;
(BONN); [EMBL:FN773542]
Halleria elliptica L.; [GenBank:AJ621108]
Harpagophytum grandidieri Baill.; [GenBank:
AF482610]
Harveya alba Hepper; E. Fischer 11547; (BONN);
[EMBL:FN794078]
Hydrotriche hottoniaeflora Zucc.; E. Fischer 10264;
(BONN); [EMBL:FN794059]
Halleria lucida L.; [GenBank:AJ609181]
Harpagophytum grandidieri Baill.; Kai Müller KM707;
(BONN); [EMBL:FN794094]
Harveya alba Hepper; E. Fischer 11547; (BONN);
[EMBL:FN794095]
Hydrotriche hottoniaeflora Zucc.; E. Fischer 10264;
(BONN); [EMBL:FN794096]
Ibicella lutea v.Eselt; Kai Müller KM735; (BONN);
[EMBL:FN794060]
Ibicella lutea v.Eselt; Kai Müller KM735; (BONN);
[EMBL:FN794097]
Halleria
Stilbaceae
Harpagophytum Pedaliaceae
Harveya
Orobanchaceae
Hydrotriche
Plantaginaceae
Ibicella
Martyniaceae
Ibicella lutea v.Eselt; [GenBank:AF531778]
Jacaranda
Jasminum
Bignoniaceae
Oleaceae
Jacaranda mimosifolia D.Don; [GenBank:AJ429328.1] Jacaranda mimosifolia D.Don; [GenBank:EF105070.1] Jacaranda mimosifolia D.Don; [GenBank:AJ431039.1]
Jasminum nudiflorum Lindl.; [GenBank:AF531779.1] Jasminum nudiflorum Lindl.; [GenBank:AF531779.1] Jasminum nudiflorum Lindl.; [GenBank:AF531779.1]
Jovellana
Calceolariaceae
Jovellana violacea G.Don; [GenBank:AJ580487.1]
Jovellana violacea G.Don; K.H. & W. Rechinger
63014; (M); [EMBL:FN794061]
Jovellana violacea G.Don; K.H. & W. Rechinger
63014; (M); [EMBL:FN794098]
Kigelia
Bignoniaceae
Kigelia africana Benth.; [GenBank:AF051988]
Kigelia africana Benth.; [GenBank:EF105072]
-
Kohleria
Gesneriaceae
Kohleria spicata Oerst.; [GenBank:AJ580486.1]
Kohleria spicata Oerst.; [GenBank:AJ439820.1]
Kohleria ocellata Fritsch in Engl. & Prantl; B.
Schäferhoff 70; (BONN); [EMBL:FN794099]
Lamiaceae
Lamium maculatum L.; [GenBank:AF531780]
Lamium amplexicaule L.; [GenBank:AB266235]
Lamium album L.; [GenBank:AJ431044]
Verbenaceae
Lantana camara L.; [GenBank:AF315303.1]
Lantana camara L.; [GenBank:AF231884.1]
Lantana camara L.; [GenBank:EU348856.1]
Limnophila
Plantaginaceae
Limnophila aromatica (Lam.) Merr.; Schäferhoff 52;
(BONN); [EMBL:FN773543]
Limnophila aromatica (Lam.) Merr.; Schäferhoff 52;
(BONN); [EMBL:FN794062]
Limnophila aromatica (Lam.) Merr.; Schäferhoff 52;
(BONN); [EMBL:FN794100]
Limosella
Scrophulariaceae
Limosella aquatica L.; Kai Müller & Andreas Worberg Limosella aquatica L.; Kai Müller & Andreas
258; (BONN); [EMBL:FN773544]
Worberg258; (BONN); [EMBL:FN794063]
Limosella grandiflora Benth.; [GenBank:AJ609170]
Lindenbergia
Orobanchaceae
Lindenbergia philippinensis Benth.; [GenBank:
AF051990]
Lindenbergia philippinensis Benth.; [GenBank:
AJ608586.1]
Lindenbergia sp.; [GenBank:AJ431049]
Lindernia
Linderniaceae
Lindernia brevidens Skan; E. Fischer 8022; (BONN);
[EMBL:FN773545]
Lindernia brevidens Skan; [GenBank:AY492182]
Lindernia brevidens Skan; [GenBank:AY492213]
Littorella
Plantaginaceae
Littorella uniflora (L.) Asch.; N. Korotkova, K.
Lewejohann & W. Lobin 2; (BONN); [EMBL:
FN773546]
Littorella uniflora (L.) Asch.; N. Korotkova, K.
Lewejohann & W. Lobin 2; (BONN); [EMBL:
FN794064]
Littorella uniflora (L.) Asch.; N. Korotkova, K.
Lewejohann & W. Lobin 2; (BONN); [EMBL:
FN794101]
Page 7 of 22
Lamium
Lantana
Schäferhoff et al. BMC Evolutionary Biology 2010, 10:352
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Table 1 Taxa, specimens and GenBank acession numbers for sequences used in the present study (Continued)
Mazus
Phrymaceae
Mazus rugosus Lour.; E. Fischer 10250; (BONN);
[EMBL:FN773547]
Mazus rugosus Lour.; E. Fischer 10250; (BONN);
[EMBL:FN794065]
Mazus stachydifolius Maxim.; AJ609167
Mecardonia
Plantaginaceae
Mecardonia procumbens Small; [GenBank:
AY492152.1]
Mecardonia procumbens Small; [GenBank:AY492184] Mecardonia procumbens Small; [GenBank:AY492215]
Micranthemum
Linderniaceae
Micranthemum umbrosum (J.F.Gmel.) Blake;
Schäferhoff 43; (BONN); [EMBL:FN773548]
Micranthemum umbrosum (J.F.Gmel.) Blake;
[GenBank:AY492186]
Micrargeria
Orobanchaceae
Micrargeria filiformis (Schum. Thonn.) Hutch. Dalziel; Micrargeria filiformis (Schum. Thonn.) Hutch. Dalziel; Micrargeria filiformis (Schum. Thonn.) Hutch. Dalziel;
E. Fischer 10299; (BONN); [EMBL:FN773549]
E. Fischer 10299; (BONN); [EMBL:FN794066]
E. Fischer 10299; (BONN); [EMBL:FN794102]
Micranthemum umbrosum (J.F.Gmel.) Blake;
[GenBank:AY492217]
Mimulus
Phrymaceae
Mimulus guttatus D.C.; [GenBank:AY667471]
Mimulus micranthus A. Heller; [GenBank:AY575534]
Mimulus aurantiacus Curtis; [GenBank:AJ609163]
Mitraria
Gesneriaceae
Mitraria coccinea Cav.; B. Schäferhoff 65; (BONN);
[EMBL:FN773550]
Mitraria coccinea Cav.; B. Schäferhoff 65; (BONN);
[EMBL:FN794067]
Mitraria coccinea Cav.; B. Schäferhoff 65; (BONN);
[EMBL:FN794103]
Myoporum
Scrophulariaceae
Myoporum montanum R.Br.; [GenBank:AF531808]
Myoporum montanum R.Br.; [GenBank:AJ296513]
Myoporum mauritianum A.DC.; [GenBank:AJ609161]
Ocimum
Lamiaceae
Ocimum basilicum L.; [GenBank:AY177670.1]
Ocimum basilicum L.; [GenBank:AY570462.1]
Ocimum basilicum L.; [GenBank:AJ505351.1]
Oftia
Scrophulariaceae
Oftia africana Bocq. Ex Baill.; Schäferhoff 66.;
(BONN); [EMBL:FN773551]
Oftia africana Bocq. Ex Baill.; Schäferhoff 66.;
(BONN); [EMBL:FN794068]
Oftia africana Bocq. Ex Baill.; [GenBank:AJ609156.1]
Olea
Orobanche
Oleaceae
Orobanchaceae
Olea europaea L.; [GenBank:AM229542.1]
Orobanche hederae Duby; [GenBank:AJ431050]
Otacanthus
Plantaginaceae
Olea europaea L.; [GenBank:AM229542.1]
Olea europaea L.; [GenBank:AM229542.1]
Orobanche caryophyllacea Sm.; [GenBank:AF051992] Orobanche coerulescens Stephan; [GenBank:
AY881137]
Otacanthus coeruleus Lindl.; [GenBank:AY667459]
Otacanthus sp.; [GenBank:AY492188]
Paulownia
Paulowniaceae
Pedicularis
Orobanchaceae
Paulownia tomentosa (Thunb.) Steud.; [GenBank:
AF051997]
Pedicularis sylvatica L.; [GenBank:AF531781]
Paulownia tomentosa (Thunb.) Steud.; [GenBank:
AJ431051]
Pedicularis attollens A. Gray; [GenBank:EF103821]
Petrea
Verbenaceae
Philcoxia
Plantaginaceae
Phryma
Phrymaceae
Phyla
Verbenaceae
Petrea racemosa Nees; Schäferhoff 55; BG Bonn
11113; (BONN); [EMBL:FN773552]
Philcoxia minensis V.C.Souza & Giul.; [GenBank:
EF467901]
Phryma leptostachya L.; [GenBank:AJ429341.1]
Paulownia tomentosa (Thunb.) Steud.; [GenBank:
AY423122]
Pedicularis cheilanthifolia Schrenk; [GenBank:
AY881133]
Petrea racemosa Nees; Schäferhoff 55; BG Bonn
11113; (BONN); [EMBL:FN794069]
Philcoxia minensis V.C.Souza & Giul.; [GenBank:
EF467889.1]
Phryma leptostachya L.; [GenBank:DQ532471.1]
Schäferhoff et al. BMC Evolutionary Biology 2010, 10:352
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Table 1 Taxa, specimens and GenBank acession numbers for sequences used in the present study (Continued)
Otacanthus sp.; [GenBank:AY492219]
Petrea racemosa Nees; Schäferhoff 55; BG Bonn
11113; (BONN); [EMBL:FN794104]
Phryma leptostachya L.; [GenBank:AJ431053.1]
Phyla nodiflora (L.) Greene; Schäferhoff 56; BG Bonn Phyla nodiflora (L.) Greene; Schäferhoff 56; BG Bonn Phyla nodiflora (L.) Greene; Schäferhoff 56; BG Bonn
4146; (BONN); [EMBL:FN773553]
4146; (BONN); [EMBL:794070]
4146; (BONN); [EMBL:FN794105]
Pinguicula agnata Casper; [GenBank:AF531782]
Pinguicula agnata Casper; [GenBank:AF482617]
Pinguicula agnata Casper; [GenBank:AF482543.1]
Pinguicula
Lentibulariaceae
Pinguicula
Lentibulariaceae
Pinguicula alpina L.; [GenBank:AF531783]
Pinguicula alpina L.; [GenBank:AF482618]
Pinguicula alpina L.; [GenBank:AF482544.1]
Pinguicula
Lentibulariaceae
Pinguicula lusitanica L.; [GenBank:DQ010661]
Pinguicula lusitanica L.; [GenBank:AF482625.1]
Pinguicula lusitanica L.; [GenBank:AF482551.1]
Plantago
Plantaginaceae
Plantago media L.; [GenBank:AY667474.1]
Plantago media L.; [GenBank:AY101920]
Plantago argentea Chaix; [GenBank:AJ431056.1]
Plocosperma
Plocospermataceae Plocosperma buxifolium Benth.; [GenBank:AJ429315]
Plocosperma buxifolium Benth.; T.Borsch, H.Flores, S.
Zumaya 377; (BONN); [EMBL:FN794071]
Plocosperma buxifolium Benth.; T.Borsch, H.Flores, S.
Zumaya 377; (BONN); [EMBL:FN794106]
Tetrachondraceae
Polypremum procumbens L.; [GenBank:AJ429351.1]
Polypremum procumbens L.; [GenBank:AJ430938.1]
Polypremum procumbens L.; [GenBank:AJ431063.1]
Martyniaceae
Proboscidea louisiana (Mill.) Thell.; [GenBank:
AF531809]
Proboscidea louisiana (Mill.) Thell.; [GenBank:
AJ608573]
Proboscidea louisiana (Mill.) Thell.; Kai Müller KM706;
BG Bonn 17132; (BONN); [EMBL:FN794107]
Rehmannia
Rehmannia elata N.E.Br.; Hong-Qing Li 2004-0607;
(HSNU); [EMBL:FN773554]
Rehmannia glutinosa Steud.; [GenBank:AY423124]
Rehmannia angulata (Oliv.) Hemsl.; [GenBank:
AJ609145]
Rhynchoglossum Gesneriaceae
Rhynchoglossum gardneri Theobald & Grupe; B.
Schäferhoff 67; (BONN); [EMBL:FN773555]
Rhynchoglossum obliquum Blume; [GenBank:
AY423133.1]
Rhynchoglossum gardneri Theobald & Grupe; B.
Schäferhoff 67; (BONN); [EMBL:FN794108]
Page 8 of 22
Polypremum
Proboscidea
Salvia
Lamiaceae
Salvia coccinea Juss. ex Murr.; [GenBank:AY840147.1] Salvia coccinea Juss. ex Murr.; [GenBank:AY506617.1] Salvia guaranitica A.St.-Hil. ex Benth.; [GenBank:
AJ505421.1]
Schlegelia
Schlegeliaceae
Schlegelia parviflora (Oerst.) Monach.; [GenBank:
AJ429345.1]
Schlegelia parviflora (Oerst.) Monach.; [GenBank:
AJ608570.1]
Schlegelia parviflora (Oerst.) Monach.; [GenBank:
AJ431057.1]
Scoparia
Plantaginaceae
Scoparia dulcis L.; E. Fischer 10254; (BONN); [EMBL:
FN773556]
Scoparia dulcis L.; E. Fischer 10254; (BONN); [EMBL:
FN794072]
Scoparia dulcis L.; E. Fischer 10254; (BONN); [EMBL:
FN794109]
Scrophularia
Scrophulariaceae
Scrophularia chrysantha Jaub. & Spach; B.
Schäferhoff 68; (BONN); [EMBL:FN773557]
Scrophularia canina L.; [GenBank:AY423123]
Scrophularia arguta [Soland.]; [GenBank:AJ431061]
Sesamum
Pedaliaceae
Sesamum indicum L.; [GenBank:AJ429340.1]
Sesamum indicum L.; [GenBank:AF479010.1]
Sesamum indicum L.; [GenBank:AJ609226.1]
Seymeria
Orobanchaceae
Seymeria pectinata Pursch; [GenBank:AF051999.1]
Seymeria laciniata Standl.; [GenBank:EF103898.1]
Seymeria laciniata Standl.; [GenBank:EF103820.1]
Stachytarpheta
Verbenaceae
Stachytarpheta cayennensis (L.C. Rich.) Vahl; E.
Martínez S. 37128; (M); [EMBL:FN773558]
Stachytarpheta cayennensis (L.C. Rich.) Vahl;
[GenBank:AJ608567.1; (M)
Stachytarpheta cayennensis (L.C. Rich.) Vahl;
[GenBank:AJ299259.1; (M)
Stemodia
Plantaginaceae
Stemodia durantifolia Sw.; [GenBank:AY492164.1]
Stemodia glabra Spreng.; [GenBank:AJ608566.1]
Stemodia durantifolia Sw.; [GenBank:AY492225]
Stemodiopsis
Linderniaceae
Stemodiopsis ruandensis Eb.Fisch.; E. Fischer 10352;
(BONN); [EMBL:FN773559]
Stemodiopsis ruandensis Eb.Fisch.; E. Fischer 10352;
(BONN); [EMBL:794073]
Stemodiopsis ruandensis Eb.Fisch.; E. Fischer 10352;
(BONN); [EMBL:FN794110]
Stilbe
Streptocarpus
Stilbaceae
Gesneriaceae
Tetrachondra
Tetrachondraceae
Stilbe ericoides L.; [GenBank:AJ429350.1]
Stilbe ericoides L.; [GenBank:AJ430937.1]
Streptocarpus bindseili Eb.Fisch.; [GenBank:AF531810] Streptocarpus bindseili Eb.Fisch,; E. Fischer 1006;
Ruanda; (KOBL, BR); [EMBL:794074]
Tetrachondra patagonica Skotsb.; [GenBank:
Tetrachondra patagonica Skotsb.; [GenBank:
AJ429352.1]
AJ430939.1]
Stilbe ericoides L.; [GenBank:AJ431062.1]
Streptocarpus bindseili Eb.Fisch,; E. Fischer 1006;
Ruanda; (KOBL, BR); [EMBL:FN794111]
Tetrachondra patagonica Skotsb.; [GenBank:
AJ431064.1]
Tetranema
Plantaginaceae
Tetranema roseum (M.Martens & Galeotti) Standl. &
Steyerm.; [GenBank:AY667475]
Tetranema roseum (M.Martens & Galeotti) Standl. &
Steyerm.; [GenBank:AY492192]
Tetranema roseum (M.Martens & Galeotti) Standl. &
Steyerm.; [GenBank:AY492226.1]
Thomandersia
Thomandersiaceae Thomandersia hensii De Wild. Et T. Durand; D.
Champluvier 5351; (M); [EMBL:FN773560]
Thomandersia hensii De Wild. Et T. Durand; D.
Champluvier 5351; (M); [EMBL:794075]
Thomandersia hensii De Wild. Et T. Durand; D.
Champluvier 5351; (M); [EMBL:FN794112]
Thunbergia
Torenia
Acanthaceae
Linderniaceae
Thunbergia alata Sims; [GenBank:AF061820]
Torenia stolonifera Boj. Ex Benth.; E. Fischer 10257;
(BONN); [EMBL:794076]
Utricularia subulata L.; [GenBank:AF482676]
Thunbergia alata Sims; [GenBank:AJ609131]
Torenia stolonifera Boj. Ex Benth.; E. Fischer 10257;
(BONN); [EMBL:FN794113]
Utricularia subulata L.; [GenBank:AF482599.1]
Thunbergia alata Sims; [GenBank:AF531811]
Torenia stolonifera Boj. Ex Benth.; E. Fischer 10257;
(BONN); [EMBL:FN773561]
Utricularia subulata L.; [GenBank:AF531821]
Utricularia
Lentibulariaceae
Utricularia
Lentibulariaceae
Utricularia multifida R.Br.; [GenBank:AF531848]
Utricularia multifida R.Br.; [GenBank:AF482659]
Utricularia multifida R.Br.; [GenBank:AF482583]
Utricularia
Lentibulariaceae
Utricularia biloba R. Br.; B. Schäferhoff 69; cult. BG
Bonn 19853; (BONN); [EMBL:FN773561]
Utricularia biloba R. Br.; [GenBank:AF482634]
Utricularia biloba R. Br.; [GenBank:AF482561.1]
Verbena
Verbenaceae
Verbena rigida Spreng.; [GenBank:AF531820]
Verbena rigida Spreng.; Kai Müller KM742; BG Bonn
4147; (BONN); [EMBL:794077]
Verbena rigida Spreng.; [GenBank:AJ431065]
Vitex
Lamiaceae
Vitex trifolia L.; [GenBank:AB284175.1]
Vitex trifolia L.; [GenBank:AJ505539.1]
Vitex trifolia L.; [GenBank:AJ505416.1]
Coffea
Rubiaceae
Coffea arabica; [GenBank:EF044213]
[GenBank:EF044213]
[GenBank:EF044213]
Nicotiana
Solanaceae
Nicotiana tabacum; [GenBank:NC001879.2]
[GenBank:NC001879.2]
[GenBank:NC001879.2]
Solanum
Solanaceae
Solanum tuberosum; [GenBank:DQ231562]
[GenBank:DQ231562]
[GenBank:DQ231562]
Schäferhoff et al. BMC Evolutionary Biology 2010, 10:352
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Table 1 Taxa, specimens and GenBank acession numbers for sequences used in the present study (Continued)
outgroup
Page 9 of 22
Key: Voucher information (collector and number, garden accession number if from living collection, herbarium acronym in braces) is provided for sequences newly generated in this study.
Schäferhoff et al. BMC Evolutionary Biology 2010, 10:352
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Table 2 Primers used in the present study
Name
Sequence 5’-3’
Design
trnK3914Fdi
GGGGTTGCTAACTCAACGG
Johnson and Soltis
[120]
LE1R
LE4R
ATAGAAATAGATTCGTTC
TTCGCCTGAAAATCCGTAACC
Müller et al. [13]
Müller et al. [13]
Page 10 of 22
factor approached 1 for all parameters, and that no supported conflicting nodes were found among the consensus trees generated from each run. Convergence and
effective sampling sizes (ESS) of all parameters were
assessed with halp of Tracer v1.5 [73].
LE5R
CAAGGTTCCTTGCRCCAACC
this study
Maximum likelihood analyses
ACmatK500F
TTCTTCTTTGCATTTATTACG
Müller and Borsch
[121]
For maximum likelihood (ML) analyses RAxML v7.0.0
[74] was used. During the search for the best tree, the
GTRGAMMA model was used, while the slightly simpler GTRCAT model was employed by RAxML during
the 500 bootstrap replicates. Support values from all
types of analysis were mapped on the tree topology
from the ML analysis and conflicting nodes were identified with help of TreeGraph2 [75].
LindmatK1714R CTCCAAAGAAAGYCAGTTCCTCTT
this study
LindmatK1580F TCAATTCATTCAACWTTTCCC
this study
LE2F
TGGTACGGAGTCAAAKTC
Müller et al. [13]
trnK2R
AACTAGTCGGATGGAGTAG
Johnson and Soltis
[120]
trntC2
TATGGCGAAATTGGTAGACGC
this study
trntF
ATTTGAACTGGTGACACGAG
Taberlet et al.
[122]
rpsF
GTGTGTAGAAAGCAACGTGCGACTT Oxelman et al.
[123]
rpsR2
TCGGGATCGAACATCAATTGCAAC
Topological tests
performed with 10,000 replicates, each using TBR
branch swapping and holding only one tree [70]. We
measured the additional information provided by SICcoded indels by the difference in decay indices (computed with PRAP2) for each node, comparing analyses
with and without indels.
Topological tests were used to see whether alternative
topologies could be rejected with confidence. Specifically
it was tested whether evidence against Byblidaceae being
sister to Lentibulariaceae was strong. Under parsimony,
the Templeton and Winning-sites (sign) tests were used
("NonparamTest” option in Paup*), while under the likelihood criterion, the Approximately Unbiased test
(AU-Test) [76] along with the more classical Shimodaira-Hasegawa test (SH-test [77]), as implemented in
consel 0.1j [78], were employed.
Bayesian Inference of Phylogeny
Ancestral state reconstruction
Bayesian inference (BI) of phylogeny was done with help
of MrBayes v3.1.2 [71]. The model of best fit for the
combined dataset as well as for each of the three partitions (trnK/matK, rps16 and trnL-F) was found to be
GTR+G+I model was found as the optimal one using
jModelTest v.0.1.1 [72]. The indel partition was coanalyzed together with the DNA partition, with the
restriction site (binary) model applied to the gap characters and the ascertainment (coding) bias set to “variable”. Default priors were used, i.e. flat dirichlets (1.0,
1.0) for state frequencies and instantaneous substitution
rates, a uniform prior (0.0, 50.0) for the shape parameter
of the gamma distribution, a uniform prior (0.0, 1.0) for
the proportion of invariable sites, a uniform topological
prior, an exponential prior Exp (10.0) for branch
lengths. Four categories were used to approximate the
gamma distribution. Two runs with 5 million generations each were run, and four chains were run in parallel for each run, with the temperature set to 0.2. The
chains were sampled every 100th generation, and the
burnin was set to 5000. To check for convergence of the
independent runs under a given model, it was ensured
that the plots of both runs indicated that the stationary
phase was reached, that the potential scale reduction
We inferred ancestral states for ten selected morphological characters. Information on character states was compiled from different sources [79,1,27,80] and is given in
Table 3. We took the fully resolved best tree from the
RAxML search, and traced the evolution of these characters on that topology via maximum likelihood, using the
“multistate” command in BayesTraits [81].
Oxelman et al.
[123]
Results
Sequence statistics and results from tree searches
Sequences of trnK/matK, trnL-F and rps16 yielded an
alignment of 7809 characters, of which 1739 were
excluded from subsequent analysis because of uncertain
homology. The alignment is available from TreeBase
(http://purl.org/phylo/treebase/phylows/study/TB2:
S10963); detailed sequence statistics are given in Table
4. Consensus trees from parsimony analyses were well
resolved and supported. The MP trees from substitutions only were 13118 steps long (CI 0.419, RI 0.504,),
those based on substitution and indel characters had a
length of 14719 steps (CI 0.453, RI 0.507,). Comparison
of decay values of substitution data versus substitutions
plus SIC-coded indels showed higher decay values for
most nodes when indel information was included (see
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Page 11 of 22
Figure 2, collapsing nodes support by less than 50% in
at least one of the tree methodological approaches. BI
and ML trees generally showed slightly higher resolution
and statistical support than trees from MP searches.
Effective sampling sizes (ESS) of all parameters from the
Bayesian analysis were > 150. A phylogram from BI with
branch lengths indicating relative substitution rates is
given in Figure 3.
Table 3 Morphological characters traced in the present
study
Taxon/character
1
2
3
4
5
6
7
8
9
10
Outgroup
0
0
0
?
0
0
?
?
0
0
Plocospermataceae
0/1
0
0
0
0
0
0
0
0
0
Carlemanniaceae
Oleaceae
1
1
0
0
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Tetrachondraceae
1
0
1
0
0
?
0
?
0
0
Calceolariaceae
1
1
2
0
1
1
0
1
0
0
Gesneriaceae
0
1
1
0
1
1
0
1
0
0
Resolution of the backbone of the Lamiales phylogeny
Plantaginaceae
0
1
0/1/2
0
0
1
1
?
0
0
Gratiolaceae
0
1
1
0
0
1
1
0
0
0
Scrophulariaceae
0
1
1
0
0
1
1
0/1
0
0
Byblidaceae
Linderniaceae
0
0
0
1
0
1
0
1
0
0
1
1
1
1
0
0/1
1
0
0
0
Stilbaceae
0
1
1
0
0
1
1
0
0
0
Lamiaceae
0
1
1
0
0
1
1
0
0
0
Mazoideae
0
1
1
0
0
1
1
0
0
0
Phrymoideae
0
1
1
0
0
1
1
0
0
0
Paulowniaceae
0
1
1
0
0
1
1
0
0
0
Rehmannia
0
1
1
0
0
?
1
1
0
0
Orobanchaceae
Thomandersiaceae
0
0
1
1
1
1
0
0
0
0
1
1
1
1
1
0
0
0
1
0
Pedaliaceae
0
1
1
0
0
1
1
0
0
0
The precise branching pattern of the nine first-branching families in the Lamiales tree (Plocospermataceae,
Carlemanniaceae, Oleaceae, Tetrachondraceae, Calceolariaceae, Gesneriaceae, Plantaginaceae (incl. Gratioleae),
Scrophulariaceae) is inferred with very high or maximum (most cases) support (Figure 2). A total of 16
nodes determining this branching pattern among
families along the spine of the basal Lamiales grade
receive very high or maximum support by all (most
cases) or at least two out of three inference methods.
An additional 19 of the nodes indicating delimitation
and relative position of the remaining 15 more derived
families receive very high or maximum support by at
least one out of three analytic approaches.
Bignoniaceae
0
1
1
0
0
1
1
0
0
0
Verbenaceae
0
1
1
0
0
1
1
0
0
0
Phylogenetic position of Hydrostachys
Schlegeliaceae
0
1
1
0
0
1
1
0
0
0
Martyniaceae
0
1
1
0
0
1
1
0
0
0
Acanthaceae
0
1
1
0
0
1
1
0
0
0
Lentibulariaceae
0
1
2
0
0
1
1
0
1
0
In our blastn searches, all sequences (rbcL, atpB, 18s
rDNA, 26s rDNA, ndhF, matK) reached highest similarity scores to other Hydrostachys sequences, followed by
sequences from Cornales taxa (Hydrangeaceae, Cornaceae, Loasaceae), with the exception of the matK
sequence of Hydrostachys multifida (AY254547) of Hufford et al. [82] used in the study of Burleigh et al. [31].
This sequence showed highest similarity with Hydrangea
hirta and a number of sequences from Avicennia. When
included in the present trnK/matK alignment, the high
similarity of sequence AY254547 to Avicennia is
obvious. A blast search of the newly generated matK
sequence of Hydrostachys [EMBL: FN8112689] resulted
in best matches with taxa from Cornales. Aligning and
analyzing the newly generated trnK/matK, rps16 and
trnL-F sequences, Hydrostachys is resolved outside
Key: 1: merosity 0 = pentamerous 1 = tetramerous; 2: symmetry 0 =
polysymmetric 1 = monosymmetric; 3: number of stamens 0 = 5 1 = 4 2 = 2;
4: geniculate stamens 0 = absent 1 = present; 5: pair flowered cymes 0 =
absent 1 = present; 6: Anthraquinones from shicimic acid metabolism 0 =
absent 1 = present; 7: biosynthetic route II decarboxylated iridoids 0 = absent
1 = present; 8: alveolated seeds 0 = absent 1 = present; 9: Carnivory 0 =
absent 1 = present; 10: Parasitism 0 = absent 1 = present.
Additional file 2, Figure S1). Trees from coding rbcL
and ndhF seqences were far less resolved than those
from our three marker combined analysis (Additional
file 3 Figure S2 and Additional file 4, Figure S3).
The tree topology from the ML analysis is shown in
Table 4 Sequence statistics for the rapidly evolving chloroplast markers used
charset
#chars
#chars*
length range
mean
S.D.
%divergence*
S.E.*
%variable*
%informative*
%GC
dataset
trnK/matK
7809
3699
6070
3035
2211-4503
454-2645
3.926.44
2.228.78
482.561
446.491
10.15
10.367
0.187
0.264
51.417
60.362
36.063
43.229
34.212
43.229
trnLF
1997
1577
489-1104
882.881
72.353
9.086
0.402
40.076
28.155
28.155
rps16
2113
1458
0-929
814.772
122.607
10.792
0.464
45.062
29.698
29.698
* calculated based on the alignment with hotspots excluded.
Standard errors calculated based on 100 bootstrap replicates.
Key: Characters = number of characters in the alignment matrix; Length range = actual sequence length in nucleotides (including hotspots; minimal and maximal
value observed); SD = standard deviation of mean length; S.E. = Standard error; % divergence (range) = pairwise sequence distance in percent (uncorrected p
distance, overall mean); % variable = percentage of variable positions; % informative = percentage of parsimony informative positions; % GC = GC content.
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Page 12 of 22
Figure 2 Phylogeny of Lamiales inferred from parsimony, likelihood and Bayesian analysis of sequences from plastid trnK/matK, trnL-F
and rps16. Topology from the maximum likelihood tree depicted, collapsing nodes not supported by > = 50% in at least one of the three
analyses. Bold numbers above branches are posterior probabilities from Bayesian inferences, italic numbers above branches are MP bootstrap
values, number below branches indicate ML bootstrap proportions. Numbers in brackets indicate that the respective node was not supported by
all three methodological approaches. The bracketed number then indicates the strongest support found for any node that contradicts the
shown node [69]. Familial annotation according to APG III [28]. For Phrymaceae monophyly is not confirmed, so subfamilies are annotated;
Rehmannia is currently not assigned to a family.
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Page 13 of 22
Figure 3 Phylogram from Bayesian Inference of phylogeny with branch lengths giving the relative substitution rates using the
GTR+G+I model.
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Lamiales. Parsimony analysis of the newly generated
matK sequence in the context of the angiosperm matK
data set [35] evidently places the newly generated matK
sequence of Hydrostachys outside Lamiales, although its
precise position within asterids remains unresolved in
the 50%-majority-rule-bootstrap tree (Additional file 5,
Figure S4).
Position of carnivorous lineages
In neither the Bayesian nor the maximum likelihood
analysis Byblidaceae were found closely related to Lentibulariaceae. In MP analyses, the position of Byblidaceae
receives no bootstrap support; interestingly, however,
the strict consensus from all shortest trees depicts Byblidaceae as sister to Lentibulariaceae, regardless of the
inclusion of indels. Because of this incongruence, albeit
unsupported, topological tests were employed to further
investigate the position of Byblidaceae. Under a parsimony framework, the Templeton and sign tests find the
ML topology (Byblidaceae not closely related to Lentibulariaceae) not to be significantly less parsimonious than
the shortest tree (Table 5), indicating that even under
parsimony there is no significant evidence against the
ML position of Byblidaceae or for its sister-group relationship to Lentibulariaceae. The AU-Test and SH-Test
indicate that a sister-group relationship of Byblidaceae
and Lentibulariaceae is significantly less likely than the
maximum likelihood and Bayesian consensus topology.
Results from ancestral state reconstruction
Ancestral state reconstruction indicated the probabilities
of the individual character states to be expected along
branches as shown in Figure 4.
Discussion
Lamiales sensu APGIII [28] (including Carlemanniaceae
and Plocospermataceae) receive maximal support in the
present study which is the first to sample taxa from
these two families in a multigene study; a single gene
study [36] did not provide support for the branching
order of the early branching lamialean families.
Page 14 of 22
The phylogenetic position of Hydrostachys
Hydrostachys as a rheophyte with tuber-like rhizomes,
fibrous roots, and no stomata is a morphologically highly
aberrant genus [32], which has always hampered inference of its phylogenetic affinities based on morphology.
Embryological characters such as endosperm development and the apical septum in the ovary [83] might be
interpreted as supporting a placement of Hydrostachys in
Lamiales [31]. The first molecular study, however, placed
it within Cornales [34]. In all previous phylogenetic studies, the genus was found on a long branch, indicating
strongly elevated substitutional rates - a fact that could
have misled previous phylogenetic inferences [33].
Burleigh et al. [31] recently used a 5-gene data matrix
to infer an angiosperm phylogeny, and resolved Hydrostachys as nested in Lamiales, branching right after Oleaceae. Results from our re-sequencing and re-analysis,
along with a blast screening of existing GenBank
sequences, strongly suggest that this placement most
likely was due to an erroneous matK sequence used in
their study. That sequence was first published by Hufford
et al. [82] but is identical to one published earlier by Hufford et al. [62], although citing a different voucher. Interestingly, Burleigh et al. [31] report that the 3-gene matrix
(rbcL, atpB, 18S) places Hydrostachys in Cornales, while
in the 5-gene matrix (additional matK and 26S data),
Hydrostachys is found in Lamiales. The authors suggest
the matK sequence to be the driving force for this result.
Indeed, the most likely incorrect matK sequence misinforms phylogenetic inference, even though only one out
of five genes provides the erroneous signal. If nothing
else, this demonstrates the strong phylogenetic signal and
potential of matK for phylogenetic analyses at the given
phylogenetic depth. Phylogenetic reconstruction using
our newly generated sequences in the context of the
three-marker matrix compiled here and in the context of
the angiosperm matK alignment clearly places Hydrostachys outside Lamiales, which is consistent with earlier
findings [36,84,85] and with the analysis of two unpublished matK sequences by Kita and Kato (AB038179,
AB038180).
Table 5 Results from topology tests
Templeton
Winning-sites
Approxiomately Unbiased
topology
Length
P
P
P
Shimodaira-Hasegawa
P
tree 1
13123
0.2971
0.4049
1.000
0.994
tree 2
13118
5e-004
0.006
Key: Maximum Parsimony: Templeton- and Winning-sites tests. Tree 1: optimal tree from RAxML search (Figure 2), tree 2: optimal tree from MP ratchet search,
where Byblidaceae appear as sister to Lentibulariaceae. P = Approximate probability of getting a more extreme test statistic under the null hypothesis of no
difference between the two trees (two-tailed test). The shortest tree (tree 2) is not significantly different from the ML topology (tree 1, Figure 2). Maximum
Likelihood: Approximatly Unbiased- and Shimodaira-Hasegawa tests. The ML topology (tree 1, Figure 2) is significantly different from and more likely than the MP
alternative (tree 2).
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Page 15 of 22
Figure 4 Evolution of selected morphological characters in Lamiales. ML ancestral state reconstruction on the ML topology (Figure 2)
simplified to represent families by only one OTU and collapsing nodes not supported by > = 50% in at least one of the analyses. Pie charts give
probabilities of character states; white indicates absence in case of binary (presence-absence) characters, while color indicates presence.
Otherwise, colors indicate states as shown in legend.
A robust hypothesis on the basal grade in Lamiales
The Central American Plocospermataceae branch first in
Lamiales (Figure 2), a scenario also found earlier in all
studies that sampled this monotypic family [26,35,36]. A
clade consisting of Carlemanniaceae plus Oleaceae
branches second. A close relationship between these two
families was found weakly supported (64% BS) previously
[36] based on rbcL sequences, and was also observed in a
study dealing with plastome rearrangements in Oleaceae
[35], when Carlemanniaceae appeared sister to Oleaceae
despite being set to as outgroup. We find the sister group
relationship between Carlemanniaceae and Oleaceae with
maximum support.
Tetrachondraceae are recovered with maximum support in all three analyses as third branch in Lamiales.
While this relationship has been observed previously
[36,26], statistical support for it has increased significantly in our study (59% MP BS support in Savolainen
et al. [36] versus PP 1.00, 100% ML BS, 94% MP BS,
support in our tree). The family comprises two genera,
Tetrachondra and Polypremum, both of which were
sampled here. The genus Tetrachondra has a disjunct
distribution (New Zealand/South America) and comprises the two aquatic or semi-aquatic species, while the
monotypic Polypremum is found from southern U.S. to
the northern part of South America.
Relationships within core Lamiales
The core Lamiales (sensu [35], all Lamiales excluding
Carlemanniaceae, Oleaceae, Plocospermataceae, and
Tetrachondraceae; Figure 2) are unambiguously recovered by our analysis. As first branch within this core
group a maximally supported clade composed of Calceolariaceae and Gesneriaceae (Figure 1f, g) is found.
The phylogenetic affinities of both families had
remained unclear so far [45,38,2] but both share the
presence of cornoside and absence of iridoids [86]. Gesneriaceae are a large (ca. 3200 species), predominantly
pantropical family of herbaceous perennials (rarely
woody shrubs and small trees), about one fifth of them
growing as epiphytes [87]. In contrast to many other
lamialean families, molecular phylogenetics confirmed
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their traditional circumscription, as proposed by Bentham in 1876 [88].
Plantaginaceae
Next in the basal grade of core Lamiales is a clade comprising Plantaginaceae as currently defined [28] (PP
1.00, 100% ML BS, 84% MP BS), in which a major split
separates two groups from each other. All former studies focusing on Plantaginaceae relationships found a
major dichotomy within this family [38,22,39,89].
Rahmanzadeh et al. [2] argued that the finding of a well
supported clade including genera from Gratioleae
together with unclear relationships of this group to
other families is handled best with the recognition of a
separate family. Thus, Gratiolaceae were resurrected [2].
Current phylogenies allow both the recognition of two
families, as well as the treatment of Plantaginaceae with
two major subfamilies. Since the taxon sampling is still
far from being complete, and clear morphological characters for either of the groups are lacking, we solely
accept Plantaginaceae throughout this manuscript. Rahmanzadeh et al. [2] tentatively assigned 36 genera to
their Gratiolaceae, 13 of which were included in our
phylogenetic study. Among the genera proposed to be
part of Gratiolaceae, the widespread genus Limosella
was found in Scrophulariaceae [22,39], and the present
analysis confirms placement of Limosella in Scrophulariaceae. Stemodiopsis is found in Linderniaceae, while
Lindenbergia is sister to the remaining Orobanchaceae.
According to Olmstead et al. [38] and Rahmanzadeh et
al. [2], Angelonieae (two genera: Angelonia and Monopera) appears closely related to Gratioleae. Gratioleae
have an integument 3-6 cells across, with large, transversely elongated endothelial cells in vertical rows; this
causes its seeds to have longitudinal ridges. The exotestal cells have hook-like thickenings [1]. Stevens et al. [1]
suggest Angelonieae (integument 5-12 cells across)
should also be included in Gratioleae. However, a denser
taxon sampling will be needed to further test what
belongs in this clade-regardless of the taxonomic level
on which it might be recognized.
Scrophulariaceae
Scrophulariaceae in their new circumscription, including
former Buddlejaceae and Myoporaceae, are the sister to
all other higher core Lamiales (PP 1.00, 100% ML BS,
79% MP BS). This was already indicated by previous
studies [2,39] and is confirmed here with high confidence. A vastly expanded circumscription of Scrophulariaceae that was presented as a possibility in APGIII [28]
would thus mean that all higher core Lamiales would
have to be included in order to respect the principle of
monophyletic families. Such a classification would have
to include a morphologically very heterogeneous assemblage of lineages with more than 17.000 species and
does therefore not appear as very helpful.
Page 16 of 22
Higher core Lamiales (HCL) and the evolution of carnivory
The remaining families Acanthaceae, Bignoniaceae,
Byblidaceae, Lamiaceae, Lentibulariaceae, Linderniaceae,
Orobanchaceae, Paulowniaceae, Pedaliaceae, Phrymaceae, Schlegeliaceae, Stilbaceae, Thomandersiaceae, and
Verbenaceae form a clade strongly supported by BI (PP
1.00) and ML (100% ML BS) analysis, but only moderately supported (76% MP BS) in MP trees (referred to
as “higher core Lamiales”, or HCL clade, in the following). There is no morphological synapomorphy known
for this clade.
A monophyletic origin of carnivory in Lamiales has
been discussed since the introduction of molecular phylogenetics to the field of angiosperm systematics (see
chapter on Lamiales in [90]). In the earliest analyses of
rbcL sequences, the genus Byblis was found sister to
Lentibulariaceae, but this placement gained only weak
statistical support [19]. Later, an analysis of three coding
plus three non-coding chloroplast markers [26] found
Byblidaceae as sister to Lentibulariaceae with 65% jackknife support. This is the highest statistical support ever
reported for this relationship, but only one Byblis species and one Pinguicula species were sampled in that
study.
Based on our data, a close relationship of carnivorous
Byblidaceae and Lentibulariaceae is extremely unlikely.
The placement of Byblidaceae next to Lentibulariaceae,
as found in previous studies and even in single MP tree
topologies of the current study, has been rejected at
highest significance levels by our topological tests and is
contradicted with substantial statistical support by our
ML and BI trees. It might be due to long branch attraction, to which MP is much more susceptible than the
other two approaches [91].
Accordingly, carnivory evolved at least twice within
Lamiales, in congruence with Müller et al. [13]. Our
data still do not provide enough resolution to identify
the immediate sister group of Lentibulariaceae. The
family appears in a weakly supported group together
with Acanthaceae, Thomandersiaceae and Martyniaceae/
Schlegeliaceae and Bignoniaceae, Pedaliaceae and Verbenaceae. An earlier study, sampling only one species
from Lentibulariaceae (Pinguicula), found Elytraria
(Acanthaceae) as sister to Lentibulariaceae [39] with
52% parsimony BS. In contrast, the monophyly of
Acanthaceae, including Elytraria, was strongly supported in a more recent study sampling 85 taxa from
Acanthaceae [92]. In congruence with that, we find Elytraria sister to remaining Acanthaceae.
The lack of resolution in higher core Lamiales still
hampers a clear identification of the precise degree of
relatedness to Martyniaceae, two strongly glandular
members of which (Ibicella and Proboscidea) have been
reported to attract and catch numerous arthropods, and
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thus have been classified as “protocarnivorous”. Recent
tests for protease activity of glands of the two respective
genera were negative [93]; however, putatively mutualistic arthropods have been reported to be associated with
each genus [94], from which the plant might benefit
in a manner similar to the symbiosis observed in the
African Roridula (Roridulaceae, Ericales) [93].
Next relatives to the supposedly carnivorous or “protocarnivorous” genus Philcoxia are found in Gratioleae,
as previously suggested [21]. Without any doubt, Gratioleae have no close connection to Lentibulariaceae,
despite some morphological similarity. Should further
tests identify Philcoxia as a truly carnivorous plant, this
would be the third independent origin of the syndrome
within the order.
Further insights into the family circumscriptions in higher
core Lamiales
Linderniaceae
The exact position of Linderniaceae within higher core
Lamiales remains unclear. It is found unresolved in tritomy together with Byblidaceae and a clade including
Acanthaceae, Bignoniaceae, Lamiaceae, Lentibulariaceae,
Martyniaceae, Orobanchaceae, Paulowniaceae, Pedaliaceae, Phrymaceae, Schlegeliaceae, Stilbaceae, Thomandersiaceae, and Verbenaceae. Only the maximum
likelihood tree depicts Linderniaceae and Byblidaceae
forming a poorly supported clade. The centers of diversity of this family are in Southeast Asia and tropical
Africa. Among them, desiccation tolerant plants like
Craterostigma are found.
Stilbaceae and remaining families
Within the remaining families, the African Stilbaceae
branch first; this scenario gains convincing support from
Bayesian Inference (PP 0.93), weak support from ML
bootstrapping (62% ML BS), and lacks parsimony bootstrap support. Molecular phylogenetic studies had
expanded the traditional circumscription of Stilbaceae
[38,39,95,96] to 11 genera (3 of which we sampled here)
with a predominantly South African distribution. Only
Nuxia extends to tropical Africa and the Arabian
Peninsula.
One of two major clades in this assembly comprises
Lamiaceae, Phrymaceae, Paulowniaceae, Rehmannia, and
Orobanchaceae. Although this clade also was recovered
previously [39], this is the first time it receives support
from BI and ML. Within that group, Lamiaceae are sister to the remaining taxa, supported by 50% ML BS
(our study), and PP 0.92 and 58% MP BS value [39]. We
find subfamily Mazoideae of Phrymaceae sister to a
clade including Paulownia, Phrymaceae subfamily Phrymoideae, Rehmannia and Orobanchaceae. Herein,
Rehmannia is weakly linked to Orobanchaceae, while
the relationship between Paulownia and Phrymoideae
Page 17 of 22
remains unresolved. Previous studies dealing with the
next relatives of Orobanchaceae found either Paulownia
[38], or Phryma and Paulownia together, but as unresolved tritomy [26], or Mimulus and Paulownia as successive sisters to Orobanchaceae [2] but did not include
Rehmannia and/or Triaenophora.
With regard to Orobanchaceae relationships, the most
extensive sampling in terms of both taxa and character
number are that of Xia et al. [43] and Albach et al. [50].
The authors found Rehmannia and Triaenophora
together as sister clade to Orobanchaceae, which should
either be included in Orobanchaceae, as suggested by
Albach et al. [50], or be recognized as a new family. As a
morphological synapomorphy, Orobanchaceae, Rehmannia and Triaenophora share alveolated seeds [43].
Although a well resolved phylogeny of Orobanchaceae
exists, it still remains to be tested using plastid sequence
data whether the non-parasitic Lindenbergia alone is sister to the remaining Orobanchaceae, or if Lindenbergia
plus the hemiparasitic genera Siphonostegia, Schwalbea,
Monochasma, Cymbaria and Bungea are in the respective
position [49].
Including taxa from both subfamilies of Phrymaceae in
a context of putative relatives, no evidence for the
monophyly of Phrymaceae was found [37,39]. Only
Beardsley and Olmstead [51] found weak support for a
monophyletic Phrymaceae, but this result is probably
due to the specific sampling used. In that study [51],
chloroplast data alone did not support this clade, while
nuclear data and the combined analysis did so. The
incongruence might be caused by a plastid-nuclear genome incongruity, which must be confirmed by additional
data.
The two subfamilies of Phrymaceae, Phrymoideae and
Mazoideae, do not form a clade in any of the trees in
Xia et al. [43] or Albach et al. [50], and the branching
order of Mazoideae, Phrymoideae and Paulownia is
inconsistent in different analyses of these studies.
Hence, the authors abstain from assigning these groups
to families. In the light of our data we suggest to segregate Mazoideae from Phrymaceae and elevate it to
family rank.
The position of Lamiaceae distinct from Verbenaceae
(Figure 2) is an important and noteworthy finding. It
ends a century-old discussion on close relationships of a
Lamiaceae-Verbenaceae complex [88,97,98]. Molecular
phylogenetic analysis rather concluded that Lamiaceae
may not be monophyletic with respect to Verbenaceae
[99]. However, analyses of rbcL [100,99] were not conclusive about their relationships and even a combined
matK/trnK analysis [2] did not provide sufficient support for Lamiaceae and Verbenaceae.
The families Acanthaceae, Bignoniaceae, Lentibulariaceae, Martyniaceae, Pedaliaceae, Schlegeliaceae,
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Thomandersiaceae, and Verbenaceae form a clade in our
Bayesian and ML analyses (PP 1.00, ML BS 48%). For all
families for which more than one taxon was sampled,
monophyly is confirmed, but there is only little resolution of intra-familial relationships in that clade, especially in MP trees. In the work of Oxelman et al. [39], a
corresponding clade was found, including the families
mentioned above, except Pedaliaceae. We find weak
support for Schlegeliaceae to be sister to Martyniaceae,
while Oxelman et al. [39] found Martyniaceae, Verbenaceae and Schlegeliaceae in a clade (PP 0.82). Wortley et
al. [42] found Thomandersia weakly linked to Schlegeliaceae, however, our data do not exhibit evidence for
support such a relationship. A close examination of the
floral anatomy of Thomandersia [101] could not
improve the knowledge on its relationships.
Implications for the evolution of floral symmetry and
other characters
Within Lamiales, both polysymmetric and monosymmetric (zygomorphic) flowers occur. Next to the typical
pentamerous flowers, some groups exhibit tetramerous
morphology. With the most highly resolved phylogeny
of Lamiales to date, the evolution of floral symmetry
and flower merosity within the order can be studied in
more detail than previously possible. Assuming the
ancestral asterid flower to be pentamerous and polysymmetric, Plocospermataceae as the most basal family of
Lamiales, share this plesiomorphic character state
(Figure 4). Regarding the evolution of tetramery, there
are two possible scenarios. In the first, tetramery evolved
once after the branching of Plocospermataceae in
Lamiales, with two reversals to pentamery in both Gesneriaceae and then independently in all Lamiales
branching after the Calceolariaceae/Gesneriaceae clade,
this possibility is the one which is favoured by our ML
ancestral state reconstruction. In the second scenario,
tetramery evolved three times independently in (i) Oleaceae/Carlemanniaceae clade, (ii) Tetrachondraceae, and
(iii) Calceolariaceae. Both options require three changes
in flower merosity, and thus are equally parsimonious.
However, there are details in floral development that
differ among the tetramerous families. In Oleaceae,
sepals are initiated in orthogonal positions, and petals
are in diagonal position, whereas in Tetrachondraceae,
sepals are initiated in diagonal, and petals in orthogonal
position [102]. Initiation in Calceolariaceae follows that
in Oleaceae; data for Carlemanniaceae are missing.
Because tetramery in the early branching lineages of
Lamiales is different for each group on more detailed
level, independent gains seem more likely than a general
shift towards tetramery and two independent reversals
to pentamery. Tetramerous flowers are also found in the
more derived Gratioleae, Veroniceae and Plantagineae
Page 18 of 22
(Plantaginaceae). Based on mixed evidence for fusion
and loss of flower parts in these groups, multiple origins
of tetramery within Plantaginaceae have been assumed.
For the Plantaginaceae, Bello et al. [103] hypothesize
two shifts from pentamery to tetramery: (i) in
Amphianthus, which has recently been shown to be
nested in Gratiola [89], and (ii) in a clade consisting of
Aragoa, Plantago and Veronica. An independent shift to
tetramery has been suggested by Albach et al. [104]
based on loss of a sepal in Veroniceae and fusion in
Plantago and Aragoa. But in these taxa the upper lip is
composed out of two petals. Evidence for this is vascularization with two midribs, teratologic, pentamerous
flowers, and an evolutionary row from pentamerous to
tetramerous flowers within this tribe [98,82]. The evolution of flower symmetry can be easily reconstructed.
Lamiales descended from a polysymmetric ancestor, and
early branching lineages in Lamiales share this character
state. After branching of Tetrachondraceae, the ancestor
of the following taxa once acquired monosymmetric
flowers, accompanied by a reduction from five stamens
to four stamens plus one staminode. There are multiple
transitions back to actinomorphic flowers in Lamiales,
e.g. in the case of Plantago (Plantaginaceae) [103,105],
in some taxa in Lamiaceae, Scrophulariaceae, Gesneriaceae, and in all Byblidaceae. The corolla of Byblidaceae
is treated here as actinomorphic, although the curved
stamens introduce a slight element of zygomorphy.
Further morphological characters
Several morphological or biochemical characters lend
further support to some of our hypothesized phylogenetic relationships in Lamiales. Carlemanniaceae and
Oleaceae share the characteristic of having only two stamens, while the first-branching Plocospermataceae have
five stamens, and the lineages branching later in the
evolution of Lamiales generally have four stamens. The
sister-group relationship between Calceolariaceae and
Gesneriaceae is further confirmed by two morphological
characters shared by these families (see Figure 4): (i) the
thyrsic inflorescence with pair flowered cymes, and (ii)
aulacospermous alveolated seeds [102]. Aulacospermous
seeds are otherwise only found in Linderniaceae (Crepidorhopalon, Hartliella). However, an aberrant type of
aulacospermous seeds is found in some genera of Scrophulariaceae s.str.. Here not all cells of the endothelium
protrude into the endosperm and the ontogeny is different from Calceolariaceae, Gesneriaceae and Linderniaceae [44,106]. With regard to chemical compounds,
Plocospermataceae, Oleaceae and Carlemanniaceae have
no anthraquinones from the shikimic acid metabolism,
Tetrachondraceae have not been examined for the
occurrence of these compounds, and all other lineages
in Lamiales possess them. Consequently, these anthraquinones have evolved immediately before or
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immediately after branching of Tetrachondraceae.
Group II decarboxylated iridoids most likely evolved
once after the branching of Calceolariaceae + Gesneriaceae, since they are shared by all taxa branching after
this clade [1]. The close relationship between Rehmannia and Orobanchaceae is supported by the shared
occurrence of alveolated seeds.
Divergence ages in Lamiales
There have been several attempts to estimate Asterid
divergence ages, using fossil calibration points outside
Lamiales. By means of the earliest relaxed clock dating
method NPRS [107], Wikström et al. [108] provided
estimates for Lamiales stem group (sga) and crown
group ages (cga) of 74 mya and 64 mya, respectively.
Using a more sophisticated approach (PL, [107]), the
later results of Bremer at al. [109] and Janssens et al.
[110] were quite congruent, estimating the stem group
age at 106 and 104 mya, and the crown group age at 97
and 95 mya, respectively. The recent study of Magallon
and Castillo [111] presents a diversification hypothesis
for all angiosperms derived from constraining minimal
ages of 49 nodes with fossil data. This setup resulted in
a sga of 80 mya and a cga of 63 mya for Lamiales,
maybe because of the strongly reduced taxon sampling
among Lamiales compared to Bremer et al. [109].
Furthermore, the highest diversification rates among
angiosperms were found in Lamiales [112]. This rapid
radiation could be a reason for the difficulty in untangling the relationships in Lamiales, as previously
supposed [2]. The very short branches among the representatives of Higher Core Lamiales (see Figure 3) are
putatively indicative of a rapid radiation. So far, reliable
relaxed-clock estimates for the age of major Lamiales
lineages have been lacking for two reasons, one of
which is the scantiness of useful fossil calibration points.
Only few fossils, sometimes with questionable assignment [113], are known from Lamiales. They include a
mummified Byblis seed (middle Eocene[114]), a fruit
from Bignoniaceae (middle Eocene, [115]), Justicia-like
pollen (Neogene, [116]), and vegetative parts from Hippuris (Hippuridaceae), Fraxinus (Oleaceae), and Chilopsis (Bignoniaceae) from Oligocene [117]. The second
reason for the absence of dating attempts in Lamiales
has been the uncertainty with respect to the phylogenetic position of the families within Lamiales. We
believe that our study represents good progress with
regard to this second problem. Nevertheless, we refrain
from trying to obtain divergence age estimated based on
our data at this point, because (i) the sparseness of reliable and useful fossil calibration points would force us
to either use an insufficient number of calibration points
or use calibration points that themselves are molecularclock based estimates with a substantial error margin,
Page 19 of 22
and (ii) because the remaining uncertainties in the
branching order within Lamiales would translate into
inferring clade ages with unsatisfyingly wide confidence
intervals.
Conclusions
Utility of chloroplast markers for Lamiales phylogenetics
Phylogenetic analysis of combined trnK/matK, trnL-F
and rps16 intron sequences enhanced both resolution
and statistical support compared to previous studies.
Addition of the more slowly evolving protein coding
rbcL and ndhF genes to our three-marker dataset did
not increase resolution and support values of trees to
the slightest degree (Additional file 6, Figure S5), and
analyses of each of the coding markers alone yield
highly unresolved topologies.
Despite the step forward reported here, more data
need to be compiled to clarify the affinities within the
derived Lamiales, especially for finding the next relatives
of carnivorous lineages and a better understanding of
the path to carnivory in the order. A recent simulation
study argued for accumulating many more characters
from slow evolving markers, and recommends 10,00020,000 characters for Lamiales [40]. Apart from the
much greater effort required by this strategy, the simulation approach taken by the authors does not allow a
rejection of the utility of non coding markers. This is
because the distribution of rates and homoplasy at individual sites, which seems to be a very important factor
determining phylogenetic utility [57], was not taken into
account by the authors. Moreover, simulations were
exclusively based on substitutional patterns derived
from functionally highly constrained ndhF and rbcL data
sets with a scarce taxon sampling and a very rough estimation of phylogeny by neighbor-joining. A currently
popular approach in large scale angiosperm phylogenetics takes this idea one step further and uses concatenated coding sequences extracted from complete cp
genome sequences (e.g. [118]).
However, regardless of the markers and number of
characters used, it has emerged as highly crucial to
maintain a high taxon sampling density while accumulating more characters [40,112,119]. Although the cost
for complete cp genome sequences have dropped dramatically in the past years, in particular when only protein coding regions are targeted and no assembly is
aimed at, the cost/benefit ratio so far has prevented
researchers from taking this avenue for resolving the
Lamiales phylogeny. For such an approach, it is currently unclear whether an appropriate number of taxa
could be upheld while keeping costs at a reasonable
level, and whether the information content in even a
large number of slowly evolving protein coding genes
would significantly exceed that in just a few more
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quickly evolving cp genome regions. In view of the substantial progress made here with this kind of marker,
adding further data from non-protein coding chloroplast
regions seems a promising strategy that, alone or in
combination with phylogenomic approaches, might
finally provide us with a clear picture of Lamiales
evolution.
Additional material
Additional file 1: Table S1: Taxa, specimens and GenBank acession
numbers for sequences used in the 5 gene analysis. Voucher
information.
Additional file 2: Figure S1: A comparison of decay values. Numbers
above branches give decay values from nucleotide data matrix; numbers
below branches that from nucleotides plus coded indels.
Additional file 3: Figure S2: Tree from rbcL analysis. Strict consensus
of 100 MP bootstrap replicates performed.
Additional file 4: Figure S3: Tree from ndhF analysis. Strict consensus
of 100 MP bootstrap replicates performed.
Additional file 5: Figure S4: Tree from angiosperm-wide matK
analysis of the Hilu et al. 2003 dataset plus our newly generated
Hydrostachys sequence. Strict consensus of 100 MP bootstrap replicates
performed.
Additional file 6: Figure S5: Tree from combined trnK/matK, trnL-F,
rps16, rbcL, ndhF analysis. 100 bootstrap replicates performed.
Acknowledgements
This study was funded by DFG grant “Carnivory in Lamiales: understanding
character evolution, substitution rate plasticity, and genome miniaturization”,
MU2875/2, to K.F.M. Additional funding was obtained from the DFG project
“Mutational dynamics of non-coding genomic regions and their potential for
reconstructing evolutionary relationships in eudicots” (BO1815/2-1 and/-2;
QU153/2-1 and/2-2) to T.B. and Dietmar Quandt. Thanks to Nadja Korotkova
for a photograph used in Figure 1. The authors would like to thank the staff
of the Bonn Botanical Gardens for cultivating plants analyzed in the present
study, and the curators of the respective herbaria (BONN, M) for providing
material for DNA extraction. We also want to thank Richard Olmstead and
two anonymous reviewers for very helpful comments that helped to
improve the manuscript.
Author details
1
Institute for Evolution and Biodiversity, University of Muenster, Hüfferstraße
1, 48149 Münster, Germany. 2Department Biology, Systematic Botany and
Mycology, Ludwig-Maximilians-Universität München, Menzinger Straße 67, D80638 Munich, Germany. 3Institut für Integrierte Naturwissenschaften Biologie, Universität Koblenz-Landau, Universitätsstraße 1, 56070 Koblenz,
Germany. 4Institut für Biologie und Umweltwissenschaften (IBU), Carl von
Ossietzky Universität Oldenburg, Carl von Ossietzky-Str. 9-11, 26111
Oldenburg, Germany. 5Botanischer Garten und Botanisches Museum BerlinDahlem and Institute for Biology, Dahlem Center of Plant Sciences (DCPS),
Freie Universität Berlin, Königin Luise-Straße 6-8, 14195 Berlin, Germany.
Authors’ contributions
B.S. generated data and drafted the manuscript. K.F.M. was responsible for
the conception of the study and helped writing the manuscript. D.C.A.
provided data and improved the manuscript. A.F. and T.B. provided plant
material. T.B. contributed during manuscript preparation. A.F., E.F. and G.H.
improved the manuscript. T.B., E.F., and D.C.A. contributed to the conception
of the study during its initial phase, G.H. in its final phase. A.F. contributed
during manuscript preparation. All authors have given final approval of the
version to be published.
Page 20 of 22
Received: 25 May 2010 Accepted: 12 November 2010
Published: 12 November 2010
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doi:10.1186/1471-2148-10-352
Cite this article as: Schäferhoff et al.: Towards resolving Lamiales
relationships: insights from rapidly evolving chloroplast sequences. BMC
Evolutionary Biology 2010 10:352.