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1269
Scheunert & al. • Phylogeny of Rhinantheae
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61 (6) • December 2012: 1269–1285
1269Version of Record (identical to print version).
IntroductIon
The cosmopolitan angiosperm family Orobanchaceae
(broomrape family) is a morphologically diverse group of al-
most exclusively parasitic plants, which form a well-supported
monophyletic lineage in the Eudicot order Lamiales (APG III,
2009). Except for the non-parasitic East Asian genus Linden-
bergia Lehm., which is sister to all remaining genera (Young
& al., 1999), members of the family are either holoparasites
lacking chlorophyll, or green, photosynthetic hemiparasitic
plants (either obligate hemiparasites, which means they require
a host plant for successful growth, or facultative hemiparasites,
which are able to complete their life cycle independent of a
host). Orobanchaceae form a monophyletic group (e.g., Young
& al., 1999; Olmstead & al., 2001; Wolfe & al., 2005; Bennett
& Mathews, 2006); hence it can be assumed that parasitism
evolved only once in this lineage (dePamphilis & al., 1997;
Nickrent & al., 1998; Young & al., 1999).
Circumscription of Rhinantheae. —
Rhinantheae were
traditionally recognized as comprising hemiparasitic plants of
former Scrophulariaceae s.l. (i.e., subfamily Rhinanthoideae
sensu Wettstein, 1891), based on Bentham (1846, 1876). After
the disintegration of Scrophulariaceae based on molecular data
(Young & al., 1999; Olmstead & al., 2001; Oxelman & al., 2005),
the parasitic members of this family were transferred to Oro-
banchaceae s.l. (Young & al., 1999; Wolfe & al., 2005; Bennett
& Mathews, 2006; Tank & al., 2006; APG III, 2009). These taxa
had previously been placed in the two large tribes Rhinantheae
Benth. and Gerardieae Benth. (= Buchnereae Benth.) while the
third tribe, Digitaleae Benth., completing the Rhinanthoideae
according to Wettstein’s treatment (1891), consists of non-
parasitic plants and was recently transferred to Plantaginaceae
(Olmstead & al., 2001). Members of the two parasitic tribes
were distinguished based on a different pattern of the imbri-
cate ascending corolla aestivation. This so-called “rhinanthoid
aestivation” is a synapomorphy of all Orobanch aceae, however
with some variation concerning the arrangement of the corolla
lobes in bud: in flowers of Buchnereae, the central lobe of the
three lobes of the lower corolla lip is folding over the two lateral
ones, whereas in all Rhinantheae, the two lateral lobes clasp
the median one (Thieret, 1967; Armstrong & Douglas, 1989).
The morphology-based assignment of the hemiparasitic taxa to
Phylogeny of tribe Rhinantheae (Orobanchaceae) with a focus on
biogeography, cytology and re-examination of generic concepts
Agnes Scheunert,1 Andreas Fleischmann,1 Catalina Olano-Marín,1 Christian Bräuchler1,2 & Günther Heubl1
1 Systematic Botany and Mycology, Ludwig-Maximilians-University (LMU), Menzinger Strasse 67, 80638 Munich, Germany
2 Botanische Staatssammlung München, Menzinger Strasse 67, 80638 Munich, Germany
Author for correspondence: Agnes Scheunert, agnes.scheunert@lrz.uni-muenchen.de
Abstract
A molecular systematic approach using DNA sequences of two non-coding chloroplast loci (trnK, rps16) and the
nuclear ITS region was applied to reconstruct phylogenetic relationships within the tribe Rhinantheae (Orobanchaceae). This
tribe includes approximately 19 genera of hemiparasitic plants predominantly occurring in the Old World. An exception is the
genus Bartsia which, accordi ng to previous taxonomic treatments, includes a rema rk able radiation (ca. 45 spec ies) in the Andes,
two species distributed in Afromontane regions, and only one species (Bartsia alpina) ranging from the alpine mountains of
northern and central Europe to northeastern North America. The present phylogenetic study includes the most comprehensive
taxon sampling of Rhinantheae to date, with main focus on the relationships of the Mediterranean genera. Both nuclear and
plastid datasets reveal a core group of Rhinantheae comprising four major lineages. Our analyses suggest that (1) the northern
temperate Bartsia alpina is sister to the rest of the core group; (2) African Bartsia are more closely related to the monotypic
African genus Hedbergia t han to other congener ic taxa; (3) Sout h America n Bartsia are nested within a high ly sup porte d clade
including Parentucellia and Bellardia; (4) Nothobartsia and Odontitella are likely to be the results of at least one intergeneric
hybridization event. Despite topological conflicts regarding some taxa, the polyphyly of Bartsia and a broadly circumscribed
Odontites are unambiguously supported by our results. Our tree topologies indicate that the importance of certain morpho-
logical characters traditionally used for generic delimitation (such as shape and indumentum of corolla, anthers, and capsules)
has been overestimated, and that some of these characters are presumably convergent. Available information on chromosome
numbers corroborates the results presented here.
Keywords
hemiparasitic plants; nrITS; Orobanchaceae; Rhinantheae; rps16; trnK
Supplementary Material
The alignment is available in the Supplementary Data section of the online version of this article
(http://www.ingentaconnect.com/content/iapt/tax).
This publication is dedicated to Dr. Markus Bolliger on the occasion of his 60th birthday.
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these two tribes has not changed since the first proposal of this
taxonomic concept by Wettstein (1891); however it is not fully
supported by molecular data (Young & al., 1999; Wolfe & al.
2005; Bennett & Mathews, 2006; Tank & al., 2006). None of the
tribes is monophyletic, and in phylogenetic reconstructions sev-
eral members of each tribe are part of the respective other, rather
following biogeographic patterns than the classical taxonomic
concept (Young & al., 1999; Bennett & Mathews, 2006; Tank
& al., 2006). Genera such as Pedicularis L., Castilleja Mutis ex
L. f. (both previously Rhinantheae), and Agalinis Raf. (previ-
ously Buchnereae) are now placed in a common clade, which is
referred to as tribe Pedicularideae Duby (Bennett & Mathews,
2006; Tank & al., 2009). However, as no modern phylogeny-
based taxonomic concept for all taxa of Rhinantheae has been
proposed yet, the traditional system of Wettstein (1891) is fol-
lowed here, with the exception of Pedicularis as a member of
Pedicularideae following Tank & al. (2009).
Morphological characters, generic concepts, and distri-
bution of Rhinantheae. —
Apart from petal aestivation and
parasitic habit, Rhinantheae share no other generative or veg-
etative synapomorphy (Fischer, 2004). The plants are annual
or perennial herbs, sometimes even small subshrubs with a
woody base, and have racemose inflorescences in which the
flowers are subtended by scale-like, leaf-like, or showy bracts.
The corolla is bilabiate and consists of five connate petals, two
forming the upper lip and three forming the lower lip. The two
lobes of the upper lip are usually fused into a helmet-like or
rostrate galea in which the anthers are inserted (most notably in
Rhynchocorys Griseb., Odontites Ludw. and Bartsia L. (Fig. 1).
However, bifid or to various degree bilobate lobes can also be
found (e.g., Euphrasia L.), and the lobes of the upper lip are
even free and expanded in Hedbergia Molau, Bornmueller-
antha Rothm. and Toz zia L. (Fischer, 2004). For generic de-
limitation in Rhinantheae, mainly corolla morphology, but also
palynological characters (pollen size, shape, exine ornamenta-
tion) were often used (Rothmaler, 1943; Inceoğlu, 1982; Molau ,
1988; Bolliger & Wick, 1990; Bolliger, 1996; Lu & al., 2007).
Some of the recently segregated genera of the tribe, such
as Macrosyringion Rothm., Odontitella Rothm., Bartsiella Bol-
liger, Bornmuellerantha, and Nothobartsia Bolliger & Molau
have been questioned by taxonomists, but—with the exception
of Nothobartsia (Těšitel & al., 2010)—have not been included
in preceding molecular studies. All have been separated from
Odontites (or Bartsia in case of Nothobartsia) based on rather
minor morphological and palynological characters (Rothmaler,
1943; Bolliger & Molau, 1992; Bolliger, 1996), yet are still
classified as Odontites (viz. Bartsia) in a broader circumscr ip-
tion in several flora treatments (e.g., Webb & Camarasa, 1972;
Davis, 1978; Valdés & al., 1987; Jahn & Schönfelder, 1995;
Mabberley, 2008).
Rhinantheae are of worldwide distribution, but the high-
est generic and species diversity is found in the Northern
Hemisphere. Main centers of species richness are located in
the Mediterranean area (Odontites) and the holarctic region
(Melampyrum L., Rhinanthus L.). Some genera also have their
center of alpha diversity in South America (Bartsia), Asia and
Oceania (Euphrasia; Fischer, 2004; Bennett & Mathews, 2006).
Preceding molecular studies on Rhinantheae. —
Rather
few phylogenetic studies have addressed generic-level relation-
ships within Rhinantheae exclusively; most of the work has
focused on the phylogenetic framework and evolution of holo-
parasitism in Orobanchaceae (dePamphilis & al., 1997; Young
& al., 1999; Olmstead & al., 2001; Schneeweiss & al., 2004;
Wolfe & al., 2005; Bennett & Mathews, 2006; Morawetz & al.,
2010). These studies provide good insight into intergeneric re-
lationships in the family, however, some of the taxonomically
difficult groups of Rhinantheae remained underrepresented.
A recent phylogenetic study on Rhinanthoid Orobanchaceae
(Těšitel & al., 2010) corroborated this group as monophy-
letic and identified certain major lineages within the tribe.
Though based on a larger taxon sampling than any previous
study, several key taxa such as Macrosyringion, Bornmueller-
antha, Odontitella, Bartsiella and the African representatives
of Bartsia were not included, thus leaving import ant quest ions
unanswered.
The present study is based on an extensive taxonomic
sampling of Rhinantheae to comprise taxa absent in previous
studies. Based on an enlarged dataset, our goals are to test
the previously published phylogenetic hypotheses in a more
comprehensive context, particularly investigating the impact of
narrowly endemic and poorly studied genera (Bartsiella, Born-
muellerantha, Macrosyringion, Nothobartsia, Odontitella) on
phylogenetic reconstruction, and to infer phylogenetic relation-
ships within and between taxa of Bartsia and Odontites to test
existing taxonomic concepts.
MaterIals and Methods
Plant material. —
The t
axon sampling follows the treat-
ment of Fischer (2004), who recognized Orobanchaceae within
Scrophulariaceae, and covers a representative number of spe-
cies from 16 of the 20 genera included in tribe Rhinantheae.
For the ingroup (tribe Rhinantheae), a total of 34 accessions
representing 29 species from 16 genera were included in the
analyses. Selection of outgroup taxa was based on the com-
prehensive molecular phylogeny of Orobanch aceae by Ben-
nett & Mathews (2006) and the phylogeny of Rhinanthoid
Orobanchaceae by Těšitel & al. (2010). Striga Lour. (tribe
Buchnereae) and Pedicularis (tribe Pedicula rideae) were cho-
sen as outgroups. Voucher specimen data, including sources
and accession numbers, are provided in the Appendix. For
sequences obtained from NCBI’s GenBank, references to the
place of original publication are given
. Herbarium specimens
used for DNA extraction were identified with the keys provided
by Molau (1990) for Bartsia, and Bolliger (1996) for Odontites
s.l. A single Bartsia voucher representing sterile specimens
from Peru used in the present study (“Bartsia sp. Peru”) could
not be fully determined to species level due to the lack of flow-
ers. Nevertheless, it was included in the study so as to increase
the number of South American Bartsia species.
DNA extraction and amplification. —
Total genom ic DNA
was extracted either from fresh leaf material (three taxa) or
from herbarium specimens (30 taxa) using the NucleoSpin Plant
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Fig. 1.
Selected species of representative genera of Rhinantheae (Orobanchaceae).
A,
Melampyrum nemorosum L.;
B,
Parentucellia latifolia ( L.)
Caruel;
C,
Bartsia alpina L.;
D,
Bellardia trixago (L.) A ll.;
E,
Rhinanthus alectorolophus (Scop.) Pollich;
F,
Euphrasia officinalis L.;
G,
Paren-
tucellia viscosa (L.) Caruel;
H,
Odontites vernus Dumort.;
I,
Lathraea squamaria L. — Photographs A, E & F, F. Brambach; B–D, G–H, A.
Fleischmann; I, C. Olano-Marín.
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Kit (Macherey-Nagel, Düren, Germany) following the manu-
facturer’s standard protocol for genomic DNA extraction. An
additional phenol/chloroform extraction step was performed to
remove proteins and potentially interfering secondary metabo-
lites. The DNA was dissolved in 50 μl elution buffer (10 mM
Tris / HCl) and checked for quality on a 1.4% agarose gel. A
standard amount of 2 μl DNA template was used for PCR.
Two non-coding chloroplast regions (part of the trnK re-
gion, comprising the partial matK gene and the 3′-terminal
end of the trnK intron, and the rps16 intron) plus one nuclear
ribosomal region (the internal transcribed spacer region, ITS)
were chosen for phylogenetic analyses. These markers have
been previously used in Rhinantheae and closely related la-
mialean groups (Schäferhoff & al., 2010; Těšitel & al., 2010).
PCRs were performed with total genomic DNA using Taq-
polymerase (Hybaid, AGS, Heidelberg, Germany) and prim-
ers LEU1 (Vargas & al., 1998) and ITS4 (White & al., 1990)
for ITS; trnK-2R (Johnson & Soltis, 1994) and Sat2-1200F
(Bräuchler & al., 2010) for partial trnK; and rps-F and rps-R2
(Oxelman & al., 1997) for rps16.
The cycling profile for ITS and the trnK region consisted
of an initial denaturation step at 94°C (2 min 30 s) followed by
40 cycles of 30 s (ITS) or 1 min (trnK) denaturation at 94°C,
30 s (ITS) or 1 min (trnK) annealing at 53°C and 1 min 15 s
(ITS) or 1 min 30 s (trnK) elongation at 72°C, and a 10 min
final extension step at 72°C.
The PCR amplification
prof ile
used for the rps16
intron consisted of an initial denaturation
step at 94°C (5 min) followed by 40 cycles of 30 s denaturation
at 94°C, 1 min annealing at 50°C and 1 min 30 s elongation at
72°C, and a 7 min final extension step at 72°C. PCR products
were purified using the NucleoSpin Extract II Kit (Macherey-
Nagel) following the manufacturer’s protocol.
Sequencing. —
Direct sequencing, employing the
DYEnamic ET Terminator Cycle Sequencing Kit (Amersham
Biosciences, Freiburg, Germany) followed the manufacturer’s
protocol. Products were purified by Sephadex filtration (G50-
Superfine, Amersham Biosciences) and were run on an ABI
3730 DNA analyzer (Applied Bio Systems, Foster Cit y, Califor-
nia, U.S.A.). All markers were sequenced bidirectionally using
the same primer pairs as for amplification. For the trnK region,
the internal primers Sat2-1780F/Sat16-1780R (Bräuchler & al.,
2010) and Sat16-2150R (Bräuchler & al., 2005) were used in
addition to cover sequence gaps.
Phylogenetic analysis. —
All sequences generated in
the study were assembled and aligned automatically with the
MUSCLE v.3.8.31 softwa re (Edgar, 2004) and adjusted manu-
ally using BioEdit v.7.0.5.1 (Hall, 1999); mononucleotide re-
peats and ambiguously aligned regions were excluded from
further analysis. Before incorporating nuclear and chloroplast
indels in the analyses, their phylogenetic information content
was assessed. This is regarded essential because within the
parasitic lineages of Lamiales (which contain fast-evolving
groups), nuclear indel data in particular are suspected to be
highly homoplasious and thus could
distort the inferred results
(Schäferhoff & al., 2010). Ing roup indels were coded
according
to the
simple indel-coding
method
(Simmons & Ochoterena,
2000), as implemented in SeqState v.1.4.1 (Müller, 2005) and
added to the data as a binary matrix. Including indels into the
combined chloroplast dataset increased support values in all
but one case, while ITS indels improved node support values in
only six cases but weakened them in 14 cases, especially in the
basal part of the tree; an alternative topology was suggested in
one case when using indels, but this received almost no support
(results not shown). As this suggests a perturbingly high level
of homoplasy in the nuclear indel dataset, these were excluded
from further analyses, while chloroplast indels were coded as
single mutation events.
The three markers were analyzed in a combined matrix
as well as in two separate datasets (ITS and chloroplast). All
analyses were conducted using both a Bayesian and maximum
likelihood (ML) approach. Bayesian analyses were performed
with MrBayes v.3.2 for 64bit systems (Ronquist & al., 2012),
applying a GTR + Γ substitution model with four rate catego-
ries to the chloroplast partition and a SYM + Γ substitution
model to the ITS partition, as suggested by MrModelTest v.2.3
(Nylander, 2004) as best fit to the DNA data. The binary indel
data were analyzed separately using the model settings recom-
mended by Ronquist & al. (2009); for chloroplast and combined
analyses, a mixed dataset was defined (one/two DNA parti-
tions, one binary partition), using the best-fit model settings
for each partition. Two Markov chain Monte Carlo (MCMC)
runs with four chains each (one cold, three hot chains with
default temperature t = 0.2) were started from independent
random trees and computed 10 million generations, with trees
sampled every 2000th generation. After discarding a burn-in of
500 trees (1/10 of all sampled trees) from each run, a consensus
tree was calculated.
ML analyses were performed with RAxML v.7.2.8 (Stamat-
ak is & al., 2008) using rax mlGUI v.0.95 (Silvestro & Michalak,
2011). Ten thousand rapid bootstrap replicates were computed
using the GTR + Γ substitution model (GTRGAMMA, replaced
automatically by BINGAMMA for indel characters); these
were subjected to a thorough ML search with Striga asiatica
(L.) Kuntze as outgroup, and without a constraint tree defined.
Each analysis provided one fully resolved best-scoring ML tree.
Assessing incongruence. —
Before combining the nuclear
and chloroplast markers, these were tested for incongruence
following Bull & al. (1993). Whether or not datasets with a po-
tentially different phylogenetic history should be combined has
been the issue of extensive debates (see reviews by Miyamoto
& Fitch, 1995; Queiroz & al., 1995; Huelsenbeck & al., 1996).
In Rhinantheae, several examples of intrageneric reticulate
evolution caused by introgression and hybridization have been
reported, e.g., in Euphrasia, Rhinanthus, Melampyrum, and
Odontites (e.g., Yeo, 1968; Kwak, 1978; Bolliger & al., 1990;
Wesselingh & Van Groenendael, 2005; Liebst, 2008;
Těšitel
& al., 2010
). As the amount of heterogeneity present on the
intergeneric level cannot be assumed to be neglectible, assess-
ment of incongruence prior to any combined analysis must
be considered particularly important in this group. Following
the “conditional combination approach” (Huelsenbeck & al.,
1996; Johnson & Soltis, 1998), taxa displaying considerable
incongruence between nuclear and chloroplast data should be
excluded from a combined dataset.
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We applied several methods to assess levels of data hetero-
geneity: first, phylograms obtained from the chloroplast and
nuclear datasets alone were visually examined and compared
for well-supported discrepancies (“hard incongruence”; Mason-
Gamer & Kellogg, 1996) using a cut-off of 70% ML bootstrap
support. As reticulate relat ionships were to be expected and sup-
port values might be artificially lowered in fast-evolving groups
due to raised levels of homoplasy (especially in the ITS dataset,
as pointed out, e.g., by Albach & Chase, 2004), we employed
an additional, more liberal threshold of 85% Bayesian support,
to reliably identify all cases of incongruence. The respective
taxa were then further analyzed using both a statistical and a
network approach. The incongruence length difference (ILD)
test, implemented in PAUP* v.4.0b10 (Swofford, 2003) as parti-
tion homogeneity test, was performed with 1000 replicates and
the MAXTREES option set to 100. The chloroplast markers
were tested against each other, and the combined chloroplast
dataset against ITS, always including outgroups. Previously
identified taxa showing hard incongruence were then succes-
sively excluded, the ILD tests were repeated and the results
compared to those of the complete dataset. Employing again
a conservative approach, we decided to assess the degree of
incongr uence introduced by a single taxon based on the increase
of the ILD P-value when excluding that taxon. In addition, a
split network was constructed in order to visualize contradict-
ing signals contained in the Bayesian chloroplast and ITS trees.
This was done with SplitsTree v.4.12.3 (Huson & Bryant, 2006)
and by using the trees from the first of two runs each of the
chloroplast and ITS analysis (discarding 1/10 burn-in trees). A
consensus network (CN) applying a threshold of 0.25 (which
presents branches appearing with a frequency of 25% or higher
of all trees obtained) was generated using mean edge weights;
splits were transformed with the equal angle transformation
method followed by a convex hull optimization (Dress & Huson,
2004), using weights, and no filter for the resulting splits. From
this network, one or more taxa were then removed using the
“exclude selected taxa” option of SplitsTree, and the resulting
CNs were compared. Finally, only taxa showing hard incongru-
ence in their placements as well as significant results in the ILD
test were excluded from the combined analysis, to avoid loss of
valuable information due to false positives.
res u lt s
Sequencing and alignment. —
A total of 98 sequences
were generated for this study, 33 each for the trnK region and
the rps16 intron, and 32 for ITS. As the sequence pherograms
for the ITS marker region provided a clear, unambiguous
signal without any signs of polymorphisms, no cloning was
performed. In a few taxa where sequencing failed, sequences
were obtained from GenBank (http://www.ncbi.nlm.nih.gov)
to complete the taxon sampling: these include Melampyrum
nemorosum L. (ITS), Euphrasia stricta J.P. Wolf f ex J.F. Lehm.
(trnK, r ps16, ITS), Pedicularis sylvatica L. / P. attollens A. Gray
(trnK, rps16, ITS) and Striga asiatica (trnK, ITS). For Striga,
no rps16 intron sequence was available in GenBank; however,
as the outgroups were intended to be in accordance with those
in
Bennett & Mathews (2006) and Těšitel & al. (2010), the
genus was nevertheless used, and rps16 was
coded as missing
data for the combined analysis. For the same reason, we used
GenBank sequences for Pedicularis originating from two spe-
cies (P. attollens for rps16 and ITS and P. sylvatica for trnK,
as this marker sequence was not available for P. attollens).
The
combined DNA data matrix of trnK, rp s16, and ITS contained
2800 aligned characters. The average sequence length was
1032 basepairs (bp) for trnK, 784 bp for rps16, and 689 bp
for ITS. Detailed information on alignment statistics for all
markers including the proportions of parsimony-informative
characters is provided in Table 1.
Table 1.
Alignment characteristics and statistics for ITS, trnK region, rps16 intron, combined chloroplast dataset, and combined dataset. Number
of constant characters, parsimony-informative characters and % parsimony-informative characters refers to non-excluded characters; number of
excluded characters includes peripheral regions of the alignment not suitable for analysis, mononucleotide repeats and regions which could not be
aligned unambiguously; proportion of unknown characters calculated without peripheral regions of the alignment.
ITS trnK rps16 Comb. Chloroplast Combined
Number of taxa 36 36 34 36 30
Sequence length
(average)
537–737 bp
(689 bp)
813–1082 bp
(1032 bp)
701–848 bp
(784 bp)
1044–1898 bp
(1772 bp)
1588–2594 bp
(2461 bp)
Aligned length 767 bp 1147 bp 886 bp 2033 bp 2800 bp
Excluded characters 197 bp 108 bp 153 bp 261 bp 458 bp
Constant characters 275 bp 708 bp 561 bp 1269 bp 1544 bp
Parsimony-informative characters 191 bp 133 bp 74 bp 207 bp 398 bp
% parsimony-informative characters 33.51% 12.80% 10.10% 11.68% 16.99%
Unknown characters within alignment
(average)
0–14.63%
(0.96%)
0–22.81%
(1.05%)
0–6.76%
(0.84%)
0–44.20%
(3.40%)
0–35.65%
(2.79%)
Average G + C content 55.24% 34.24% 34.54% 34.39% 40.27%
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Chloroplast and ITS analyses. —
The combined chloro-
plast dataset—including coded indels—showed a standard de-
viation of split frequencies of 0.002 after the Bayes runs. ML
optimization resulted in a final likelihood of −7324.158667,
the length of the best tree found was 0.571629, and the alpha
parameters were estimated at 0.754251 for the DNA data parti-
tion, and at 2.602716 for the binary indel data partition. The ITS
dataset, at the end of the Bayes analysis, also had a standard de-
viation of split frequencies of 0.002. ML optimization resulted
in a final likelihood of −4653.464574, with a best tree length
of 2.041682, and an alpha parameter of 0.459579. Bayesian
and ML analyses resulted in highly similar topologies; there-
fore the ML bootstrap support values (BS) were plotted onto
the respective Bayesian 80% consensus tree. Nodes from the
ML tree not supported by Bayesian analysis were added to the
consensus tree only if their support equalled or exceeded 75%.
Individual phylogenetic reconstructions from the combined
chloroplast (trnK, rps16 ) and the single nuclear marker (ITS)
are shown in Figs. 2 and 3.
The topology of the ITS tree is largely similar to that of
the combined chloroplast tree. ML bootstrap support values
are considerably lower than Bayesian posterior probabilities
Striga asiatica
Pedicularis spp.
Melampyrum nemorosum
Rhynchocorys orientalis
Rhynchocorys kurdica
1.00
Rhinanthus alectorolophus
Lathraea squamaria
1.00
1.00
Bartsia alpina Germany
Bartsia alpina Italy
Bartsia alpina Finland
Bartsia alpina Canada
Euphrasia stricta
Tozzia alpina
Odontitella virgata
Nothobartsia asperrima
Nothobartsia spicata
Hedbergia abyssinica
Bartsia decurva
Bartsia longiflora
93
85
1.00
100
99
100
100
1.00
1.00
1.00 100
100
71
1.00
1.00
100
1.00
88
100
1.00
100
1.00
99
Rhinantheae
Core group of
Rhinantheae
Bellardia trixago
Parentucellia viscosa
Parentucellia latifolia
Bartsia mutica
Bartsia sp. Peru
1.00
Bartsia canescens
1.00
1.00
1.00
Macrosyringion glutinosum
Bornmuellerantha aucheri
Bornmuellerantha alshehbaziana
1.00
Odontites corsicus
Odontites himalayicus
1.00
1.00
Odontites bocconei
Odontites cyprius
1.00
Odontites viscosus
Bartsiella rameauana
Odontites pyrenaeus
1.00
1.00
96
98
77
98
96
100
100
100
100
93
0.78
81
x
x
Bartsia longiflora subsp. macrophylla
0.88
80
1.00
100
1.00
99
OG I. Bartsia s.str. II. Hedbergia IV. Odontites s.l. III. BellardiaRRL clade
Fig. 2.
Baye sian consensus tre e (cladogram) fr om the combined chloroplast dataset (rps16 intron and trnK region). Poste rior probabil ities (PP) are
given above each node, maximum likelihood (ML) bootstrap support values (BS) for the corresponding node are indicated below. Only nodes
equalling or exceeding either 80% in Bayesian or 75% in ML analysis are shown. Branches sufficiently supported by ML only are represented by
dashed lines (corresponding PP values are given, no support in Bayesian inference is denoted by an “x”). PP values obtained from 9002 trees, BS
values obtained from a best-scoring ML tree from 10,000 bootstrap replicates with subsequent maximum likelihood optimization (not shown).
Divergence of the “Rhinantheae” and “core group of Rhinantheae” (as defined in the text) is marked by arrows. Taxa which were excluded in the
combined analysis are highlighted in bold. OG, outgroup; RRL clade, Rhynchocorys-Rhinanthus-Lathraea clade.
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(PP). Melampyrum is revealed as highly supported sister to
an equally supported clade which comprises all remaining in-
group taxa in both analyses; however, support for these two
nodes is weak in the nuclear ML tree (BS: 66 and 64, respec-
tively). A clade comprising Rhynchocorys, Rhinanthus and
Lathraea L. (referred to as the “RRL” clade) receives high
support in the chloroplast topology (PP: 1.00, BS: 99), but is
only moderately to weakly supported in the nuclear tree (PP:
0.81, BS: 50). The remaining genera of the ingroup constitute
the “core group of Rhinantheae”, a clade with high support in
almost all analyses (chloroplast PP: 1.00, BS: 100; ITS PP: 1.00,
BS: 68). Within this group, four main clades can be identified:
“Bartsia s.str.” (clade I), “Hedbergia” (clade II), “Bellardia”
(clade III) and “Odontites s.l.” (clade IV), see Figs. 2 and 3. In
both datasets, clade I is sister to a clade containing clades II–IV
in addition to Euphrasia stricta and Tozzia alpina L. as well
Striga asiatica
Pedicularis spp.
Melampyrum nemorosum
Rhynchocorys orientalis
Rhynchocorys kurdica
Lathraea squamaria
Rhinanthus alectorolophus
Bartsia alpina Germany
Bartsia alpina Italy
Bartsia alpina Finland
Bartsia alpina Canada
Euphrasia stricta
Tozzia alpina
Odontites cyprius
Bartsiella rameauana
Odontites bocconei
1.00
1.00
0.84
43
74
68
core group of
Rhinantheae
1.00
0.98
64
66
Rhinantheae
96
1.00
0.95
74
0.81
50
Hedbergia abyssinica
1.00
Bartsia decurva
Bartsia longiflora
Bartsia longiflora subsp. macrophylla
Odontitella virgata
Nothobartsia asperrima
Nothobartsia spicata
Odontites corsicus
Odontites himalayicus
0.92
97
1.00
100
99
Macrosyringion glutinosum
Bornmuellerantha aucheri
Bornmuellerantha alshehbaziana
0.97
0.91
61
87
Bellardia trixago
Parentucellia viscosa
Parentucellia latifolia
Bartsia mutica
Bartsia sp. Peru
0.93
Bartsia canescens
1.00
71
94
1.00
95
Odontites pyrenaeus
Odontites viscosus
1.00
96
0.89
68
0.83
35
1.00
100
1.00
95
1.00
83
0.85
40
0.99
62
0.99
83
1.00
100 0.62
90
OG I. Bartsia s.str. II. Hedbergia IV. Odontites s.l.III. BellardiaRRL clade
Fig. 3.
Bayesian consensus tree (cladogram) from the nuclear internal transcribed spacer region (ITS). Posterior probabilities (PP) are given
above each node, maximum likelihood (ML) bootstrap support values (BS) for the corresponding node are indicated below. Only nodes equalling
or exceeding either 80% in Bayesian or 75% in ML analysis are shown. Branches sufficiently supported by ML only are represented by dashed
lines. PP values obtained from 9002 trees, BS values obtained from a best-scoring ML tree from 10,000 bootstrap replicates with subsequent
maximum likelihood optimization (not shown). Divergence of the “Rhinantheae” and “core group of Rhinantheae” (as defined in the text) is
marked by arrows. Taxa which were excluded in the combined analysis are highlighted in bold. OG, outgroup; RRL clade, Rhynchocorys-
Rhinanthus-Lathraea clade.
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as a group of Odontitella virgata (Link) Rothm., Nothobartsia
asperrima (Link) Benedí & Herrero and N. spicata (Ramond)
Bolliger & Molau. In the chloroplast tree, Euphrasia is highly
supported as sister to the remaining taxa, which in turn are part
of an unresolved polytomy; the latter consists of clade II plus a
clade comprising Nothobartsia and Odontitella; clades III–IV;
and Tozz ia. In the ITS tree, the position of Euphrasia remains
unresolved. Information about the relationships among clades
II–IV is more easily obtained from the combined analyses of
all three markers (see below), and detailed information on each
clade is also given there.
While the monophyly of clades II, III and IV is highly
supported in most reconstructions, several taxa within the
clades show contradicting positions in the chloroplast and
nuclear phylogenies (hard incongruence as defined above):
Macro syringion glutinosum (M. Bieb.) Rothm. is moderately
supported as sister to the remaining taxa of clade IV in the
chloroplast tree (PP: 0.78, BS: 81), while it is deeply nested
within the clade in the ITS tree, forming a clade with the two
species of Bornmuellerantha (PP: 0.91, BS: 61). Bartsia sp.
Peru is sister to B. mutica (Kunth) Benth. in the chloroplast
analysis (PP: 1.00, BS: 98), but sister to B. canescens Wedd .
in the ITS analysis (PP: 0.93, BS: 71). Bartsia alpina L. from
Italy is sister to the accessions from Finland and Canada in the
chloroplast tree (PP: –, BS: 93), but groups with the specimen
from Germany in the ITS tree (PP: 0.95, BS: 74). However, the
most obvious contradictory placement concerns Nothobartsia,
the two species of which are revealed as monophyletic with
maximum support. Nothobartsia is part of a highly supported
clade with Odontitella virgata in both analyses, but this clade
is sister to the Hedbergia clade in the chloroplast analysis (PP:
1.00, BS: 100), while it is sister to the Odontites s.l. clade in the
ITS analysis with moderate support (PP: 0.85, BS: 40).
Incongruence tests and phylogenetic reconstruction. —
The ILD test showed the two chloroplast datasets (trnK, rps16)
to be congruent for all taxa (P = 0.442), so these data were
concatenated in all analyses and analyzed as one combined
chloroplast dataset. In contrast, when testing the ITS marker
against the combined chloroplast matrix, the test displayed
significant heterogeneity (P = 0.013, with values < 0.05 consid-
ered significant following Farris & al., 1995 and Cunningham,
1997), implying conflicting signal within the data.
Sequential exclusion of the taxa showing hard incon-
gruence as defined above (the group of Nothobartsia asper-
rima / N. picata / Odontitella; Macrosyringion; Bartsia sp.
Peru; and B. alpina [Italy]) resulted in a noticeable increase
in P-values in five cases (P + 0.064 without Nothobartsia and
Odontitella, P + 0.065 when excluding Macrosyringion, and
P + 0.122 when additionally removing Bartsia sp. Peru, com-
pared to P = 0.013 when including all taxa). Excluding Bartsia
alpina did not improve congruence in the ILD test (decrease
in P by 0.003). Consequently, Bartsia alpina was maintained
in the sampling for the combined analysis of all markers, while
the other five taxa were excluded.
This is in accordance with the consensus network (CN)
constructed from 9002 Bayesian chloroplast and nuclear trees.
The CN shows tree-like as well as network-like relationships,
illustrated by sets of parallel edges (branches) which indicate
differing signals within the data (Fig. 4A). Almost no reticula-
tions are present in the Hedbergia and Bellardia clades (with
few exceptions in the latter, due to the variable positions of
Bartsia sp. Peru and Bellardia trixago), while relationships
in the Odontites s.l. clade are revealed to be complex. This is
largely attributable to Macrosyringion, while the conflicts in
the placement of the Nothobartsia / Odontitella group result in
the highly network-like central part. When the five taxa show-
ing hard incongruence are removed from the network, it takes
on a much more tree-like structure (Fig. 4B), greatly simplify-
ing from 78 splits and 18 sets of parallel edges to 63 splits and
8 sets of parallel edges. Confidence values for each remaining
set of parallel edges are given at the respective branches in
Fig. 4B. The ILD test showed this reduced dataset to be con-
gruent (P = 0.264) and thus suitable for the combined analyses.
Combined analysis. —
A combined analysis was con-
ducted using all three markers and a reduced set of 29 ingroup
taxa. The Bayesian 80%/ML 75% consensus tree is shown in
Fig. 5. The standard deviation of split frequencies at the end
of the Bayes analysis was 0.002. ML optimization resulted
in a final likelihood of −11241.558677, a best tree length of
0.837353 and an alpha parameter estimated at 0.430033 for the
DNA data partition and at 4.327658 for the binary indel data
partition. As in the chloroplast and nuclear trees, Bayesian and
ML topologies computed from the combined dataset were very
similar, so the ML bootstrap supports could be plotted onto the
Bayesian consensus tree.
Within the combined phylogeny, Melampyrum, here rep-
resented by M. nemorosum, is again revealed as sister to the
rest of the Rhinantheae (PP: 1.00, BS: 99). The RRL clade
comprising Rhynchocorys orientalis Benth. and Rhynchocorys
kurdica Nábělek (PP: 1.00, BS: 100) and a clade of Rhinanthus
alectorolophus (Scop.) Pollich and Lathraea squamaria L. (PP:
1.00, BS: 100) now receives maximum support (PP: 1.00, BS:
100). This RRL clade is sister to the highly supported “core
group of Rhinantheae” (PP: 1.00, BS: 95), which comprises four
clades, each with unambiguous support for their monophyly.
Clade I (“Bartsia s.str.”) is composed of all included acces-
sions of Bartsia alpina, covering the whole geographic range
of the species. This clade is inferred as sister to the remaining
Rhinanthoid taxa (PP: 1.00, BS: 95), in accordance with the
separate chloroplast and nuclear results. Within clade I, there
is varying support for a sister relationship between accessions
of B. alpina from Finland and Canada (PP: 0.61, BS: 95), and
Germany and Italy (PP: 0.96, BS: 77).
Euphrasia is indicated as sister to the remaining clades
plus To zzia alpina of the monotypic Tozzia with high support
(PP: 1.00, BS: 97). As in the single-marker analyses, the latter
genus remains unresolved, reflecting its two, almost equally
probable positions in the CN (Fig. 4B; confidence values 41.6
vs. 39.5).
Clade II (“Hedbergia”) is composed of the monotypic
Hedbergia and the African accessions of Bartsia (B. decurva
Hochst. ex Benth., B. longiflora Hochst. ex Benth. and B. lon-
giflora subsp. macrophylla ( Hedberg) Hedberg), with the latter
two taxa highly supported as sisters (PP: 1.00, BS: 99).
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Fig. .
Split consensus networks for the combined chloroplast and ITS data, using a 25% threshold, obtained from the collections of trees pro-
duced by the separate Bayesian analyses of the combined chloroplast dataset and the ITS dataset, respectively (yielding the consensus trees
shown in Figs. 2 and 3). Trees from the first run of each analysis (discarding a 10% burn-in) were analyzed (9002 trees). Depicted edge lengths
are proportional to mean branch lengths, values attached to edges denote corresponding confidence values. Composition of the consensus net-
work based on
A)
all 36 taxa included in the study, and
B)
with five taxa displaying high levels of incongruence (Macrosyringion glutinosum,
Bartsia sp. Peru, Nothobartsia asperrima, Nothobartsia spicata and Odontitella virgata, shown in bold italics in A), removed using the “exclude
selected taxa” command (see text). The tree from the Bayesian analysis with all markers combined and based on the reduced sampling presented
in B) is shown in Fig. 5. Clades I–IV are shaded grey: I, Bartsia s.str.; II, Hedbergia; III, Bellardia; IV, Odontites s.l. — O., Odontites; B., Born-
muellerantha.
Bartsia alpina Canada
Bartsia alpina Germany
Bartsia alpina Italy
Euphrasia stricta
Tozzia alpina
Bartsia longiflora
Bartsia longiflora subsp. macrophylla
Bartsia
decurva
Hedbergia
abyssinica
Nothobartsia asperrima
Nothobartsia spicata
Odontitella virgata
Macrosyringion
glutinosum
Bornmuellerantha
alshehbaziana
B. aucheri
O. corsicus
O. himalayicus
O. viscosus
Bartsiella rameauana
O. cyprius
Odontites
bocconei Odontites pyrenaeus
Parentucellia latifolia
Bartsia canescens
Bartsia sp. Peru
Bartsia mutica
Parentucellia
viscosa
Bellardia
trixago
Rhinanthus alectorolophus Lathraea squamaria
Rhynchocorys
kurdica Rhynchocorys
orientalis
Melampyrum
nemorosum Pedicularis spp.
Striga asiatica
Bartsia alpina Finland
Striga asiatica
Melampyrum
nemorosum
Pedicularis spp.
Rhinanthus alectorolophus Lathraea squamaria
Rhynchocorys
kurdica Rhynchocorys
orientalis
Euphrasia stricta
Parentucellia
viscosa
Bellardia
trixago
Bartsia canescens
Bartsia mutica
Parentucellia latifolia
Bartsia alpina Canada
Bartsia alpina Germany
Bartsia alpina Italy
Bartsia alpina Finland
Tozzia alpina
Bartsia longiflora
Bartsia longiflora subsp. macrophylla
Bartsia
decurva
Hedbergia
abyssinica
O. cyprius
Odontites bocconei Odontites
pyrenaeus
O. viscosus
O. corsicus
O. himalayicus
Bornmuellerantha
alshehbaziana
B. aucheri
A
B
Clade II
Clade III
Clade IV
Clade I
Parentucellia
viscosa
Bellardia
trixago
Bartsia canescens
Bartsia mutica
Parentucellia latifolia
Clade II
Clade I
Clade III
Clade IV
Bartsiella
rameauana
39.5
41.6
56.4 29.3
29.5
69.6
32.1
53.8
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Fig. 5.
Bayesian consensus tree (cladogram) from the combined dataset (ITS, rps16 intron and trnK region). Posterior probabilities (PP) are given
above each node, maximum likelihood (ML) bootstrap support values (BS) for the corresponding node are indicated below. Only nodes equalling
or exceeding either 80% in Bayesian or 75% in ML analysis are shown. Branches sufficiently supported by ML only are represented by dashed
lines. PP values obtained from 9002 trees, BS values obtained from a best-scoring ML tree from 10,000 bootstrap replicates with subsequent
Maximum Likelihood optimization (not shown). Divergence of the “Rhinantheae” and “core group of Rhinantheae” (as defined in the text) is
marked by arrows. OG, outgroup, RRL clade, Rhynchocorys-Rhinanthus-Lathraea clade.
Striga asiatica
Pedicularis spp.
Melampyrum nemorosum
Rhynchocorys orientalis
Rhynchocorys kurdica
1.00
Rhinanthus alectorolophus
Lathraea squamaria
1.00
1.00
Bartsia alpina Germany
Bartsia alpina Italy
Bartsia alpina Finland
Bartsia alpina Canada
Euphrasia stricta
Tozzia alpina
Hedbergia abyssinica
Bartsia decurva
Bartsia longiflora
1.00
100
100
100
100
1.00
100
1.00
96
Rhinantheae
1.00
1.00
1.00
1.00
97
95
95
99
core group of
Rhinantheae
1.00
99
1.00
100
Odontites corsicus
Odontites himalayicus
Odontites pyrenaeus
Odontites viscosus
Bornmuellerantha aucheri
1.00
100
Bartsia longiflora subsp. macrophylla
1.00
99
1.00
96
Bellardia trixago
Parentucellia viscosa
Parentucellia latifolia
Bartsia canescens
1.00
1.00
100
100
Bartsia mutica
Bornmuellerantha alshehbaziana
1.00
100
Odontites bocconei
Odontites cyprius
95
0.61
1.00
91
1.00
82
0.98
75
1.00
100
0.96
77
Bartsiella rameauana
0.80
50
OG I. Bartsia s.str. II. Hedbergia IV. Odontites s.l.III. BellardiaRRL clade
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The sister relationship between clade III and IV is highly
supported in the Bayesian analysis and receives moderate sup-
port in the ML analysis (PP: 0.98, BS: 75).
Clade III (“Bellardia”) comprises the two included spe-
cies of Neotropical Bartsia, two accessions of Parentucellia
Viv., and Bellardia trixago (L.) All. The monotypic Bellar-
dia All. and Parentucellia viscosa (L.) Caruel are shown in a
polytomy, while P. latifolia (L.) Caruel is sister to the South
American species of Bartsia (PP: 1.00, BS: 100). A close rela-
tionship of Parentucellia and the Neotropical Bartsia species
to Bellardia is strongly indicated in all analyses; furthermore,
Parentucellia is clearly paraphyletic, with the South American
Bartsia species nested within it. Neotropical Bartsia itself (here
represented by B. canescens from Peru and B. mutica from
Argentina) is supported as monophyletic (PP: 1.00, BS: 96).
Clade IV (“Odontites s.l.”) contains six species of Odon-
tites being part of two subclades which receive maximum to
moderate support: one subclade (PP: 1.00, BS: 82) is com-
posed of four Odontites species and the monotypic Bartsiella
(Bartsiella rameauana (Emb.) Bolliger) nested within it, be-
ing sister to O. viscosus (L.) Clairv. with weak support (PP:
0.80, BS: 50). A second group comprises O. bocconei (Guss.)
Walp. and O. cyprius Boiss.; O. pyrenaeus (Bubani) Rothm.
is indicated as sister to all taxa of the subclade. The other sub-
clade (PP: 1.00, BS: 100) consists of the sister species O. cor-
sicus G. Don and O. himalayicus Pennell, and of the monophy-
letic Bornmuellerantha, comprising B. aucheri (Boiss.) Rothm.
and the recently described B. alshehbaziana (Dönmez & Mutlu,
2010). Given the positions of Bornmuellerantha and Bartsiella,
Odontites is rendered paraphyletic in this analysis.
dIscussIon
Biogeography of Rhinantheae. —
Těšitel & al. (2010)
conducted a dispersal-vicariance analysis and identified
temperate western Eurasia as the origin of the Rhinanthoid
Orobanchaceae, in accordance with the Laurasian origin north
of the Tethyan Sea as assumed by Wolfe & al. (2005) and the
Eurasian origin inferred for the cosmopolitan hemiparasitic
Euphrasia by Gussarova & al. (2008). Most genera of the core
group of Rhinantheae sampled here have a distinct center of
diversity in the Mediterranean area. An exception are the fru-
tescent perennial Afromontane species of Bartsia sect. Longi-
florae Molau (Molau, 1990) and the perennial hemiparasitic
monotypic Hedbergia from East Africa, which form a highly
supported group in all analyses (Figs. 2, 3 & 5). Molau (1988,
1990) considered Hedbergia to represent an ancestral li neage of
Rhinantheae, based on its distinctive corolla morphology. How-
ever, the contrary is evident from our molecular phylogenetic
reconstructions, as Hedbergia is clearly revealed as member
of core Rhinantheae, closely associated with the Afromontane
species of Bartsia. Regarding their disjunct distribution pat-
tern, the core Rhinantheae agree with some other typical ele-
ments of the European alpine flora, representatives of which
can often be found in Afromontane habitats (Hedberg, 1970;
Gehrke & Linder, 2009; Emadzade & Hörandl, 2011).
The most interesting biogeographic pattern of the Bellar-
dia clade is the position of the Neotropical species of Bartsia
s.l. as a lineage derived from the Mediterranean taxa. The
New World Bartsia species are confined to Andean montane
habitats of Colombia, Bolivia, Peru, and Chile to northern Ar-
gentina, and seem to descend from a Mediterranean lineage
represented by the extant Parentucellia latifolia, which is
widespread across the Mediterranean area and beyond, rang-
ing from the Canary Islands to eastern Asia. As this species
is revealed as sister to all Neotropical species of Bartsia s.l., it
can be assumed that the ancestor of the Neotropical lineage ar-
rived from the Mediterranean via long-distance dispersal, and
diversified in an adaptive radiation to occupy the new habitats.
Examples for disjunct Mediterranean-Andean taxa with an Old
World origin are found in several other plant groups, such as
Eryngium L., Lupinus L., Menthinae, Pericallis D. Don, and
Senecio L. (Panero & al., 1999; Coleman & al., 2003; Hughes
& Eastwood, 2006; Calviño & al., 2008; Bräuchler & al., 2010;
Kadereit & Baldwin, 2012).
Taxonomic implications. —
Although the sampling of this
study is limited, our results allow some taxonomic conclusions
regarding the generic placement of several taxa. This is espe-
cially true for Bartsia s.l. (in the circumscription of Molau,
1990), and the small genera Bartsiella and Bornmuellerantha.
The latter two genera were previously included within Odon-
tites (Wettstein, 1891), but were later recognized as distinct
based on corolla shape, anther indumentum, and pollen types
(Rothmaler, 1943; Bolliger & Wick, 1990; Bolliger & Molau,
1992; Bolliger, 1996).
The type of Bartsia (Bartsia alpina L.; typ. cons.) is unam-
biguously dissociated from the remaining members of the ge-
nus in both tree and network analyses, and it is shown as sister
to the core group of Rhinantheae. As a result, Bartsia L. (nom.
cons.) should be redefined as a monotypic genus including
only B. alpina (equ all ing Bartsia sect. Bartsia in Molau, 1990),
which is geographically restricted to Arctic-alpine (montane)
Europe and northeastern North America. Thus circumscribed,
the genus consists of one hemiparasitic, perennial rhizomatous
geophyte with a perennial woody rhizome and annual aerial
shoots, whereas all other species included in Bartsia sensu
Molau (1990) are either annuals, or perennial hemicryptophytes
(with a woody base and monopodial growth). Reliable chromo-
some counts for B. alpina show a range of cytotypes including
2n = 24 (Löve & Löve, 1956; Löve, 1982; Dalgaard, 1988;
Molau, 1990; Dobeš & Vitek, 2000), the odd number of 36
(which might perhaps be from a triploid specimen), tetraploid
4x = 48 (Dobeš & Vitek, 2000) and hexaploid 6x = 72 (Taylor
& Rumsey, 2003), with 2n = 24 being the most common and
predominant number recorded throughout the European range
of the species (Molau, 1990; Dobeš & Vitek, 2000). This sug-
gests a polyploid series with a consistent base number of x =
12 in the genus. However, the report of 36 chromosomes might
also imply a base number of x = 6.
The two yellow-f lowered, hemiparasitic, frutescent Afro-
montane species of Bartsia (Bartsia sect. Longiflorae), B. de-
curva and B. longiflora (including B. longiflora subsp. macro-
phylla; Hedberg & al., 1979; Molau, 1990) are associated with
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the monotypic Afromontane Hedbergia (H. abyssinica (Benth.)
Molau) in all phylogenetic analyses, as well as in the consensus
network (Figs. 2–5). Since vegetative morphology and paly-
nology provide strong evidence for this clade to form a natu-
ral unit, we propose to transfer the respective African taxa of
Bartsia to Hedbergia. In consequence, the genus requires an
updated circumscription to include Bartsia sect. Longiflorae
(see “New Generic Circumscriptions”), and thus is no more ex-
clusively characterized by a rotate corolla symmetry (Hedberg
& al., 1980; Molau, 1988). The unique actinomorphic symmetry
of the flowers observed in Hedbergia (Molau, 1988) mig ht have
evolved in adaptation to a different pollinator. Unfortunately,
there is not much cytological data published for members of
this clade: for the African Bartsia species only a single chromo-
some count of 2n = app. 28 for B. decurva (a s B. macrocalyx
R.E. Fr.) was reported by Hedberg (Hedberg, 1957; Hedberg
& Hedberg, 1977; Hedberg & al., 1979), and no count for Hed-
bergia is available so far.
The sister relationship of the Odontites s.l. clade and the
Bellardia clade is highly supported in the consensus tree of
the combined dataset (Fig. 5). This corroborates the results
of Těšitel & al. (2010) who found a clade containing Paren-
tucellia species which was sister to several Odontites taxa.
Furthermore, both groups are well-circumscribed by several
morphological characters. All members of the Odontites s.l.
clade are characterized by one-sided racemes, whereas the rac-
emose inflorescences of the members of the Bellardia clade are
predominantly multilateral (partially unilateral in the Neotropi-
cal Bartsia sect. Diffusae Molau; Molau, 1990). Furthermore,
members of the Bellardia clade have upright ovules (Molau,
1990), whereas members of the Odontites s.l. clade share the
synapomorphy of having pendulous ovules (Rothmaler, 1943;
Bolliger, 1996).
In accordance with preceding studies (Wolfe & al., 2005;
Bennett & Mathews, 2006; Těšitel & al., 2010), our phylogeny
supports the monophyly of the New World Bartsia species and
their placement embedded in a grade of Bellardia and Parentu-
cellia. However, the results of the present study were obtained
using a sample designed to avoid misleading results caused
by the incorporation of incongruent data, a fact that was not
accounted for by Těšitel & al. (2010). While the monotypic
Bellardia recently has been re-included in Bartsia in several
treatments (e.g., Molau, 1990; López-Sáez & al., 2002), it is
clearly not supported as part of Bartsia s.str. here. Instead, it
is part of a highly supported clade together with Parentucellia
and the New World species of Bartsia s.l. Its phylogenetic po-
sition underlines the close affinity to Parentucellia: Bellardia
trixago, especially the yellow-flowered variant (Benedí, 2002),
is not only frequently confused with Parentucellia viscosa due
to morphological similarity and overlapping distribution ranges
(Benedí, 1998), but apparently is also naturally hybridizing with
the latter (Valdés & al., 1987; Benedí, 1998)—however, the
identity of the putative hybrids is somewhat dubious. Consid-
ering morphological, molecular and biogeographical evidence,
including Parentucellia in Bellardia see ms reasonable; in doing
so, the generic name Bellardia All. has nomenclatural priority
over the younger name Parentucellia Viv. As the New World
species of Bartsia s.l. are nested within Parentucellia and the
Bellardia clade, these species also should be included in Bel-
lardia. The cytological data available for the group give some
additional support, as both Bellardia and Parentucellia, as wel l
as New World Bartsia share a chromosome base number of
x = 12 (alt hough this is a common base number in Rhina ntheae):
Bellardia trixago is a diploid with 2n = 24 (Spet a, 1971; Molau,
1990), while Parentucellia is tetraploid (2n = 48; Speta, 1971;
Molau, 1990), except for one report of a diploid karyotype in
an introduced invasive population of P. viscosa in California
(Chuang & Heckard, 1992). This count, however, could have
resulted from misidentification of a Bellardia trixago specimen.
The Neotropical Bartsia species studied by Molau (1990) were
either diploids with 2n = 24, or tetraploids with 2n = 48, the
latter restricted to the Andean sections.
The Odontites s.l. clade, irrespective of inter nal topological
conflicts, is supported in the chloroplast, ITS and combined
analyses as well as in the consensus network, arguing for a
broader definition of the genus. Our results strongly support
Odontites to include Bornmuellerantha and Bartsiella, while
the position of Macrosyringion remains doubtful in this respect
(see below). The divergent corolla and anther morphology ob-
served in Bornmuellerantha (Rothmaler, 1943; Bolliger, 1996;
Dönmez & Mutlu, 2010) is a synapomorphy of its two species.
However, both are deeply nested within Odontites s.l. in all
analyses. The karyotype of Bornmuellerantha aucheri (2n = 24;
Bolliger & Molau, 1992; Bolliger, 1996) fits the common base
number of x = 12 shared by most taxa of Odontites. Therefore,
we propose to reclassify Bornmuellerantha as Odontites, ap-
plying a broad definition of the genus as mentioned above. The
small monotypic Bartsiella has been segregated from Odontites
s.l. based mainly on palynological evidence (Bolliger & Wick,
1990; Bolliger, 1996). However, here it is also revealed as nested
in Odontites s.l. Thus, recognizing Bartsiella as distinct does
not seem justified, and the name should be transferred back to
Odontites, as originally envisioned by Emberger (1932). To date,
no chromosome counts are available for Bartsiella.
Macrosyringion shows considerable incongruence rega rd-
ing its placement in the chloroplast and ITS phylogenies: it
is nested within the Odontites s.l. clade in the ITS topology,
but is sister to all other taxa of clade IV in the chloroplast
tree. Although ancient hybridization could possibly produce a
pattern like this (Joly & al., 2006), the process which created
the incongruence cannot be clearly elucidated here. Further-
more, support values for a clade IV including Macrosyringion
are low (PP: 0.78, BS: 81) in the chloroplast phylogeny, rais-
ing the question whether the genus should be included into a
more broadly defined Odontites. Chromosome numbers can-
not provide additional evidence as the reported number of 2n
= 24 for Macrosyringion (with the dysploid number 2n = 26
occasionally observed in M. longiflorum; Bolliger & Molau,
1992; Bolliger, 1996) is found in Odontites as well as other taxa
of Rhinantheae. Morphological characteristics are ambigu-
ous as well: the prolonged corolla tubes, which could possibly
represent a synapomorphy for the genus, were considered not
significant enough to support it as distinct (Rothmaler, 1943).
The shortly bifid corolla lip, however, additionally used by
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Rothmaler to exclude Macrosyringion from Odontites, could
also represent a transitional link to the deeply bilobate upper
lip of Bornmuellerantha, which is sister to Macrosyringion
in the ITS topology (Fig. 3) and clearly part of Odontites s.l.
Given the available evidence, a formal inclusion of the ge-
nus in Odontites is not advisable without further evidence by
analyses applying a broader sample of Odontites taxa as well
as additional markers.
The taxonomic placement of the two species of Nothobart-
sia has frequently changed in the past, as the plants unite mor-
phological characters of both Bartsia and Odontites (Molau,
1990; Bolliger & Molau, 1992). Both taxa were originally de-
scribed as Bartsia (e.g., Wet tstei n, 1891) before a sepa rate genus,
Nothobartsia, was proposed by Bolliger & Molau (1992). In the
present study, the genus is found as sister to Odontitella virgata,
with which it forms a highly supported clade in all analyses.
This clade, like Macrosyringion, features conspicuous in-
congruence in the single-marker reconstructions and almost
exclusively accounts for the high ly networked central par t of the
CN in Fig. 4A. In the chloroplast tree, it is sister to Hedbergia
and the Afromontane Bartsia species with maximum support.
This relationship is supported palynologically by a highly simi-
lar retipilate exine sculpture, which differs from the reticulate
pollen of Odontites and the majority of Rhinantheae (Hedberg
& al., 1979, 1980; Bolliger & Wick, 1990; Molau, 1990; Bolliger
& Molau, 1992; Bolliger, 1996). However, a retipilate pollen
sculpture is also found in the only distantly related Macrosyrin-
gion. In the ITS phylogeny, the Nothobartsia/Odontitella group
is sister to the Odontites s.l. clade, yet with moderate to weak
support (PP: 0.85, BS: 40). This position is supported by the
common synapomorphy of a pendulous ovule position (Roth-
maler, 1943; Molau, 1990; Bolliger & Molau, 1992; Bolliger,
1996) which is not found in the remainder of core Rhinantheae.
The latter character could be considered derived within Rhi-
nantheae, and thus is more likely to represent a synapomorphy
of the Odontites s.l. clade and the Nothobartsia/Odontitella
group (as suggested by the ITS topology), rather than having
evolved in parallel as indicated by the chloroplast topology.
The affinity of Nothobartsia and Odontitella to Odontites s.l. is
further confirmed by the fact that seeds and capsules of the two
genera closely resemble those of Odontites s.l. (Molau, 1990;
Bolliger & Molau, 1992; Bolliger, 1996).
The incongruent single -marker phylogenies, and the highly
supported relationship to the Hedbergia clade in the chloro-
plast tree, as opposed to strong morphological similarities with
Odontites s.l., argue for the presence of a reticulate pattern in
the origin of the Nothobartsia/Odontitella group. Assuming
ancient hybridization between ancestral lineages leading to the
Odontites s.l. clade and the Hedbergia clade would provide a
possible explanation for the origin of the putative ancestor of
Nothobartsia and Odontitella. A hybrid origin could explain
the morphological characteristics of the two perennial spe-
cies of Nothobartsia, which are intermediate between Bartsia
and Odontites s.l. (Bolliger & Molau, 1992). Equally, the an-
nual Odontitella shares characters with both Odontites s.l. and
Bartsia s.l. (Rothmaler, 1943; Bolliger, 1996), the latter sug-
gesting a possible relationship to the Hedbergia clade.
new generIc cIrcuMscrIptIons
As evident from our molecular phylogenetic reconstruc-
tions and as already discussed above, some combinations
are required to maintain monophyly of certain genera. The
paraphyly of Bartsia L., which has been known since Bennett
& Mathews (2006) and is evident from our tree topologies, re-
quires to circumscribe it as monotypic genus, comprising only
the type species Bartsia alpina L. Therefore, we propose the
following new combinations for the African and Neotropical
taxa which have hitherto been assigned to Bartsia:
Hedbergia longiflora (Hochst. ex Benth.) A. Fleischm. &
Heubl, comb. nov. ≡ Bartsia longiflora Hochst. ex Benth.
in Candolle, Prodr. 10: 545. 1846 – Holotype: [Ethiopia],
inter frutices in rupium rimis medio regionis ad latus sep-
tentrionale montis Kubbi, 12 Dec 1837, Schimper 418 (K
photo!; isotype: M!).
Hedbergia longiflora subsp. macrophylla (Hedberg) A.
Fleischm. & Heubl, comb. nov. ≡ Bartsia macrophylla
Hedberg in Symb. Bot. Upsal. 15: 174. 1957 ≡ Bartsia
longiflora subsp. macrophylla (Hedberg) Hedberg in Nor-
weg. J. Bot. 26: 7. 1979 – Holotype: Uganda, Ruwenzori,
Bujuku Valley near Bigo camp, at small steam, 3400 m, 21
Mar 1948, Hedberg 349 (UPS; isotype: K photo!).
Hedbergia decurva (Hochst. ex Benth.) A. Fleisch m. & Heubl,
comb. nov. ≡ Bartsia decurva Hochst. ex Benth. in Can-
dolle, Prodr. 10: 545. 1846 – Holoty pe: [Ethiopia], in latere
boreali montis Silke [Mt. Selki], 22 Feb 1840, Schimper
1329 (K photo!; isotype: M!).
For Parentucellia latifolia (L.) Caruel and P. viscosa (L.)
Caruel used in the present study, there are prior combinations
to include them in Bellardia All. (this generic name has no-
menclatural priority over Parentucellia Viv.), which should be
used for these taxa in order to avoid paraphyly of Bellardia.
Bellardia latifolia (L.) Cuatrec. in Trab. Mus. Ci. Nat. Barcelona
12: 428. 1929 ≡ Euphrasia latifolia L., Sp. Pl. 2: 604. 1753
– Lectotype (designated by Sutton in Jarvis, Order Out Of
Chaos: 514. 2007): [icon.] Euphrasia pratensis Italica lati-
folia in Morison, Pl. Hist. Univ. 3: 431, s. 11, t. 24, f. 8. 1699.
Bellardia viscosa (L.) Fisch. & C.A. Mey in Index Seminum
[St. Petersburg] 2: 4. 1836 ≡ Bartsia viscosa L., Sp. Pl. 2:
602. 1753 – Lectotype (designated by Fischer in Feddes
Repert. 108: 113. 1997): [icon.] Euphrasia lutea latifolia
palustris in Plukenet, Phytographia: t. 27, f. 5. 1691.
A third, yet taxonomically doubtful species of Parentucel-
lia (P. floribunda Viv., representing the generic type), endemic
to Libya, is accepted in numerous treatments (e.g. Qaiser, 1982).
Since we were neither able to consult the type nor to obtain
recently collected material for molecular analysis, we refrain
from making a new combination here.
1282
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P. & Sáez, L. (eds.), Plantas parásitas de la Península Ibérica e
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Although it is very likely that the Neotropical species of
Bartsia (sensu Molau, 1990) are monophyletic, we hesitate to
propose new combinations for the unsampled taxa, and exem-
plarily refer to the two representatives used in our study:
Bellardia canescens (Wedd.) A. Fleischm. & Heubl, comb.
nov. ≡ Bartsia canescens Wedd., Chlor. Andina 2: 123.
1860 – Lectot ype (designated by Molau in Opera Bot. 102:
76. 1990): Peru, Lima, without date, Dombey s.n. (P; iso-
type: PH photo!).
Bellardia mutica (Kunth) A. Fleischm. & Heubl, comb. nov.
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1818 ≡ Bartsia mutica (Kunth) Benth. in Candolle, Prodr.
10: 548. 1846 – Lectotype (designated by Molau in Opera
Bot. 102: 58. 1990): Peru, crescit locis siccis Peruviae inter
Lucarque et Ayavaca, 1300 hex [ca. 2400 m], without date,
Bonpland 3466 (P photo!; isotype: H photo!).
A broad circumscription of Odontites Ludw., including the
small genera Bornmuellerantha Rothm. and Bartsiella Bolliger,
is in agreement with the phylogenetic results, and requires the
following new combination:
Odontites alshehbazianus (Dönmez & Mutlu) A. Fleischm.
& Heubl, comb. nov. ≡ Bornmuellerantha alshehbaziana
Dönmez & Mutlu in Novon 20: 265. 2010 – Holotype: Tur-
key, Antalya, Gazipaşa, 1760 m, 23 Sep 2006, Dönmez
& Mutlu AAD 14036 (HUB; isotypes: E, INU, M!, MO).
conclusIon
The Rhinantheae as studied here are characterized by some
obvious topological incongruences between the plastid and nu-
clear datasets. Nothobartsia constitutes the most conspicuous ex-
ample of reticulation in the evolutionary history of Rhinantheae,
explaining part of the incongruent patterns observed. Hybrid-
ization is likely to play a major role in Rhinantheae, especially
in Odontites s.l. However, incongruence in general may have
several other reasons, including sampling artefacts, incomplete
lineage sorting, or low sequence divergence in closely related
groups with ongoing speciation (which is likely to apply in case
of the incongruent placement of the South American Bartsia
sp. Peru). The sample and focus of this study do not allow us to
discriminate unequivocally among the different processes and
to determine those actually involved here. Nevertheless, our
findings allow some reliable conclusions concerning the circum-
scription of Bartsia, Bellardia, and Hedbergia, and corroborate
a broader circumscription of Odontites. Fur ther extension of the
results presented here seems advisable, using a comprehensive
sample including several specimens of all taxa known to account
for overall genetic diversity within the group. Analysis of sev-
eral nuclear DNA sequence regions and a thorough assessment
of hybridization on the inter- and intrageneric level (applying
cloning techniques where necessary) can surely complement
our understanding of this important tribe of Orobanchaceae.
acknowledgeMents
The authors would like to thank Ulf Molau and Markus Bolliger
for helpful correspondence; the curators of the Munich Herbarium
(M), especially Franz Schuhwerk for providing important material
for this study; Ali A. Dönmez and Birol Mutlu for making available
material from the newly described Bornmuellerantha alshehbaziana;
Eberhard Fischer for providing material of African Bartsia; Daniel
Pinto Carrasco for his supporting correspondence; and two anonymous
reviewers for helpful comments on the manuscript. Tanja Ernst is
acknowledged for technical assistance.
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Appendix.
List of taxa used in the phylogenetic analyses with voucher information. Previously published sequences of ITS, trnK and rps16 are provided
with reference citations. Key to references: (1): Těšitel & al., 2010; (2): Schäferhoff & al., 2010; (3): Tank & Olmstead, 2008; (4): Müller & al., 2004; (5):
Morawetz & Wolfe, 2009; (6): Young & al., 1999.
Taxon name; region of origin, coll. date, collector, coll. no. (voucher location); acc. no. ITS; acc. no. trnK; acc. no. rps16
Bartsia alpina L.; Germany, 2000, Förther, H., 10816 (M); JF900502; JF900567; JF900535; – Bartsia alpina L.; Canada, 1965, Doppelbaur, H., 203 (M)
JF900505; JF900570; JF900538; – Bartsia alpina L.; Finland, 1971, Federley, B., s.n.; JF900504; JF900569; JF900537; – Bartsia alpina L.; Italy, 1972, Lip-
pert, W. & Podlech, D., 11758 (M); JF900503; JF900568; JF900536; – Bartsia canescens Wedd.; Peru, 1998, Beenken, L., 1033 (M); JF900518; JF900582;
JF900550; – Bartsia decurva Hochst. ex Benth.; Kenya, 1983, Gebauer, G., s.n. (M); JF90 0511; JX629749; JF900543; – Bartsia longiflora Hochst. ex Benth.;
Kenya, 1978, Grau, J., 1899 (M); JF900510; JF900575; JX629747; – Bartsia longif lora subsp. macrophylla (Hedberg) Hedberg; Rwanda, 2010, Fischer &
Th iel , 20 415 (private herb. E. Fischer); JF900519; JF900583; JF900551; – Bartsia mutica (Kunth) Benth.; Argent ina, 2008, Bräuchler, C., 5170 (M); JF900517;
JF900581; JF900549; – Bartsia sp.; Per u, 20 01, Henning, T. & Schneider, C., 18 (M); JF900516; JF900580; JF900548; – Bartsiella rameauana (Emb.) Bolliger;
Morocco, 1951, Rauh, W., 393 (M); JF900523; JF900587; JF900555; – Bellardia trixago (L.) All.; Spain, 2010, Schuhwerk, F., 27 (M); JF900513; JF900577;
JF900545; – Bornmuellerantha alshehbaziana Dönmez & Mutlu; Turkey, 2006, Dönmez, A. & Mutlu, B., 14036 (HUB); JF900522; JF900586; JF900554; –
Bornmuellerantha aucheri (Boiss.) Rothm.; Iran, 2001, Podlech, D. & Zarre, Sh., 55243 (M ); JF900521; JF900585; JF900553; – Euphrasia stricta J.P. Wolff
ex J.F. Lehm.; Czech Republic, 2007, Svobodova, S., 5091 (CBFS); FJ 790051(1); FJ790111(1); n/a; – Euphrasia stricta J.P. Wolff ex J.F. Lehm.; n/a, n/a, Borsch,
T., 3785 (BON N) n/a; n/a; FN794093(2); – Hedbergia abyssinica (Bent h.) Molau; Et hiopi a, 1973, Ash, J.W., 2054 (M ); JF900509; JF9 00574; JF90 0542; – Lath-
raea squamaria L.; Germany, 2010, Olano-Marín, C., 1 (M); JF900500; JF900565; JF90 0533; – Macrosyringion glutinosum ( M. Bieb.) Roth m.; France, 1997,
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Dutartre, G., 18417 (M); JF900520; JF900584; JF900552; – Melampyrum nemorosum L.; Germany, 1998, Lippert, W., 27890 (M); FJ797592 (1); JF900562;
JF900530; – Nothobartsia asperrima (Link) Benedí & Herrero; Portugal, 1977, Malato-Beliz, J. & Guerra, J. A., 14115 (M) JF900508; JF900573; JF900541;
– Nothobartsia spicata (Ramond) Bolliger & Molau; France, n/a, Bordère, 5908 (M); JX629746; JX629748; JX629750; – Odontitella virgata (Link) Rothm.;
Spain, 1988, Montserrat, P. & al., 15513 (M); JF90 0507; JF9 00572; JF900540 ; – Odontites bocconei (Guss.) Walp.; Italy, 1996, Certa, G., 18421 (M); JF900528;
JF900592; JF900560; – Odontites corsicus G. Don; France, 1998, Lambinon, J., 98/765 (M); JF900525; JF900589; JF900557; – Odontites cyprius Boiss.;
Cyprus, 2004, Vitek, E., Abr-61 (M); JF900529; JF900593; JF900561; – Odontites himalayicus Pennell; Pakistan, 1955, Webster, L. & Nasir, E., 6290 (M)
JF900526; JF900590; JF900558; – Odontites pyrenaeus (Bubani) Rothm.; Spain, 1996, Nydegger, M., 35183 (M); JF900527; JF900591; JF900559; – Odon-
tites viscosus (L.) Clair v.; France, 1985, Perrin, F., 12515 (M); JF900524; JF900588; JF900556; – Parentucellia latifolia (L.) Car uel; Greece, 2007, Tillich,
H.J., 5333 (M); JF900515; JF900579; JF900547; – Parentucellia viscosa (L.) Caruel; Greece, 2003, Tillich, H. J., 4488 (M); JF900514; JF900578; JF900546;
– Pedicularis attollens A. Gray; n/a, n/a, Tank, D., 01-50 (WTU); EF103743(3); n/a; EF103821(3); – Pedicularis sylvatica L.; n/a, n/a, Müller, K., 744 (BONN);
n/a; AF531781(4); n/a; – Rhinanthus alectorolophus (Scop.) Pollich; Germany, 2010, Olano-Marín, C., 3 (M); JF900501; JF900566; JF900534; – Rhynchocorys
kurdica Nábělek; Iraq, 1957, Rechninger, K.H., 11069 (M); JF900499; JF900564; JF900532; – Rhynchocorys orientalis Benth.; Armenia, 2003, Fayvush, G.
& al., 03-1382 (M); JF900498; JF900563; JF900531; – Striga asiatica (L.) Kuntze; n/a, n/a, Morawetz, J. 116 (OS); EU253604(5); AF052000(6); n/a; – To zz ia
alpina L.; Austria, 1998, Panzer, R., s.n. (M); JF900512; JF900576; JF900544.
Appendix.
Continued.
