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Phylogeny of tribe Rhinantheae (Orobanchaceae) with a focus on biogeography, cytology and re-examination of generic concepts

  • SNSB Staatliche Naturwissenschaftliche Sammlungen Bayerns (Bavarian Natural History Collections)
  • Botanische Staatssammlung München, Munich, Germany

Abstract and Figures

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, according to previous taxonomic treatments, includes a remarkable radiation (ca. 45 species) 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 than to other congeneric taxa; (3) South American Bartsia are nested within a highly supported 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 morphological 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.
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Scheunert & al. • Phylogeny of Rhinantheae
61 (6) • December 2012: 1269–1285
1269Version of Record (identical to print version).
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,
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.
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
This publication is dedicated to Dr. Markus Bolliger on the occasion of his 60th birthday.
61 (6) • December 2012: 1269–1285Scheunert & al. • Phylogeny of Rhinantheae
1270 Version of Record (identical to print version).
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. —
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
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
Scheunert & al. • Phylogeny of Rhinantheae
61 (6) • December 2012: 1269–1285
1271Version of Record (identical to print version).
Fig. 1.
Selected species of representative genera of Rhinantheae (Orobanchaceae).
Melampyrum nemorosum L.;
Parentucellia latifolia ( L.)
Bartsia alpina L.;
Bellardia trixago (L.) A ll.;
Rhinanthus alectorolophus (Scop.) Pollich;
Euphrasia officinalis L.;
tucellia viscosa (L.) Caruel;
Odontites vernus Dumort.;
Lathraea squamaria L. — Photographs A, E & F, F. Brambach; B–D, G–H, A.
Fleischmann; I, C. Olano-Marín.
61 (6) • December 2012: 1269–1285Scheunert & al. • Phylogeny of Rhinantheae
1272 Version of Record (identical to print version).
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. (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
(Scferhoff & al., 2010). Ing roup indels were coded
to the
simple indel-coding
(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;
& 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.
Scheunert & al. • Phylogeny of Rhinantheae
61 (6) • December 2012: 1269–1285
1273Version of Record (identical to print version).
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 (
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
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).
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
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 G + C content 55.24% 34.24% 34.54% 34.39% 40.27%
61 (6) • December 2012: 1269–1285Scheunert & al. • Phylogeny of Rhinantheae
1274 Version of Record (identical to print version).
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
Rhinanthus alectorolophus
Lathraea squamaria
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
1.00 100
Core group of
Bellardia trixago
Parentucellia viscosa
Parentucellia latifolia
Bartsia mutica
Bartsia sp. Peru
Bartsia canescens
Macrosyringion glutinosum
Bornmuellerantha aucheri
Bornmuellerantha alshehbaziana
Odontites corsicus
Odontites himalayicus
Odontites bocconei
Odontites cyprius
Odontites viscosus
Bartsiella rameauana
Odontites pyrenaeus
Bartsia longiflora subsp. macrophylla
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.
Scheunert & al. • Phylogeny of Rhinantheae
61 (6) • December 2012: 1269–1285
1275Version of Record (identical to print version).
(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
core group of
Hedbergia abyssinica
Bartsia decurva
Bartsia longiflora
Bartsia longiflora subsp. macrophylla
Odontitella virgata
Nothobartsia asperrima
Nothobartsia spicata
Odontites corsicus
Odontites himalayicus
Macrosyringion glutinosum
Bornmuellerantha aucheri
Bornmuellerantha alshehbaziana
Bellardia trixago
Parentucellia viscosa
Parentucellia latifolia
Bartsia mutica
Bartsia sp. Peru
Bartsia canescens
Odontites pyrenaeus
Odontites viscosus
100 0.62
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.
61 (6) • December 2012: 1269–1285Scheunert & al. • Phylogeny of Rhinantheae
1276 Version of Record (identical to print version).
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).
Scheunert & al. • Phylogeny of Rhinantheae
61 (6) • December 2012: 1269–1285
1277Version of Record (identical to print version).
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
all 36 taxa included in the study, and
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-
Bartsia alpina Canada
Bartsia alpina Germany
Bartsia alpina Italy
Euphrasia stricta
Tozzia alpina
Bartsia longiflora
Bartsia longiflora subsp. macrophylla
Nothobartsia asperrima
Nothobartsia spicata
Odontitella virgata
B. aucheri
O. corsicus
O. himalayicus
O. viscosus
Bartsiella rameauana
O. cyprius
bocconei Odontites pyrenaeus
Parentucellia latifolia
Bartsia canescens
Bartsia sp. Peru
Bartsia mutica
Rhinanthus alectorolophus Lathraea squamaria
kurdica Rhynchocorys
nemorosum Pedicularis spp.
Striga asiatica
Bartsia alpina Finland
Striga asiatica
Pedicularis spp.
Rhinanthus alectorolophus Lathraea squamaria
kurdica Rhynchocorys
Euphrasia stricta
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
O. cyprius
Odontites bocconei Odontites
O. viscosus
O. corsicus
O. himalayicus
B. aucheri
Clade II
Clade III
Clade IV
Clade I
Bartsia canescens
Bartsia mutica
Parentucellia latifolia
Clade II
Clade I
Clade III
Clade IV
56.4 29.3
61 (6) • December 2012: 1269–1285Scheunert & al. • Phylogeny of Rhinantheae
1278 Version of Record (identical to print version).
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
Rhinanthus alectorolophus
Lathraea squamaria
Bartsia alpina Germany
Bartsia alpina Italy
Bartsia alpina Finland
Bartsia alpina Canada
Euphrasia stricta
Tozzia alpina
Hedbergia abyssinica
Bartsia decurva
Bartsia longiflora
core group of
Odontites corsicus
Odontites himalayicus
Odontites pyrenaeus
Odontites viscosus
Bornmuellerantha aucheri
Bartsia longiflora subsp. macrophylla
Bellardia trixago
Parentucellia viscosa
Parentucellia latifolia
Bartsia canescens
Bartsia mutica
Bornmuellerantha alshehbaziana
Odontites bocconei
Odontites cyprius
Bartsiella rameauana
OG I. Bartsia s.str. II. Hedbergia IV. Odontites s.l.III. BellardiaRRL clade
Scheunert & al. • Phylogeny of Rhinantheae
61 (6) • December 2012: 1269–1285
1279Version of Record (identical to print version).
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.
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
61 (6) • December 2012: 1269–1285Scheunert & al. • Phylogeny of Rhinantheae
1280 Version of Record (identical to print version).
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
Scheunert & al. • Phylogeny of Rhinantheae
61 (6) • December 2012: 1269–1285
1281Version of Record (identical to print version).
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.
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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.
Euphrasia mutica Kunth, Nov. Gen. Sp. [quarto] 2: 334.
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).
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.
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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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 alshehbaziananmez & 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,
Scheunert & al. • Phylogeny of Rhinantheae
61 (6) • December 2012: 1269–1285
1285Version of Record (identical to print version).
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.
... Later, Bolliger (1996) distinguished one more monospecific genus, Bartsiella Bolliger. Subsequent studies based on the sequencing of nuclear and chloroplast DNA regions demonstrated that only Odontitella should be treated as an independent genus while the rest should be returned to Odontites (Těšitel et al., 2010;Scheunert et al., 2012;Gaudeul et al., 2016;Pinto-Carrasco et al., 2017). The most recent circumscription recognizes 32 species (47 taxa, including subspecies) grouped into five main lineages (Pinto-Carrasco et al., 2017). ...
... 2.4 Chloroplast DNA: Amplification, editing, and statistical analysis For the 301 samples, we amplified and sequenced two regions of chloroplast DNA ( 3′ rps16-5′ trnK and the rps16 intron) that showed sufficient variability in an initial screening. For the rps16 intron region, we used the same PCR program as in Scheunert et al. (2012), while the one used for 3′ rps16-5′ trnK consisted of an initial denaturation at 94°C (1 min), followed by 35 cycles of 40 s of denaturation at 94°C, 1 min of annealing at 52°C, and 2 min of extension at 72°C, ending with a final extension of 8 min at 72°C. The primers for the rps16 intron (rpsR2 and rpsF) were published by Oxelman et al. (1997), and those of the region 3′ rps16-5′ trnK (trnK(UUU)x1 and rps16x2F2) by Shaw et al. (2007). ...
... The primers for the rps16 intron (rpsR2 and rpsF) were published by Oxelman et al. (1997), and those of the region 3′ rps16-5′ trnK (trnK(UUU)x1 and rps16x2F2) by Shaw et al. (2007). In both cases, the quantities and concentrations of the different reagents were similar to those used for the rps16 intron in Scheunert et al. (2012). The samples were sequenced preferentially with the forward primers used for the amplification, but some of them were sequenced bidirectionally. ...
The Odontites vernus group is the most widespread of the genus Odontites, occupying the temperate regions of Eurasia and northern Morocco. The group contains three species, all inhabiting the Iberian Peninsula, where Odontites vernus s.l. (sensu lato) exhibits remarkable morphological variability and includes diploid and tetraploid individuals corresponding to the two subspecies that occur there. We collected 301 individuals from 100 sampling sites covering the entire distribution of O. vernus in the Iberian Peninsula and genotyped them using 12 SSR markers. Their ploidy level was estimated by flow cytometry, and two cpDNA regions (rps16 intron and trnK-rps16) were sequenced. We found 129 diploids and 172 tetraploids distributed following a mosaic parapatry model, while only two mixed-ploidy populations were discovered. The 20 haplotypes found fit two well-defined haplogroups, to some extent correlated with estimated ploidy levels. The frequencies of the SSR alleles shared by both cytotypes, as well as those of the private alleles corresponding to the tetraploid cytotype, indicate that tetraploids likely originated at least twice through autopolyploidy. Additionally, the results from SSR markers were structured in a higher number of groups than did the cpDNA sequences. Thus, the genetic distance analysis detected 4 groups, but the Bayesian analysis of population structure identified 7, with only low levels of gene flow detected among groups. The distributions of the 7 genetic groups coincide with well-known refugium areas within the Iberian Peninsula during the climatic oscillations of the Quaternary. Thus, the results give additional support to the "refugia within refugia" hypothesis. This article is protected by copyright. All rights reserved.
... In his protologue, Hong (1979) distinguished Pseudobartsia from Bartsia Linnaeus and Odontites Ludwig; the latter two genera are resolved as members of the tribe Rhinantheae in molecular phylogenies (Tesitel et al., 2010;Scheunert et al., 2012;McNeal et al., 2013). ...
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The millions of herbarium specimens in collections around the world provide historical resources for phylogenomics and evolutionary studies. Many rare and endangered species exist only as historical specimens. Here, we report a case study of the monotypic Pseudobartsia yunnanensis (= Pseudobartsia glandulosa: Orobanchaceae) known from a single Chinese collection taken in 1940. We obtained genomic data of Pseudobartsia glandulosa using high-throughput short-read sequencing, and then assembled a complete chloroplast genome and nuclear ribosome DNA region in this study. We found that the newly assembled three plastid DNA regions (atpB-rbcL, rpl16, and trnS-G) and nrITS of Pseudobartsia glandulosa were more than 99.98% similar to published sequences obtained by target sequencing. Phylogenies of Orobanchaceae using 30 plastomes (including ten new plastomes), using both supermatrix and multispecies coalescent (MSC) approaches following a novel plastid phylogenomic workflow, recovered seven recognized tribes and two unranked groups, both of which were proposed as new tribes, i.e., Brandisieae and Pterygielleae. Within Pterygielleae, all analyses strongly supported Xizangia as the first diverging genus, with Pseudobartsia as sister to Pterygiella + Phtheirospermum (excluding Phtheirospermum japonicum); this supports reinstatement of Pseudobartsia and Xizangia. Though elements of Buchnereae-Cymbarieae-Orobancheae and Brandisieae-Pterygielleae-Rhinantheae showed incongruence among gene trees, the topology of the supermatrix tree was congruent with the majority of gene trees and functional-group trees. Therefore, most plastid genes are evolving as a linkage group, allowing the supermatrix tree approach to yield internally consistent phylogenies for Orobanchaceae.
... Total genomic DNA was extracted from dried leaf tissue, and PCR of the ITS region was performed as described in Bräuchler et al. (2010) by using the primer pairs aITS1 and aITS4 (Bräuchler et al., 2004). Purification of PCR products and sequencing on an ABI 3730 DNA analyzer (Applied Bio Systems, Foster City, California) used the PCR primers described in Scheunert et al. (2012). Alignment of sequences was done in Bioedit (Hall, 1999), and the alignment including outgroups had a total length of 652 base pairs. ...
Chondrilla chondrilloides (Asteraceae) is a rare and endangered early-successional plant species endemic to the Eastern European Alps. Its distribution is restricted to near-natural braided rivers and to alluvial fans. The species was common along Alpine gravel rivers, but has declined markedly due to river regulation and degradation in the 19th and 20th century, while some recent restoration projects benefit the plant. Its population declines were caused by habitat fragmentation and destruction as a consequence of extensive hydro-engineering. This paper summarises the published material on taxonomy, morphology, habitat requirements and distribution of the species. The review is complemented by own research data and a phylogenetic assessment of extant and extinct populations within the infrageneric context. A summary on location, size and structure of the remaining populations in the north-eastern and south-eastern Alps is combined with data on seed germination and the habitat niche of the species, with a particular focus on differences between northern and southern populations. Chondrilla chondrilloides forms meta-populations on consolidated gravel bars and older terraces, with extinction and recolonisation due to floodplain dynamics; small populations quickly recover from few founder individuals. Populations in the southern parts of the species’ range are larger with bigger plants and more reproduction, while germination is very high in all populations. Thus, C. chondrilloides has characteristics that allow it to respond rapidly to degradation and restoration of its habitats along gravel rivers in the Eastern Alps.
... Odontitella virgata (Link) Rothm. was used as outgroup based on previous phylogenetic analysis (Scheunert et al., 2012;Pinto-Carrasco et al., 2017). Additionally, a principal coordinate analysis (PCoA) based on Dice's coefficient was performed with NTSYS-pc version 2.02 (Rohlf, 2009). ...
Premise of the study: Ecological drivers for genetic differentiation in Mediterranean climates are still underexplored. We have used the strictly Mediterranean endemic Odontites recordonii as a model species to address this question. This species is one of the three Iberian representatives of the O. vernus group, which are morphologically similar. Thus, it was additionally necessary to clarify their phylogenetic relationships. Methods: We used AFLPs to reveal phylogenetic relationships within O. vernus group, and to reconstruct the phylogeographic patterns within O. recordonii. Additionally, ENMs were generated to detect refugia along the Quaternary climatic oscillations. And finally, alleles under natural selection were identified, and correlations between allele presences and environmental variables were calculated in order to shed light on the ecological drivers promoting differentiation. Key results: The three species from O. vernus group were recovered as distinct species. Three genetic groups were found within O. recordonii and a putative refugium was detected for each one. Eighty-one alleles could be under diversifying selection, and 58 alleles showed significant correlations with environmental variables, especially with temperature and precipitation seasonality and summer drought. Conclusions: The three Iberian species of the Odontites vernus group are reciprocal monophyletic taxa. The three genetic groups of O. recordonii could have been restricted to narrow refugia during the Quaternary and displayed present distributions in accordance with bioclimatic conditions. Temperature and precipitation seasonality and the intensity of summer drought are definitory climatic parameters of Mediterranean type climates, and they could have acted as drivers of genetic differentiation on O. recordonii. This article is protected by copyright. All rights reserved.
... We supplemented these previous data with newly generated sequence data from 11 additional UK samples representing a wider range of species and geographical locations, including 11 UK Euphrasia samples, an Austrian sample of E. cuspidata intended as a close outgroup to UK species, and B. alpina as an outgroup to the full sample set (Těšitel et al., 2010;Scheunert et al., 2012;A.D.T., unpubl. res.). ...
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Background and aims: Genome size varies considerably across the diversity of plant life. Although genome size is, by definition, affected by genetic presence/absence variants, which are ubiquitous in population sequencing studies, genome size is often treated as an intrinsic property of a species. Here, we studied intra- and interspecific genome size variation in taxonomically complex British eyebrights (Euphrasia, Orobanchaceae). Our aim is to document genome size diversity and investigate underlying evolutionary processes shaping variation between individuals, populations and species. Methods: We generated genome size data for 192 individuals of diploid and tetraploid Euphrasia and analysed genome size variation in relation to ploidy, taxonomy, population affiliation, and geography. We further compared the genomic repeat content of 30 samples. Key results: We found considerable intraspecific genome size variation, and observed isolation-by-distance for genome size in outcrossing diploids. Tetraploid Euphrasia showed contrasting patterns, with genome size increasing with latitude in outcrossing Euphrasia arctica, but with little genome size variation in the highly selfing Euphrasia micrantha. Interspecific differences in genome size and the genomic proportions of repeat sequences were small. Conclusions: We show the utility of treating genome size as the outcome of polygenic variation. Like other types of genetic variation, such as single nucleotide polymorphisms, genome size variation may be affected by ongoing hybridisation and the extent of population subdivision. In addition to selection on associated traits, genome size is predicted to be affected indirectly by selection due to pleiotropy of the underlying presence/absence variants.
... There are only four endemic genera (Dendrosenecio, Haplosciadium, Hedbergia (cf. Scheunert et al. 2012), and Oreophyton; the former endemic genus Uebelinia is embedded in Lychnis; Popp et al. 2008). ...
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The monumental work of Olov Hedberg provided deep insights into the spectacular and fragmented tropical alpine flora of the African sky islands. Here we review recent molecular and niche modelling studies and re-examine Hedberg’s hypotheses and conclusions. Colonisation started when mountain uplift established the harsh diurnal climate with nightly frosts, accelerated throughout the last 5 Myr (Plio-Pleistocene), and resulted in a flora rich in local endemics. Recruitment was dominated by long-distance dispersals (LDDs) from seasonally cold, remote areas, mainly in Eurasia. Colonisation was only rarely followed by substantial diversification. Instead, most of the larger genera and even species colonised the afroalpine habitat multiple times independently. Conspicuous parallel evolution occurred among mountains, e.g., of gigantism in Lobelia and Dendrosenecio and dwarf shrubs in Alchemilla . Although the alpine habitat was ~ 8 times larger and the treeline was ~ 1000 m lower than today during the Last Glacial Maximum, genetic data suggest that the flora was shaped by strong intermountain isolation interrupted by rare LDDs rather than ecological connectivity. The new evidence points to a much younger and more dynamic island scenario than envisioned by Hedberg: the afroalpine flora is unsaturated and fragile, it was repeatedly disrupted by the Pleistocene climate oscillations, and it harbours taxonomic and genetic diversity that is unique but severely depauperated by frequent bottlenecks and cycles of colonisation, extinction, and recolonisation. The level of intrapopulation genetic variation is alarmingly low, and many afroalpine species may be vulnerable to extinction because of climate warming and increasing human impact.
... This binary matrix was then manually concatenated with the end of each haplotype. For these gap coding haplotypes, "ML + rapid bootstrap" analyses were performed with the following settings: Data Type = 'Mixed' with two partitions [DNA and BIN (binary)], number of bootstraps = 1,000, GTRGAMMA model (replaced automatically by BINGAMMA for binary data; Scheunert et al. 2012). A phylogenetic tree was produced using FigTree ver. ...
The Japanese archipelago exhibits a notable difference in snow depth in winter, deep snow on the Sea of Japan side and low snow cover on the Pacific Ocean side. This contrasting pattern has shaped the distribution of infraspecific taxon pairs in a range of woody plants, with taxa found on the Sea of Japan side typically exhibiting a stunted shrub form with multiple decumbent stems. The phylogenetic origin of these taxon pairs is unknown, i.e., whether the two taxa diverged from the same species or if they have different origins. This study aimed to reveal the phylogenetic origin of two varieties of Torreya nucifera (Taxaceae); var. nucifera is a tree found on the Pacific Ocean side, whereas var. radicans is a shrub found on the Sea of Japan side. We examined the phylogenetic relationships of the two varieties and worldwide Torreya taxa using whole chloroplast genomes, chloroplast DNA fragments, and the nuclear ribosomal internal transcribed spacer (ITS). The whole chloroplast genome phylogeny indicated that T. nucifera var. radicans was a sister taxon to Chinese T. grandis, rather than to var. nucifera. In contrast, the nuclear ITS phylogeny indicated that while several haplotypes of T. nucifera var. radicans were closely related to T. grandis, most haplotypes of T. nucifera var. radicans formed a single clade with those of var. nucifera. This implies that the homogenization of the ITS has occurred between the two taxa, while taxon-specific chloroplast DNA haplotypes were retained. These discordant phylogenies suggested that the two taxa have different phylogenetic origins, but have an intricate evolutionary history, involving inter-taxa hybridization and gene flow, possibly when their distributions were confined to sympatric refugia. Given the genetic evidence and distinct difference in growth form, we propose that T. nucifera var. radicans should be taxonomically treated as a distinct species, T. fruticosa.
In the present study, the chemical composition of the essential oil from aerial parts of Parentucellia latifolia (L.) Caruel collected in Central Sicily was analyzed by gas chromatography and gas chromatography–mass spectrometry. The results showed the presence of sesquiterpene hydrocarbons, with germacrene D and germacrene B accounting, respectively, for 59.2% and 24.3% of the total oil. Different colonies of bacteria and fungi frequently affect cellulosic objects such as books stored in libraries and museums. The antibacterial and antifungal activity against some microorganisms infesting historical-artistic craftsmanship was determined, demonstrating that the essential oil was particularly active against Bacillus subtilis, Staphylococcus aureus, and Proteus vulgaris.
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Premise of the study: Clade specific bursts in diversification are often associated with the evolution of key innovations. However, in groups with no obvious morphological innovations, observed upticks in diversification rates have also been attributed to the colonization of a new geographic environment. In this study, we explore the systematics, diversification dynamics, and historical biogeography of the plant clade Rhinantheae in the Orobanchaceae, with a special focus on the Andean clade of the genus Bartsia L.. Methods: We sampled taxa from across Rhinantheae, including a representative sample of Andean Bartsia species. Using standard phylogenetic methods, we reconstructed evolutionary relationships, inferred divergence times among the clades of Rhinantheae, elucidated their biogeographic history, and investigated diversification dynamics. Key results: We confirmed that the South American Bartsia species form a highly supported monophyletic group. The median crown age of Rhinantheae was determined to be ca. 30 Ma, and Europe played an important role in the biogeographic history of the lineages. South America was first reconstructed in the biogeographic analyses around 9 Ma, and with a median age of 2.59 Ma, this clade shows a significant uptick in diversification. Conclusions: Increased net diversification of the South American clade corresponds with biogeographic movement into the New World. This happened at a time when the Andes were reaching the necessary elevation to host an alpine environment. Although a specific route could not be identified with certainty, we provide plausible hypotheses to how the group colonized the New World.
The tropical clade of Orobanchaceae contains approximately forty genera, typically with fewer than ten species each, and contributes significantly to the variation in floral morphology found within the family. Despite the economic importance of this clade, which contains three of four most important genera of crop parasites within the family, it has been under–sampled in previous phylogenies. We tested the monophyly of the tropical clade and its major genera using DNA sequences from the nuclear (internal transcribed spacer) and plastid (rpl16, trnT–L) genomes. The tropical clade was strongly supported as monophyletic in all analyses, and four main clades were recovered. The earliest diverging lineage from the remainder of the tropical clade is comprised of the shrubby genera Asepalum and Cyclocheilon, previously placed within Cyclocheilaceae. The atypical holoparasitic Alectra alba was shown to belong within the primarily holoparasitic Harveya, and the hemiparasitic Harveya obtusifolia was shown to belong to an otherwise holoparasitic lineage within Harveya. Both New World Melasma species were included here for the first time, and these were shown to be more closely related to the Neotropical hemiparasitic Escobedia than the African Melasma lineage. These results support a previous study recognizing Nesogenes within the tropical clade of Orobanchaceae rather than the separate family Nesogenaceae.