Dissertation zur Erlangung des Doktorgrades der
Naturwissenschaften (Dr. rer. nat.) an der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
Evolutionary history and biogeography of
the genus Scrophularia (Scrophulariaceae) and
hemiparasitic Orobanchaceae (tribe Rhinantheae)
with emphasis on reticulate evolution
vorgelegt von
Agnes Scheunert
München, Dezember 2016
II
Diese Dissertation wurde angefertigt
unter der Leitung von Prof. Dr. Günther Heubl
an der Fakultät für Biologie, Department I,
Institut für Systematische Botanik und Mykologie
an der Ludwig-Maximilians-Universität München
Erstgutachter:
Prof. Dr. Günther Heubl
Zweitgutachter: Prof. Dr. Jochen Heinrichs
Tag der Abgabe: 15.12.2016
Tag der mündlichen Prüfung: 22.03.2017
III
IV
Eidesstattliche Versicherung und Erklärung
Eidesstattliche Versicherung
Ich, Agnes Scheunert, versichere hiermit an Eides statt, daß die vorgelegte Dissertation
von mir selbständig und ohne unerlaubte Hilfe angefertigt ist.
München, den 14.12.2016
______________________________________
Agnes Scheunert
Erklärung
Diese Dissertation wurde im Sinne von § 12 der Promotionsordnung von Prof. Dr.
Günther Heubl betreut. Hiermit erkläre ich, Agnes Scheunert, dass die Dissertation nicht
ganz oder in wesentlichen Teilen einer anderen Prüfungskommission vorgelegt worden
ist, und daß ich mich anderweitig einer Doktorprüfung ohne Erfolg nicht unterzogen
habe.
München, den 14.12.2016
______________________________________
Agnes Scheunert
V
VI
Declaration of author contribution
In this cumulative thesis, I present the results of my doctoral research, which was
conducted at the Ludwig-Maximilians-Universität of Munich under the supervision of
Prof. Dr. Günther Heubl. The results of my research have been published or accepted for
publication in international peer-reviewed journals. The four articles included in this
dissertation are presented in chapter 4; all of them have resulted from collaboration
with other scientists, and my contributions to each of them were as follows:
Article I:
Scheunert A, Fleischmann A, Olano-Marín C, Bräuchler C, Heubl G. 2012. Phylogeny of
tribe Rhinantheae (Orobanchaceae) with a focus on biogeography, cytology and reexamination of generic concepts. Taxon 61(6): 1269-1285.
The study was designed by G. Heubl and C. Bräuchler. I did the conceptual design of the
methodical treatment of incongruence, and conducted most of the respective analyses. I
revised the manuscript, coordinated generation of complementary sequences and did
their editing and alignment, re-analyzed most of the data, did the final phylogenetic
analyses and interpreted the results. I wrote large parts of the Material and Methods and
Results sections and created most of the figures. A. Fleischmann co-supervised the
study, selected the taxon sampling, determined the plant material and interpreted the
results. He helped to draft the manuscript, wrote large parts of the Introduction and
Discussion including the taxonomic treatment, made the taxonomic combinations, and
contributed photographs to the manuscript. C. Olano-Marín conducted the laboratory
work, did most of the initial phylogenetic analyses, provided a photograph and
contributed to the manuscript. C. Bräuchler helped with the selection of herbarium
material, improved the manuscript in its initial phase, created some of the graphics and
submitted the sequence data to NCBI's Genbank. G. Heubl supervised the work, helped
with the conception of the manuscript and improved and corrected the manuscript.
Article II:
Scheunert A, Heubl G. 2011. Phylogenetic relationships among New World Scrophularia
L. (Scrophulariaceae): new insights inferred from DNA sequence data. Plant Systematics
and Evolution 291: 69-89.
G. Heubl designed the study. I selected the taxon sampling, and did the laboratory work
including DNA extraction, PCR amplification and product purification, sequencing, and
subsequent sequence editing, alignment and phylogenetic analyses. I also planned and
performed the dating analyses and the statistical tests. I interpreted the results, wrote
the manuscript and created the figures, tables and maps. G. Heubl supervised the study,
the laboratory work and the phylogenetic analyses, helped to interpret the data and
improved the manuscript.
VII
Article III:
Scheunert A, Heubl G. 2014. Diversification of Scrophularia (Scrophulariaceae) in the
Western Mediterranean and Macaronesia - phylogenetic relationships, reticulate
evolution and biogeographic patterns. Molecular Phylogenetics and Evolution 70: 296313.
The study was designed by G. Heubl and myself. I selected the taxon sampling, cultivated
several of the plants in the greenhouses of the Botanical Garden in Munich, and
determined the used specimens. I designed the methodical concept to address
hybridization and incongruence, including the use of phylogenetic networks and the
taxon duplication approach in combination with ancestral area reconstruction. I
supervised the laboratory work, did editing and alignment of sequence data, conducted
the statistical tests, performed the phylogenetic analyses, indel coding and
biogeographic reconstruction, and created the haplotype and consensus networks.
Finally I interpreted the results, wrote the manuscript and prepared the figures, tables
and maps. G. Heubl supervised the study, helped to interpret the results and improved
and corrected the manuscript.
Article IV:
Scheunert A, Heubl G. Against all odds: reconstructing the evolutionary history of
Scrophularia (Scrophulariaceae) despite high levels of incongruence and reticulate
evolution. Organisms Diversity and Evolution, in press.
doi: 10.1007/s13127-016-0316-0
G. Heubl and myself developed the concept of the study. I generated sequence data and
supervised the laboratory work. I conducted an extensive literature search on the topics
of among-dataset incongruence, possible treatments of intra-individual-polymorphism,
and the evolutionary characteristics of ITS. Based on this I compiled the workflow used
in the study to examine hybridization and other sources of phylogenetic incongruence
and ambiguity. I conducted indel and character coding and compared the available
methods. I performed data analysis (statistical tests, phylogenetic tree reconstruction,
biogeography, divergence dating, consensus and Neighbor-Net networks) and
interpretation, wrote the manuscript and prepared the figures, tables and the dispersal
map. G. Heubl supervised the study and improved and shortened the manuscript.
Munich, 13/12/2016
____________________________________________
Agnes Scheunert
____________________________________________
Prof. Dr. Günther Heubl (supervisor)
All photographs, images and illustrations in this work were made by Agnes Scheunert
unless denoted otherwise.
VIII
Funding
My doctoral studies were financially supported for two years (November 2007 - October
2009) by a scholarship, provided by the "Universität Bayern e.V.". Graduate funding was
granted based on the "Bayerisches Eliteförderungsgesetz" (BayEFG) and included a
salary and facultatively also travel expenses.
IX
X
Für meine Mama.
XI
XII
Contents
Preface
Eidesstattliche Versicherung und Erklärung
Declaration of author contribution
V
VII
Funding
IX
Dedication
XI
Contents
XIII
1. Summary / Zusammenfassung
1
2. Introduction
5
2.1. Biology and Systematics of Scrophulariaceae Juss.
5
2.1.1. Traditional circumscription of Scrophulariaceae
5
2.1.2. Current understanding of phylogenetic relationships
6
2.2. Parasitism in plants: the example of Orobanchaceae Vent.
10
2.2.1. Parasitism
10
2.2.2. Orobanchaceae Vent.
10
2.2.3. Tribe Rhinantheae Lam. & DC.
12
2.3. The genus Scrophularia L.
13
2.3.1. Taxonomic history
13
2.3.2. Morphology, distribution and phylogenetic relationships
14
2.3.3. Pollination biology and evolution of the staminode
18
2.3.4. Anatomy and phytochemistry
19
3. Methodology
21
3.1. Testing for incongruence among phylogenetic trees
21
3.2. Combining incongruent datasets for phylogenetic tree construction
22
3.3. Character and indel coding
22
3.4. Network methods
25
4. Scientific manuscripts
29
4.1. Article I: Phylogeny of tribe Rhinantheae (Orobanchaceae) with
a focus on biogeography, cytology and re-examination
of generic concepts
31
4.2. Article II: Phylogenetic relationships among New World
Scrophularia L. (Scrophulariaceae): new insights
inferred from DNA sequence data
51
XIII
4.3. Article III: Diversification of Scrophularia (Scrophulariaceae)
in the Western Mediterranean and Macaronesia
- Phylogenetic relationships, reticulate evolution
and biogeographic patterns
75
4.4. Article IV: Against all odds: reconstructing the evolutionary
history of Scrophularia (Scrophulariaceae) despite
high levels of incongruence and reticulate evolution
5. General Discussion
95
125
5.1. Phylogenetic relationships in Rhinantheae and taxonomic implications
125
5.2. The biogeographic history of Scrophularia
128
5.2.1. Eastward migrations
130
5.2.2. Westward migrations
130
5.3. The influence of geography and topography on diversification
5.3.1. Origin and expansion of Scrophularia
132
132
5.3.2. Diversification in the Irano-Turanian region and the
Mediterranean
5.3.3. Diversification in Eastern Asia and the New World
5.4. The influence of hybridization on diversification
133
134
135
5.4.1. Phylogenetic tree incongruence and intra-individual
polymorphism
135
5.4.2. Hybrid speciation in Scrophularia - homoploid hybrid species
137
5.4.3. Allopolyploid hybrid species and hybrid lineages
139
5.4.4. Combined effects of topography, hybridization and
climate fluctuations
140
5.5. Taxonomy and morphological traits
140
5.6. Conclusions and Outlook
142
6. Literature
145
7. Acknowledgements
161
8. Curriculum vitae
163
XIV
1. Summary
This dissertation presents the results of my doctoral studies, which were
focussed on main phylogenetic relationships and important evolutionary processes
within two families of Lamiales, Scrophulariaceae Juss. (dealing with the type genus
Scrophularia L.) and Orobanchaceae Vent. (tribe Rhinantheae Lam. & DC.). Rhinantheae
are hemiparasitic plants with a predominantly Northern Hemisphere distribution,
alongside some representatives which have radiated on southern continents, e.g. the
genus Bartsia L. sensu Molau (1990) with several Andean and two afromontane species.
The figwort genus Scrophularia is widespread across the temperate Northern
Hemisphere. It comprises about 250 species of mainly herbaceous or suffrutescent
perennials, and is characterized by considerable morphological variability and
chromosomal diversity. Reticulate evolution is common in Scrophularia as well as
several genera within Rhinantheae.
The aim of this thesis was 1) to test previous phylogenetic hypotheses of
Rhinantheae, also by including several small genera; 2) to investigate phylogenetic
relationships within Bartsia sensu Molau (1990) and Odontites Ludw., and to re-evaluate
existing taxonomic concepts; 3) to verify the monophyly of Scrophularia and, for the first
time, establish a comprehensive phylogenetic framework including all relevant lineages;
4) to reconstruct the biogeographic history of the genus, to gain insight into colonization
pathways and the processes which have formed present distribution patterns; 5) to
determine possible causes for the high species diversity and endemicity in several areas
of the distribution range; 6) to assess the influence of hybridization on the evolutionary
history of Scrophularia, and to identify possible parental lineages of polyploid (and
homoploid) hybrid species; 7) to examine the impact of hybridization and other
processes on molecular phylogenetic reconstruction; and 8) to explore how the
problems resulting from this influence can be alleviated or even utilized to elucidate the
underlying processes; and to compile corresponding practical workflows.
To achieve these goals, I followed a molecular systematic approach. Nucleotide
sequences from five non-coding chloroplast loci (intergenic spacers trnQ-rps16 and
psbA-trnH, the trnL-trnF region, part of the trnK region, and the rps16 intron) as well as
nuclear ribosomal DNA (the internal transcribed spacer region, ITS) were analyzed to
infer phylogenetic relationships. This was done using Bayesian inference, Maximum
Likelihood and Maximum Parsimony approaches, and statistical parsimony networking.
Information from nuclear intra-individual sequence polymorphisms as well as plastid
indels was coded, and selected individuals were cloned for the ITS marker. Regarding
plastid and nuclear data, among-dataset incongruence and within-dataset uncertainty
and conflict were assessed with statistical tests and examined using unrooted
phylogenetic networks (split networks). The biogeographic history of the genus was
inferred using divergence time estimation and ancestral area reconstruction. Where
appropriate, a taxon duplication approach was applied to permit the incorporation of
incongruent taxa into a combined analysis of markers from both compartments. Plant
material for the studies was obtained from specimens on loan from several herbaria and
collected during herbarium studies at Vienna (W/WU) and Harvard University (A/GH);
several species were cultivated at the greenhouses at the Botanical Garden in Munich.
1
According to my phylogenetic analyses, Rhinantheae contain a core group of four
major lineages. Bartsia sensu Molau (1990) is polyphyletic; most species belong to
either the African genus Hedbergia Molau or the hitherto monotypic Bellardia All., with
the latter also including Parentucellia Viv. For Odontites, a broader circumscription was
proposed; Rhinantheae are updated to comprise app. 13 genera. Topological
incongruence among plastid and nuclear trees in some cases could be the result of
intergeneric hybridization; this process might also have created the genera Nothobartsia
Bolliger & Molau and Odontitella Rothm. However, plastid and nuclear markers can be
reconciled by pruning conflicting taxa.
By contrast, my studies on Scrophularia revealed far more substantial plastid nuclear marker incongruence. In ITS, intra-individual nucleotide polymorphism is
widespread, which argues for incomplete concerted evolution and causes topological
ambiguity. I inferred these phenomena to be caused by a combination of reticulation
(including introgression), incomplete lineage sorting ("ILS") of ancestral
polymorphisms, and, regarding ITS, possibly incipient pseudogenization and inter-array
heterogeneity. Species diversity in Scrophularia is however largely attributable to
ancient and recent hybridization and (allo-)polyploidization, for which I was able to
illustrate several examples. It is evident that these processes were one major driving
force in the evolutionary history of the genus. The other key factor likely was allopatric
speciation, promoted by the heterogeneous topography of the preferred mountain
habitats. Often, these two factors have supposedly acted in combination, also under the
influence of different climatic periods and associated temperature oscillations (including
the Pleistocene glaciations). The emergence and diversification of Scrophularia are
closely linked to geological and climatic events in the Irano - Turanian region and
Central / Eastern Asia; the genus originated in Southwestern Asia during the Miocene.
Most diversification events and further successful dispersals to its present distribution
areas occurred in the Pliocene and Pleistocene. This also involved the colonization of the
New World across the Bering Land Bridge and dispersal to the Macaronesian and
Caribbean islands. My analyses support two main species groups in Scrophularia, which
correspond to previously proposed major sections. The genus is monophyletic upon
inclusion of the Himalayan genus Oreosolen Hook.f. Like in Rhinantheae, morphological
synapomorphies are rarely present, and some morphological traits have presumably
evolved in convergence.
Scrophularia represents a useful model for studying evolutionary history in the
context of reticulation. This dissertation provides the first comprehensive phylogenetic
framework for the genus, which will serve as a solid basis for subsequent research. The
methodical workflows implemented in this thesis constitute valuable guidelines for
researchers dealing with similarly complex plant lineages.
1. Zusammenfassung
Die vorliegende Dissertation stellt die Ergebnisse meiner Doktorarbeit vor, die
die grundsätzlichen phylogenetischen Beziehungen und wichtige evolutionäre Prozesse
innerhalb zweier Familien der Lamiales zum Schwerpunkt hatte, den Scrophulariaceae
Juss. (und hierbei die Typusgattung Scrophularia L.) und den Orobanchaceae Vent.
(Tribus Rhinantheae Lam. & DC.). Die Rhinantheen sind halbparasitische Pflanzen mit
einer vornehmlich nordhemisphärischen Verbreitung, abgesehen von einigen Vertretern
mit Radiationen auf südlichen Kontinenten, wie z.B. der Gattung Bartsia L. sensu Molau
(1990) mit mehreren andinen sowie zwei afromontanen Arten. Die Gattung
2
Scrophularia, die Braunwurz, ist weitverbreitet auf der gemäßigten Nordhalbkugel. Sie
umfaßt ca. 250 hauptsächlich krautige oder halbstrauchige, mehrjährige Arten, und ist
durch
erhebliche
morphologische
Variabilität
sowie
eine
Vielfalt
an
Chromosomenzahlen gekennzeichnet. Vernetzte Evolution ("reticulate evolution") ist
häufig, wie auch in einigen Gattungen der Rhinantheae.
Das Ziel dieser Doktorarbeit war 1) bestehende stammesgeschichtliche
Hypothesen betreffend die Rhinantheae zu überprüfen, auch unter Einbeziehung
mehrerer kleiner Gattungen; 2) phylogenetische Beziehungen innerhalb Bartsia sensu
Molau (1990) und Odontites Ludw. zu erforschen, und bestehende taxonomische
Konzepte neu zu bewerten; 3) die Monophylie von Scrophularia zu bestätigen und
erstmalig ein umfassendes verwandtschaftliches Grundgerüst zu etablieren, das alle
relevanten Linien umfaßt; 4) die biogeographische Entwicklung der Gattung zu
rekonstruieren, um Einblicke in Ausbreitungswege zu erhalten sowie in die Prozesse,
welche gegenwärtige Verbreitungsmuster geformt haben; 5) mögliche Gründe für die
hohe Artdiversität und den Endemismus in mehreren Bereichen des
Verbreitungsgebiets zu finden; 6) den Einfluß von Hybridisierung auf die
Evolutionsgeschichte von Scrophularia einzuschätzen, und mögliche Elternlinien
polyploider (und homoploider) hybridogener Arten zu identifizieren; 7) die Einwirkung
von Hybridisierung und anderen Prozessen auf die molekularbasierte Rekonstruktion
von Stammesgeschichte zu untersuchen; und 8) zu erkunden wie die Probleme, die sich
aus diesem Einfluß ergeben, abgeschwächt oder sogar nutzbar gemacht werden können,
um die zugrundeliegenden Prozesse aufzuklären; und entsprechende praktische
Arbeitsabläufe zusammenzustellen.
Um diese Ziele zu erreichen, verfolgte ich einen molekularsystematischen Ansatz.
Nukleotidsequenzen von fünf nicht-codierenden Chloroplasten-Loci (intergenic spacer
trnQ-rps16 und psbA-trnH, die trnL-trnF Region, ein Teil der trnK Region, und das rps16
Intron), sowie ribosomale Kern-DNA (die internal transcribed spacer Region, ITS),
wurden analysiert, um auf Verwandtschaftsbeziehungen rückzuschließen. Dabei wurden
Bayes'sche Statistik, Maximum-Likelihood- und Maximum-Parsimony-Methoden, und
Netzwerke auf Basis statistischer Parsimonie verwendet. Information aus nukleären
intraindividuellen Sequenzpolymorphismen und plastidären Insertionen-Deletionen
(Indels) wurde codiert, und ITS-Sequenzbereiche ausgewählter Individuen wurden
kloniert. Inkongruenzen zwischen den Plastiden- und Kerndatensätzen sowie Konflikt
und Unklarheit innerhalb derselben wurden mittels statistischer Tests bewertet, und
unter Verwendung von nicht-gewurzelten phylogenetischen Netzwerken (SplitNetzwerken) untersucht. Aus der Datierung von Artaufspaltungen (divergence time
estimation) sowie der Rekonstruktion ursprünglicher Verbreitungsgebiete (ancestral
area reconstruction) wurde die historische Biogeographie der Gattung abgeleitet. Wo
nötig wurde eine Methode zur Aufteilung der Sequenzinformation einzelner
Akzessionen (taxon duplication approach) angewandt, um inkongruente Taxa in eine
kombinierte Analyse von Markern beider Kompartimente einbeziehen zu können.
Pflanzenmaterial für die Studien wurde von entliehenen Belegen diverser Herbarien
entnommen, auch wurden Herbarstudien in Wien (W/WU) und an der Universität
Harvard (A/GH) durchgeführt; mehrere Arten wurden in den Gewächshäusern des
Botanischen Gartens München kultiviert.
Meine Abstammungsanalysen zeigen, daß die Tribus Rhinantheae eine zentrale
Artengruppe aufweist, die aus vier Hauptlinien besteht. Bartsia sensu Molau (1990) ist
polyphyletisch; die meisten Arten gehören entweder der afrikanischen Gattung
Hedbergia Molau an oder der bis dato monotypischen Bellardia All.; letztere beinhaltet
auch Parentucellia Viv. Eine breitere Umschreibung wurde für Odontites vorgeschlagen;
3
die Tribus Rhinantheae besteht nun aus ca. 13 Gattungen. Inkongruenzen bezüglich der
Position von Taxa in Plastiden- versus Kernmarker-Stammbäumen könnten in einigen
Fällen das Ergebnis gattungsübergreifender Hybridisierung sein; dieser Prozess könnte
auch die beiden Gattungen Nothobartsia Bolliger & Molau und Odontitella Rothm.
erzeugt haben. Nach Ausschluß zwiespältiger Akzessionen können plastidäre und
nukleäre Marker jedoch kombiniert analysiert werden.
Im Gegensatz dazu offenbarten meine Studien an Scrophularia weit
beträchtlichere Konflikte zwischen Chloroplasten- und Kernmarkerdaten. Zusätzlich
sind innerhalb der ITS-Sequenzen häufig Nukleotid-Polymorphismen vorhanden, was zu
Unsicherheiten in der Baumtopologie führt und für eine unvollständige
Homogenisierung verschiedener Sequenzkopien spricht (incomplete concerted
evolution). Diese Phänomene sind nach meinen Schlußfolgerungen das Ergebnis einer
Kombination von retikulater Evolution (inklusive Introgression), unvollständiger
Aufteilung ursprünglicher Allelvarianten (incomplete lineage sorting, "ILS"), und, im Fall
von ITS, möglicherweise beginnender Pseudogenisierung und abweichender Kopien in
getrennten Genbereichen. Die Artenvielfalt in Scrophularia ist jedoch zum großen Teil
auf historische und rezente Hybridisierung und (Allo-)Polyploidisierung
zurückzuführen, für die ich mehrere Beispiele aufzeigen konnte. Es ist offensichtlich,
daß diese Prozesse eine der Hauptantriebskräfte in der Evolutionsgeschichte der
Gattung waren. Der andere Schlüsselfaktor war wahrscheinlich geographische
Artbildung, gefördert durch die ungleichmäßige Topographie der bevorzugten bergigen
Habitate. Diese beiden Faktoren wirkten vermutlich des Öfteren kombiniert, auch unter
dem
Einfluß
verschiedener
Klimaperioden
und
der
dazugehörigen
Temperaturschwankungen (inklusive der Vergletscherungen während des Quartärs).
Das Erscheinen und die Auffächerung von Scrophularia sind eng mit geologischen und
klimatischen Ereignissen in der irano-turanischen Florenregion sowie in Zentral- und
Ostasien verbunden; die Gattung entstand in Südwestasien, während des Miozän. Die
meisten Artaufspaltungsereignisse und die weitere erfolgreiche Ausbreitung in ihre
heutigen Verbreitungsgebiete fanden im Pliozän und Pleistozän statt. Dies beinhaltete
auch die Kolonisierung der Neuen Welt über die Bering-Landbrücke und die
Ausbreitung auf die makaronesischen und karibischen Inseln. Meine Untersuchungen
stützen zwei große Artgruppen in Scrophularia, die bisher vorgeschlagenen
Hauptsektionen entsprechen. Die Gattung ist monophyletisch nach Einbeziehung der
himalayischen Gattung Oreosolen Hook.f. Wie in den Rhinantheen sind kaum
gemeinsame abgeleitete morphologische Merkmale (Synapomorphien) vorhanden, und
einige morphologische Eigenschaften haben sich vermutlich konvergent entwickelt.
Scrophularia stellt ein geeignetes Modell dar, um Evolutionsgeschichte unter dem
Einfluß von Retikulation zu erforschen. Diese Dissertation legt das erste umfassende
Grundgerüst der Stammesgeschichte der Gattung vor, das als solide Basis für
weitergehende Forschungen dienen wird. Die methodischen Arbeitsschritte, die in
dieser Arbeit angewandt wurden, bilden wertvolle Richtlinien für Forscher, die mit
ähnlich komplexen Pflanzengruppen arbeiten.
4
2. Introduction
2.1. Biology and Systematics of Scrophulariaceae Juss.
2.1.1. Traditional circumscription of Scrophulariaceae
In his book "Genera Plantarum, secundum ordines naturales disposita", Antoine
Laurent de Jussieu in 1789 validly published the scientific name Scrophulariaceae Juss.,
the figwort family, as an order named "Scrophulariae" (Jussieu, 1789). However, the
name had been mentioned already in 1782 by Jean François Durande, who presented a
French translation of de Jussieu's classification system in his "Notions élémentaires de
botanique" (Durande, 1782). David Don (1835) and George Don (1838) provided a
description of "Scrophularineae" containing nine and eleven tribes, respectively (see
Table 1), while Verbascin(e)ae, Rhinanthaceae, Orobancheae, Cheloneae, Sibthorpiaceae
and Aragoaceae were kept as separate families. At the same time, George Bentham
presented a first small account on the family proposing 12 tribes (Bentham, 1835,
1836). For de Candolle's "Prodromus" (Bentham, 1846), he improved this concept and
also included three subfamilies; and in "Genera Plantarum" (Bentham, 1876), he restructured his classification, recognizing subfamilies Pseudosolaneae, Antirrhin(o)ideae
and Rhinanth(o)ideae. Amongst other characters, he distinguished infrafamiliar groups
based on the mode of aestivation of the corolla: in Antirrhinoideae (and
Pseudosolaneae), the posterior / upper corolla lobes or the upper lip is external in bud
(antirrhinoid aestivation, see Fig. 1), while in Rhinanthoideae the lower lip (respectively
either the anterior / lower lobe or the lateral lobes) are positioned externally
(rhinanthoid aestivation). The use of this characteristic later was adopted by many
authors; although occasionally it may be hard to determine, it has long been the basis for
distinguishing subfamilies in Scrophulariaceae (see Armstrong and Douglas, 1989).
Indeed, the latter authors also concluded that corolla aestivation may per se be suitable
for distinguishing higher taxonomic categories.
Based on Bentham (1876), von Wettstein presented a treatment of
Scrophulariaceae in Engler and Prantl's "Die natürlichen Pflanzenfamilien" (Wettstein,
1891), which also used corolla aestivation as diagnostic character, and has been widely
referred to until today. Scrophulariaceae sensu Wettstein (1891) are photosynthetic or
root parasitic (including hemiparasitic) herbs, subshrubs, shrubs or trees, with
alternate, opposite or whorled leaves, without stipules and with diverse inflorescences
bearing axillary, zygomorphic flowers, which are characterized by a persisting calyx,
mostly four stamens, and a bilocular ovary with central placentation; the fruits are
capsules (or berries), and the seeds contain endosperm. The author emphasized the
considerable morphological variability within the family, but remarked that anatomical
characters were considered helpful in separating Scrophulariaceae from other families
and in diagnosing groups of genera. However, he also stated that close relationships
often hindered reliable delimitations, and emphasized the need for using combinations
of vegetative and floral characters for distinguishing families. Twelve tribes were
recognized (Table 1): Verbasceae and Aptosimeae (Pseudosolaneae), Calceolarieae,
Hemimerideae, Antirrhineae, Cheloneae, Manuleae and Gratioleae (Antirrhinoideae),
and Digitaleae, Gerardieae and Rhinantheae (Rhinanthoideae); furthermore, compared
to Bentham (1876), Wettstein (1891) added tribe Selagineae to Antirrhinoideae.
The lack of distinct synapomorphic characters for Scrophulariaceae, together
with the striking similarities to closely related families, encouraged Hallier (1903) to
include four more tribes: Leucophylleae (already proposed within Pseudosolaneae by
5
Bentham, 1876), Plantagineae, Lentibularieae, and Orobancheae. Hallier (1903) was the
first taxonomist to abandon the concept of delimiting subfamilies based on corolla
aestivation. Further infrafamiliar concepts are
listed in the comprehensive review by Thieret
(1967). Francis W. Pennell, in two treatments
on the Scrophulariaceae of Eastern North
America (Pennell, 1919, 1935) dismissed
subfamily Pseudosolaneae (which had been
erected to accommodate taxa with a supposedly
close relationship to Solanaceae) and instead
included Verbasceae and Leucophylleae into
Antirrhinoideae. Chosen tribal names refer to
Bentham (1846), e.g. in resurrecting Veroniceae
(which had been given subtribal rank within
Digitaleae by Bentham, 1876), Buchnereae (a
subtribe of Gerardieae sensu Bentham, 1876,
but later used to replace the name Gerardieae
which is based on a synonym) or Euphrasieae
(which corresponds to Rhinantheae) of the
Rhinanthoideae; three additional tribes in
Pennell (1919; Paulownieae, Russelieae,
Angelonieae) each contain only one, introduced or cultivated genus previously assigned
to Cheloneae and Hemimerideae by Wettstein (1891).
Since then, many authors have more or less followed Wettstein (1891) in their
classifications, especially regarding more "distant" lineages: Cronquist (1981) defined
Scrophulariaceae exclusive of Plantaginaceae, Orobanchaceae, Lentibulariaceae, and
Globulariaceae, while additionally excluding Paulownia Siebold & Zucc. Takhtajan's
(1980) treatment agreed with this definition, except for Globulariaceae, which together
with Selaginaceae were included into Scrophulariaceae; the treatment comprised four
subfamilies, Scrophularioideae, Rhinanthoideae, Globularioideae, and Selaginoideae.
Later however, the author incorporated Orobanchaceae into the family as well
(Takhtajan, 1987). Thorne (1992), who recognized subfamilies Scrophularioideae,
Rhinanthoideae and Orobanchoideae, included Selagineae but not Globulariaceae; he
agreed with Hallier (1903) in including Orobanchaceae and (again) Leucophylleae, but
did not incorporate Plantaginaceae and Lentibulariaceae. Furthermore, he included
Schlegelieae into the family, which he synonymized with Paulownieae.
While several families and enigmatic genera were tentatively in- or excluded
from Scrophulariaceae, others were mostly regarded as distinct according to these
authors, e.g. Hippuridaceae, Callitrichaceae, Myoporum Banks & Sol. ex G.Forst., and
Buddleja L., although the latter had already been included into Scrophulariaceae as tribe
Buddleieae/Buddlejeae (D Don, 1835; G Don, 1838; Bentham, 1835, 1836, 1846).
Nonetheless until the early 1990's, Scrophulariaceae were one of the largest families
within Lamiales, with app. 30 tribes recognized (according to Barringer, 1993, who
himself newly described tribes Alonsoeae, Bowkerieae, Caprarieae and Freylinieae), and
with app. 4000 species according to Cronquist (1981) or 3000 species within 220
genera according to Thorne (1992).
Fig. 1. Antirrhinoid aestivation in Scrophularia
kakudensis Franch. The reddishly marginate
two upper lobes are external in bud, the two
lateral and lower median lobe are positioned
beneath
2.1.2. Current understanding of phylogenetic relationships
The classical circumscription of Scrophulariaceae was profoundly challenged
when DNA-based studies became the central means of investigating phylogenetic
6
D. Don
1835
9
G. Don
1838
11
Calceolarieae
Calceolarieae
Antirrhineae
Scrophularieae
Gratioleae
Antirrhineae
Scrophularieae
Gratioleae
Bentham
1835/1836
12
Hemimerideae
Antirrhineae
Bentham
1846
15
Antirrhinideae
Calceolarieae
Hemimerideae
Antirrhineae
Bentham
1876
12 (12 subtribes)
Antirrhinideae
Calceolarieae
Hemimerideae
Antirrhineae
von Wettstein
1891
12 (4 subtribes)
Antirrhinoideae
Calceolarieae
Hemimerideae
Antirrhineae
Cheloneae
Gratioleae
Cheloneae
Escobedieae
Gratioleae
Gratioleae
(Limosellinae)
Manuleieae
Hallier
1903
13+3
Pennell
1919
7+3
Antirrhinoideae
Pennell
1935
10
Antirrhinoideae
Calceolarieae
Hemimerideae
Antirrhineae
Antirrhineae
Antirrhineae
Cheloneae
Cheloneae
Cheloneae
Cheloneae
Gratioleae
(Limosellinae)
Manuleae
Selagineae
Gratioleae
Gratioleae
Gratioleae
Oxelman et al.
2005
8
APG website
2016
7
Hemimerideae
Hemimerideae
Scrophularieae
Scrophularieae
Limoselleae
Limoselleae
Myoporeae
Myoporeae
Leucophylleae
Aptosimeae
Leucophylleae
Aptosimeae
Buddlejeae
Buddlejeae
Manuleeae
Selagineae
Collinsieae
Verbasceae
Buddlejeae
Veroniceae
Gerardieae
Buchnereae
Euphrasieae
Buddleieae
Veroniceae
Teedieae
Hallerieae
Gerardieae
Buchnereae
Euphrasieae
Verbasceae
Salpiglossideae
Salpiglossideae Salpiglosseae
Rhinanthideae
Buddle(i)eae
Buddleieae
Digitaleae
Digitaleae
Sibthorpieae
Veroniceae
Veroniceae
Teedieae
Gerardieae
Buchnereae
Rhinantheae
Gerardieae
Buchnereae
Euphrasieae
Pseudosolaneae Pseudosolaneae
Leucophylleae
Aptosimeae
Aptosimeae
Verbasceae
Verbasceae
Rhinanthideae
Rhinanthoideae
Digitaleae
(Sibthorpiinae)
(Veronicinae)
Digitaleae
Leucophylleae
Aptosimeae
Verbasceae
Leucophylleae
Verbasceae
Verbasceae
Rhinanthoideae Rhinanthoideae
Digitaleae
Digitaleae
Veroniceae
Veroniceae
Teedieae
Gerardieae
(Buchnerinae)
Euphrasieae
Gerardieae
Gerardieae
Buchnereae
Rhinantheae
Rhinantheae
Buchnereae
Euphrasieae
Rhinantheae
Table 1. Taxonomic classification of Scrophulariaceae as provided by various authors. The number of recognized tribes is provided beneath each publication; complete
references are given in the Literature section. Subfamilies are underlined; names in bold denote the tribe the generic type Scrophularia L. was assigned to by the respective
author. Subtribes are given only if they refer to a tribe mentioned elsewhere. Hallier (1903) recognized three additional tribes not included in this table (Plantagineae,
Lentibularieae, Orobancheae); three further tribes listed by Pennell (1919) each only contained one, introduced or cultivated genus (Paulownieae, Russelieae,
Angelonieae)
7
relationships and character evolution. Olmstead and Reeves (1995) were the first
to discover that Scrophulariaceae s.l. were not monophyletic, but composed of two
distinct groups of taxa (named "scroph I" and "scroph II" by the authors). Thereby, it
also became evident that corolla aestivation in Scrophulariaceae s.l. is a homoplastic
character, as both antirrhinoid and rhinanthoid aestivation were found within "scroph
II" (see also Young et al., 1999). Further work by Olmstead et al. (2001) revealed that
Scrophulariaceae s.str. comprised Bentham's (1876) tribes Verbasceae, Aptosimeae,
Hemimerideae without Angelonia Bonpl., Manuleeae and Leucophylleae, Wettstein's
(1891) Selagineae, and surprisingly the small genera Myoporum and Buddleja (however
included into Scrophulariaceae by Bentham, 1846). As Verbasceae also comprised the
generic type Scrophularia L., the name of the tribe was consequently proposed to be
changed to Scrophularieae (a name already used by D Don, 1835). A second clade named
"Veronicaceae" (now Plantaginaceae due to nomenclatural priority reasons) included
Bentham's (1876) tribes Digitaleae, Antirrhineae, Cheloneae, and Gratioleae, tribe
Angelonieae (as recognized by Pennell, 1919), Globulariaceae without Selagineae, and
the two aquatic families Callitrichaceae and Hippuridaceae. Most importantly, the
"Veronicaceae" clade also included Plantago L. (Plantaginaceae), which in a way
corroborated Hallier (1903). The rhinanthoid tribes Buchnereae (Gerardieae) and
Rhinantheae (Euphrasieae) were found to cluster with Orobanchaceae, distant from
Scrophulariaceae s.str. and Plantaginaceae (see chapter 2.2.2.). Calceolarieae, due to
their distinct position, were given family rank, as well as Paulownieae and Schlegelieae.
Subsequent work corroborated and expanded these findings, e.g. Beardsley and
Olmstead (2002; on Phrymaceae), Müller et al. (2004; on Lentibulariaceae), Albach et al.
(2005; on Plantaginaceae), Oxelman et al. (2005; on Lamiales), Rahmanzadeh et al.
(2005; on Linderniaceae), Bennett and Mathews (2006; on Orobanchaceae), Schäferhoff
et al. (2010; on Lamiales), Barker et al. (2012; on Phrymaceae), Refulio Rodriguez and
Olmstead (2014; on Lamiidae); see also the review by Tank et al. (2006). Relationships
among families within Lamiales as accepted today are illustrated in Fig. 2.
Within the previously defined Scrophulariaceae s.str., Kornhall et al. (2001)
revealed Selagineae to be nested within Manuleeae. Kornhall and Bremer (2004) found
that the latter clade also includes Limosella L., which resulted in the name of the tribe
being changed to Limoselleae (which had been a subtribe of Gratioleae sensu Bentham,
1876). Oxelman et al. (2005) proposed eight tribes based on molecular results: apart
from tribes in accordance with Olmstead et al. (2001), i.e. Scrophularieae, Aptosimeae,
Hemimerideae, Limoselleae, Leucophylleae, Myoporeae, and Buddlejeae, Oxelman et al.
(2005) included Teedieae, consisting of four taxa closely allied to Buddlejeae (and not
identical in taxon arrangement to Teedieae sensu G Don, 1838 or Bentham, 1836). The
APG website (Stevens, 2001 and onward; see also APG IV, 2016) largely followed this
concept, but included Teedieae into Buddlejeae. A description of each tribe is available
in Tank et al. (2006), their phylogenetic relationships are shown in Fig. 2.
Scrophulariaceae in their present circumscription comprise 1880 species in 59
genera (Stevens, 2001 and onward) and have a predominantly southern hemispheric
distribution, with four of the seven tribes centered in South Africa (Tank et al., 2006).
Diagnosis of tribes by morphological characters has remained difficult; many of the
traditionally used characteristics may be plesiomorphic, which explains the large
similarities among several Lamiales families. As outlined by Tank et al. (2006),
Scrophulariaceae s.l. acted as a "repository" for typical Lamiales taxa lacking clear
synapomorphic characters (which exist e.g. in Bignoniaceae or Lamiaceae). In
consequence, Scrophulariaceae s.l. have contributed taxa to a number of other families
in the course of their reduction (see Fig. 2), mainly to Plantaginaceae and
Orobanchaceae but also to Phrymaceae (e.g. Leucocarpus D.Don from Cheloneae sensu
8
OUTGROUP
Oleaceae
Calceolariaceae
Gesneriaceae
Plantaginaceae
Hemimerideae
Androya
Myoporeae
Leucophylleae
Buddlejeae
Limoselleae
Scrophulariaceae
Aptosimeae
Scrophularieae
Stilbaceae
Linderniaceae
Pedaliaceae
Martyniaceae
Acanthaceae
Bignoniaceae
Verbenaceae
Schlegeliaceae
Lentibulariaceae
Lamiaceae
Mazaceae
Phrymaceae
Paulowniaceae
Rehmannieae
Cymbarieae
Orobancheae
Brandisia
Rhinantheae
Buchnereae
Orobanchaceae
Lindenbergieae
Pedicularideae
Fig. 2. Phylogenetic relationships in Lamiales. Main tree based on Refulio Rodriguez and Olmstead (2014)
and the APG website (Stevens, 2001 and onward). Tribal relationships in Scrophulariaceae and
Orobanchaceae are according to Oxelman et al. (2005) and McNeal et al. (2013), respectively. Families
which now contain representatives of the former Scrophulariaceae s.l. are marked in red. Selected
representative genera from top to bottom - Scrophulariaceae: Nemesia Vent., Hemimerideae (photo by
Andreas Fleischmann); Verbascum L., Scrophularieae; Orobanchaceae: Orobanche L., Orobancheae;
Rhinanthus L., Rhinantheae
Wettstein, 1891; Mimulus L. from Gratioleae), Calceolariaceae (from Calceolarieae),
9
Linderniaceae (e.g. Torenia L. from Gratioleae), Stilbaceae (e.g. Halleria L. from
Cheloneae; see also tribe Hallerieae in G Don, 1838), Mazaceae (e.g. Lancea Hook.f. &
Thomson from Gratioleae sensu Wettstein, 1891), Paulowniaceae and Schlegeliaceae
(e.g. Paulownia and Synapsis Griseb., respectively, from Cheloneae).
2.2. Parasitism in plants: the example of Orobanchaceae Vent.
2.2.1. Parasitism
Parasitism is an effective strategy found throughout the tree of life (Poulin and
Morand, 2000) including flowering plants (Kuijt, 1969). While myco-heterotrophic (i.e.,
being dependent on mycorrhizal fungi) plants rely on a fungal partner, several parasitic
plants obtain other plants as hosts. The roots of the parasites are modified into special
organs called haustoria (Kuijt, 1969), which are used to exploit a host by connecting to
its stems or roots (stem vs. root parasites). In flowering plants, parasitism is thought to
have evolved 12-13 times independently and is now known in 12 orders, with app. 1 %
of all angiosperms being parasitic (Westwood et al., 2010). Hemiparasitic plants are
photosynthetic and only extract water and nutrients from their host by connecting to the
xylem; while some of them are dependent upon a host to mature (obligate parasites),
others do not necessarily require this connection to complete their life cycle (facultative
parasites). In contrast to hemiparasites, holoparasitic plants are fully heterotrophic and,
apart from water and nutrients, also obtain all necessary carbon compounds from their
host's phloem. The transition to holoparasitism leads to a variety of changes, including
the reduction of leaves, the loss of non-haustorial roots, and in extreme cases most of the
vegetative tissues (in Rafflesiaceae) as outlined by dePamphilis et al. (1997). The loss of
photosynthesis and the associated pigments is accompanied by a reduction of the plastid
genome and a shift towards higher substitution rates in the remaining genes (e.g. in
Orobanchaceae Vent.; Wicke et al., 2013). Cusimano and Wicke (2016) found that
functional losses in Orobanchaceae occur within 10 million years ("my") after transition
to obligate parasitism, although many photosynthesis-related genes survive within the
nuclear or mitochondrial genomes. The genomes of nonphotosynthetic species of
Orobanchaceae were also shown to contain higher amounts of repetitive DNA (Piednoël
et al., 2012).
2.2.2. Orobanchaceae Vent.
In Lamiales, parasitism has evolved only within Orobanchaceae. This plant family
today comprises a full spectrum of (few) autotrophic (non-parasitic) plants, facultative
and obligate hemiparasites, as well as holoparasites (see Westwood et al., 2010). Its
members are globally distributed with a focus on temperate regions of the Northern
Hemisphere and the Old World; most genera only comprise few species with limited
distributions (Wolfe et al., 2005). It also includes some serious crop pests, e.g. Striga
Lour., Orobanche L., or Alectra Thunb. (see citations in Wolfe et al., 2005).
The taxonomic boundaries of Orobanchaceae have changed considerably
throughout their history. The first valid description of the family name (as
"Orobanchoideae") dates back to 1799 and was provided by Etienne Pierre Ventenat in
his "Tableau du Regne Végétal" (Ventenat, 1799, p. 292). G Don (1838) described the
"very natural family" as distinctive by their persistent corolla, unilocular ovary (two
characters which were partially refuted later, see below), and their usually parasitic life
strategy with the herbaceous plants lacking chlorophyll and "proper" leaves. He also
10
emphasized the minute, globular, undifferentiated embryo typical for Orobanchaceae.
Further characteristics of the family, also according to Beck von Mannagetta (1893),
included mostly racemose terminal inflorescences, and flowers with usually two-lipped
tubular corollas, four didynamous stamens and a superior ovary.
Close relationships of Orobanchaceae to Scrophulariaceae s.l. became evident
early. Morphologically intermediate taxa like Lathraea L. were placed either in
Orobanchaceae (e.g. Beck von Mannagetta, 1893) or Scrophulariaceae (e.g. Warming,
1895). Wettstein (1897) noted close relationships of Rhinantheae and other
scrophulariacean genera to Orobanchaceae. Hallier (1903), who included
Orobanchaceae as tribe Orobancheae into Scrophulariaceae s.l., discussed in detail the
similarities among Orobanche and several scrophulariacean taxa from the Rhinantheae
and Buchnereae. Boeshore (1920) postulated a morphological series leading from
hemiparasitic genera of the "pre-Olmsteadian" Scrophulariaceae s.l. via Lathraea to the
holoparasitic Orobanchaceae. He also stated that transitions occur between the typically
unilocular ovary of Orobanchaceae and the bilocular one of Scrophulariaceae.
At the time of the first molecular evidence for Scrophulariaceae s.l. being
polyphyletic (Olmstead and Reeves, 1995), other researchers aimed at revealing the
origin of parasitism within Lamiales. In a study that involved both parasitic
Orobanchaceae and Scrophulariaceae s.l., dePamphilis et al. (1997) found evidence for a
monophyletic clade containing hemi- and holoparasitic Scrophulariaceae s.l. as well as
holoparasitic Orobanchaceae. Analyses by Young et al. (1999), based on a larger
sampling and more molecular markers, yielded the same results. From these two studies
it has become evident that while there is only a single origin of parasitism within the
group, holoparasites have evolved several times independently, a finding which refutes
Boeshore's (1920) concept of a transitional series leading from hemi- to holoparasitism.
Another important result was that hemiparasites of the traditional Scrophulariaceae s.l.
are more closely related to Orobanchaceae than to Scrophulariaceae s.str. Young et al.
(1999) consequently proposed that the parasitic clade plus the non-parasitic genus
Lindenbergia Lehm. (Gratioleae sensu Wettstein, 1891) should be defined as
Orobanchaceae. Their results were corroborated by Olmstead et al. (2001, see chapter
2.1.2.) and others (Wolfe et al., 2005; Bennett and Mathews, 2006; McNeal et al., 2013).
Thus, the former rhinanthoid tribes Buchnereae and Rhinantheae were moved to
Orobanchaceae; however, neither of these tribes as traditionally defined was found to be
monophyletic (Young et al., 1999). Subsequent analyses by Wolfe et al. (2005) revealed
an additional lineage comprising the hemiparasites Castilleja Mutis ex L.f. and
Pedicularis L., while Bennett and Mathews (2006) identified another clade at a basal
position, composed of five taxa including the hemiparasitic Cymbaria L. and Schwalbea L.
Relationships among the five lineages were found to differ depending on the markers
used (Young et al., 1999; Wolfe et al., 2005; Bennett and Mathews, 2006). A
comprehensive analysis by McNeal et al. (2013), using several molecular markers and
the largest sampling to date, resulted in a new tribal classification, in which Buchnereae
are sister to Pedicularideae (which includes taxa mainly from Gerardieae and
Rhinantheae sensu Fischer, 2004). Sister to both are Rhinantheae; Brandisia Hook.f. &
Thomson (which had been included into Cheloneae, or different families) is sister to this
clade. The basalmost positions are occupied by a grade of Orobancheae, Cymbarieae,
and Lindenbergia, which is sister to all other Orobanchaceae. The APG website (Stevens,
2001 and onward) followed this concept, adding Lindenbergieae and Rehmannieae. The
latter contain the non-parasitic Rehmannia Libosch. ex Fisch. & C.A.Mey. and
Triaenophora Soler., two genera found to be sister to Lindenbergia and the remaining
Orobanchaceae by Xia et al. (2009). Thus, Orobanchaceae today comprise seven tribes
(see Fig. 2), 99 genera and 2060 species (Stevens, 2001 and onward). Apart from those
11
already mentioned at the beginning of this chapter, morphological characteristics of the
family include ascending = rhinanthoid petal aestivation (with very few exceptions), an
often galeate upper corolla lip, anther thecae which are acuminate at apex or rounded to
mucronate, and a bilocular to usually unilocular ovary (Fischer, 2004).
2.2.3. Tribe Rhinantheae Lam. & DC.
Tribe Rhinantheae Lam. & DC. as defined today has a worldwide distribution with
its main diversity center in the Northern Hemisphere. It contains facultative (e.g. the
annual Rhinanthus L., or Euphrasia L.) and obligate (e.g. the perennial Tozzia L.)
hemiparasites as well as one holoparasite (Lathraea). Further important genera are
Odontites Ludw. and Bartsia L.; the most species-rich genus within the tribe is Euphrasia
with 170-350 species (Stevens, 2001 and onward). Uribe-Convers and Tank (2015)
estimated the Rhinantheae clade to be app. 31 million years old, based on secondary
calibration using an ITS age estimate by Wolfe et al. (2005), or alternatively a geological
constraint.
Morphological synapomorphies for Rhinantheae are largely missing; the typical
mode of corolla aestivation in the tribe, rhinanthoid with the two lateral lobes of the
corolla clasping the lower median one in bud, is not exclusive within Orobanchaceae
(also found in Pedicularideae). Rhinantheae are annual or perennial herbs or subshrubs;
the racemose inflorescences feature scale-like, leaf-like or showy bracts. The corolla is
bilabiate; the two lobes of the upper lip are usually fused into a helmet-like or rostrate
galea. However, the lobes can also be bifid or variously bilobate; in few genera, the
upper lip lobes are even more or less free and expanded, resulting in almost
actinomorphic corollas, e.g. in Hedbergia abyssinica (Benth.) Molau (Fischer, 2004).
Molecular phylogenetic relationships were first revealed by Bennett and
Mathews (2006). The authors found Melampyrum L. to be sister to the remainder of
Rhinantheae, and a clade of Rhinanthus and Lathraea plus Rhynchocorys Griseb. placed
in a basal position. The remaining taxa (e.g. Euphrasia, Tozzia, Bartsia, Odontites) formed
a clade which is here referred to as the "core group of Rhinantheae". It also became clear
that Bartsia in its traditional circumscription was polyphyletic, with B. alpina L. placed
distant from the New World species, which grouped with Parentucellia Viv. Těšitel et al.
(2010) showed B. alpina to be sister to the rest of the core group of Rhinantheae. The
taxonomically difficult Odontites seemed to be paraphyletic with respect to one
(Bornmuellerantha Rothm.) of the four genera that had been segregated from it by
Rothmaler (1943) and Bolliger (1996). Phylogenetic positions of the other three taxa
(Macrosyringion Rothm., Bartsiella Bolliger, Odontitella Rothm.) remained however
uncertain, as well as the taxonomy of Bartsia; Bartsia trixago L. is now regarded (again)
as distinct genus (Bellardia All.), but African Bartsia (two species distributed in alpine
regions of Eastern Africa) had never been sampled.
Within Rhinantheae, Těšitel et al. (2010) found multiple independent transitions
from perennity to annuality in several unrelated lineages and concluded that typical
features of annuals must thus be analogous. Reconstructions of seed size evolution
revealed large seeds to be an ancestral feature; these are primarily present in basal
lineages of Rhinantheae. Furthermore, regarding the group of their RhinanthusLathraea, Rhynchocorys and Melampyrum clades, the largest seeds are found in annual
(compared to perennial) species. According to Těšitel et al. (2010), this is possibly due
to enhanced light competition pressure on the seedlings in their typical habitats.
Smaller, less competitive seeds in Odontites, Euphrasia and New World Bartsia were
correlated by the authors with their occurrence in stressful environments characterized
by open communities. The combination of reduced seed size together with a preference
12
for low-competition habitats might have promoted long distance dispersal in the latter
two genera (Těšitel et al., 2010).
The biogeographic origin of family Orobanchaceae was hypothesized north of the
Tethys Sea by Wolfe et al. (2005). Rhinantheae originated in temperate Western Eurasia
according to Těšitel et al., (2010), possibly including the Caucasian region, where
Rhynchocorys was inferred to have its origin by the authors. Other lineages have arisen
in the Mediterranean according to this analysis (e.g. Odontites, Nothobartsia Bolliger &
Molau, Bellardia). Uribe-Convers and Tank (2015) found Europe to be the ancestral
range for Rhinantheae and several of the backbone node ancestors, as well as
Rhynchocorys and Odontites. The range of the Bellardia ancestor could not be
unequivocally determined by their analyses: possible ranges included Europe, South
America and/or Eurasia.
2.3. The genus Scrophularia L.
2.3.1. Taxonomic history
The first mention of plants with this name is attributed to Dioscorides. Matthaeus
Silvaticus, in his famous pharmacopoeia "Liber / Opus pandectarum medicinae", which
was finished in 1317 and first printed in 1474, included "Scrofularia" because of its use
as a remedy for "scrofula" (Silvaticus, 1498), a disease characterized by infectional
swelling of the lymph nodes (see Mann, 2009). According to the Doctrine of Signatures,
the use of the plants against scrofula, as well as other "knots" like haemorrhoids,
anogenital warts or ulcers continued through medieval times and was based on the
knots found on the roots of S. nodosa L. (Ehrlich, 1720; Mann, 2009). The Latin genus
designation and the English name "knotted figwort" still refer to that use, as do old
german names like "Groß Feigwarzen-Kraut" or "Wurm-Kraut" (see Ehrlich, 1720;
Mann, 2009). Years before Linné published his master work "Species Plantarum" with
formal descriptions of several Scrophularia species (Linné, 1753), the Saxon-Thuringian
physician Heinrich Christian Ehrlich presented a dissertation on Scrophularia (Ehrlich,
1720), in which he compiled ancient and contemporary information on the genus. He
provided a morphological characterization himself, while also reproducing several
descriptions by former botanists (e.g. Johann Bauhin, Robert Morison, John Ray, Joseph
Pitton de Tournefort and August Bachmann/Rivinus), amongst those possibly one of the
oldest descriptions of Scrophularia, which he attributes to Adam Lonicer, Hieronymus
Bock/Tragus and Jacob Theodor/Tabernaemontanus, dating back to the 16th century:
"radicem albam, &, fabariae in modum, nodosam,
caules quadrangulos, fuscos, folia basilico ac urticae
conformia, flores cochlearum testas referentes,
vascula seminalia ex rotundo acuminata,
seminaque hyoscyamo similia"
[root pale, and, as in the Fabariae, knotty, shoots quadrangular, dark brownish, leaves
like those of basil and Urtica, flowers resembling snail shells, seed capsules acuminate
from a globe, and seeds similar to those of Hyoscyamus]
13
Apart from characterizing Scrophularia morphologically, Ehrlich (1720) mainly
concentrated on medicinal aspects. Wydler (1828) was the first to give a detailed
account on all species known at the time, and accepted 47 of them. He also cited historic
contributions on the genus (also compare Stiefelhagen, 1910), e.g. by C Bauhin (1623),
Willdenow (1800), Persoon (1806) and Sprengel (1825), who already knew eight to 48
species. His two proposed main sections were named by G Don (1838) as Scrophularia
sects. Venilia G.Don and Scorodonia G.Don (= S. sect. Scrophularia) and complemented by
a third, Canina G.Don (Table 2). The genus was placed in tribe Scrophularieae by the
author, while Bentham, after first associating it with Verbascum L. in Verbasceae
(Bentham, 1835), later sorted it into Cheloneae of Antirrhinoideae (Bentham, 1846,
1876). Bentham (1846) recognized Scrophularia sects. Venilia and Scorodonia, but
incorporated parts of the latter together with members from S. sect. Canina into a
section which he called Tomiophyllum Benth. Boissier (1879), who provided a treatment
mainly on the Asian species in his "Flora orientalis", apart from S. sects. Scorodonia and
Tomiophyllum recognized S. sect. Ceramanthe Rchb. (which had been elevated to genus
rank by Dumortier, 1834) and added S. sects. Mimulopsis Boiss. and Pycnanthium Boiss.,
each with one species only. The genus itself was kept in tribe Cheloneae by Boissier
(1879) and also by Wettstein (1891), a fact that was criticized by Hallier (1903), who
classified it into Hemimerideae while emphasizing its affinities to Verbasceae, i.e., Celsia
L. / Verbascum.
Wydler
1828
G Don
1838
Bentham
1846
Boissier
1879
Stiefelhagen
1910
143
47
60
79
78
"I" (2)
Venilia G.Don (3)
Venilia G.Don (8)
Ceramanthe Rchb. (11)
Pycnanthium Boiss. (1)
Mimulopsis Boiss. (1)
"II" (45)
Scorodonia
"A" (23)
G.Don (44)
Scorodonia G.Don (36)
Scorodonia G.Don (16)
Anastomosantes Stiefelh. (76)
Vernales Stiefelh. (9)
"B" (11)
Scorodoniae
"C" (11)
(Benth.)Stiefelh. (67)
Canina
G.Don (13)
Tomiophyllum Benth. (35) Tomiophyllum Benth. (49) Tomiophyllum Benth. (67)
Lucidae Benth. (23)
Oppositifoliae Boiss. (44)
Farinosae Stiefelh. (1)
Caninae Benth. (12)
Sparsifoliae Boiss. (5)
Orientales Stiefelh. (3)
Lucidae Stiefelh. (63)
Table 2. Intrageneric classification in Scrophularia. (Sub)sections as erected by previous authors (for
publication details refer to the Literature section). The number of recognized species is given beneath
each publication. Names in bold are main sections, subsections are listed using regular font. Numbers in
brackets after (sub)section names represent corresponding species numbers
The most recent classical monographic treatment was done by Stiefelhagen (1910). He
recognized 143 species and divided the genus into two main sections and five
subsections which are based mainly on leaf vein characteristics (clearly anastomosing or
not): Scrophularia sects. Tomiophyllum Benth. (= S. sect. Canina) and Anastomosantes
Stiefelh. (= S. sect. Scrophularia). In this thesis, to avoid confusion with other taxonomic
entities or clades, Stiefelhagen's (1910) names will be used to refer to the two main
sections in Scrophularia, instead of the nomenclaturally correct designations.
2.3.2. Morphology, distribution and phylogenetic relationships
Today, the genus Scrophularia comprises app. 250 species of mainly herbaceous
or suffrutescent perennials, and more rarely biennial or annual herbs (some examples
14
Fig. 3. Selected species of Scrophularia, from left to right and top to bottom: S. kakudensis Franch., S.
vernalis L., S. grandiflora DC., S. calliantha Webb. & Berthel., S. canina L. ssp. hoppii (W.D.J.Koch) P.Fourn., S.
villosa Pennell, S. lowei Dalgaard, S. nodosa L., S. smithii Hornem. ssp. smithii
are shown in Fig. 3). The plants are glabrous to densely villose, sometimes foetid, and
characterized by mostly quandrangular stems with opposite leaves (the upper ones
sometimes alternate) of various shapes and margin types. The inflorescence is typically
a thyrse, can be bracteolate or frondose, and wears few- or many-flowered cymes
15
arranged in mainly simple or compound dichasia. The flowers are generally
zygomorphic (now rather an exception within Scrophulariaceae, see Tank et al., 2006),
having a five-lobed calyx with or without a scarious margin, a sympetalous, bilabiate,
tubular to subglobose, typically ventricose corolla of greenish, reddish and/or brownish
color, and four fertile stamens which are more or less didynamous and have unilocular,
reniform anthers. The fifth (adaxial) stamen is generally sterile, forming a staminode
consisting of a basal part that is normally adnate to the upper corolla lip (the former
filament) and a free, scale-like part (the former anther), which can have various shapes
and sizes (clearly visible in the flower in Fig. 3i). The superior ovary is bilocular and
connected to a capitate to weakly bilobed stigma; a fleshy, nectariferous disc is found at
the base of the ovary. The fruit is a septicidal, globose to subconical, often apiculate
capsule containing numerous small seeds; these have an alveolated endosperm with
elongated alveoles (Fischer, 2004).
Similarities to the sister genus Verbascum include the general floral morphology
and leaf architecture, septicidal many-seeded capsules, often cymose inflorescences,
tricolporate pollen and single-celled subepidermal idioblasts. Further analogies are
found in habitat preferences and the temperate northern hemispheric distribution with
a Southwestern Asian center of diversity (see below), which also is unusual within
Scrophulariaceae s.str. (Hallier, 1903; Lersten and Curtis, 2001; Fischer, 2004; Oxelman
et al., 2005). Plesiomorphic characters, which are shared with other lineages, are typical
for Scrophulariaceae s.l. as described above, and are also present in Scrophularia and
Verbascum. Examples are the longitudinally ridged seeds also found in Limosella
(Scrophulariaceae s.str.), Linaria Mill. (Plantaginaceae), Lindernia All. (Linderniaceae),
Orobanchaceae and others (Hallier, 1903), or the presence of iridoids which cause the
typical blackening of plant material upon drying, and which as iridoid glycosides may
deter herbivores by their bitter taste (Fischer, 2004). In Scrophularia, morphological
characteristics in general are considered extremely variable among and within species
(Stiefelhagen, 1910; Grau, 1981a, b) and are not necessarily useful for delimitation of
species or species groups (Wydler, 1828; Grau, 1981b). The high morphological
plasticity found in the genus is also correlated with a surprisingly high tendency to
hybridization and polyploid formation (chromosome numbers range from 2n = 18 to 2n
= 96; Shaw, 1962; Goldblatt and Johnson, 1979 and onward); successful artificial
crossings were made by e.g. Goddijn and Goethart (1913), Shaw (1962), or Dalgaard
(1979), and several authors mentioned natural hybrids, e.g. Stiefelhagen (1910), Pennell
(1943) or Grau (1981a).
Species of Scrophularia occur in forests, on river banks, in scrubs and grassland,
in rock crevices or on gravelly substrates, on mountain slopes or cliffs, and also along
roadsides and in disturbed places. Several species are found in shady or moist habitats;
others are xerophytic and occur also in dry environments, while the genus comprises
only very few desert plants. Scrophularia occurs from coasts and lowlands to alpine
regions, with the majority of species inhabiting mountainous regions and highland
plateaus (Stiefelhagen, 1910; Shaw, 1962). The distribution of the genus extends
throughout the temperate zone of the Northern Hemisphere (Fig. 4), with very few
species expanding into tropical regions (these are mostly limited to higher altitudes, e.g.
in the Greater Antilles). The primary diversity center is situated in the Irano - Turanian
floristic region sensu Takhtajan (1986), with emphasis on Iran and Turkey (42 and 59
species; Flora of Turkey, Lall and Mill, 1978; Flora Iranica, Grau, 1981a; Flora of Turkey
Supplement, Davis et al., 1988), Afghanistan (Grau, 1981a), and, within the area of the
Flora of the USSR, the Caucasian and Central Asian region (Gorschkova, 1997). New
species are continuously described (e.g. Attar, 2006; Attar and Hamzeh'ee, 2006; Attar et
16
Fig. 4. Distribution of Scrophularia and assumed migration routes. The ancestral region of the genus as
inferred by biogeographic reconstructions is marked by a dashed line. The present-day distribution of the
genus is shown in light blue, centers of species diversity are highlighted in dark blue. The primary
diversity center is located close to the ancestral region of the genus. Black arrows indicate main dispersal
routes as inferred from plastid DNA data. Note that arrows in some cases may illustrate more than one
dispersal
al., 2006; Mozaffarian, 2010; Ahmad, 2014; Kandemir et al., 2014; Uzunhisarcıklı et al.,
2015). Secondary centers of species richness are located in China (36 species; Flora of
China, Hong et al., 1998) and southwards in the Himalayan region and Pakistan, as well
as the Iberian Peninsula and Macaronesia (28 species; Dalgaard, 1979; Flora Iberica,
Ortega Olivencia, 2009) and adjacent areas. African species are mostly restricted to the
Mediterranean realm (Hartl, 1965). Apart from treatments for the floras of the most
species-rich regions, morphological surveys and / or taxonomic revisions have been
provided for e.g. the Western Himalayas (Pennell, 1943), the Middle East (Eig, 1944),
North America (Pennell, 1935, 1947; Shaw, 1962), the Balkan Peninsula (Grau, 1981b),
Pakistan (Qaiser et al., 1988), Iran (Attar, 2006), and Korea (Jang and Oh, 2013).
Phylogenetic relationships had been only rarely addressed. The close relationship
of Scrophularia and Verbascum, proposed as early as 1835 by Bentham (see chapter
2.3.1.) and later also suggested by Thieret (1967) and others, was confirmed first by
Olmstead and Reeves (1995), who found Scrophularia being sister to Verbascum
including Celsia. Kornhall et al. (2001) identified a sister relationship of the
Scrophularia-Verbascum clade to the South African Antherothamnus N.E.Br. The closest
relative of Scrophularia known to date is the genus Oreosolen Hook.f. as discovered by
Albach et al. (2005) and Oxelman et al. (2005). Oreosolen is endemic to the Himalayas
and the Tibetan Plateau and is used as a traditional Tibetan medicine (Rosendal Jensen
et al., 2008). Oxelman et al. (2005) pointed out its close relation to Scrophularia in terms
of floral morphology and leaf architecture, and emphasized the unusual Northern
Hemisphere distributions of both genera, which are shared with Verbascum (see above).
Altogether, tribe Scrophularieae thus now comprises four genera in the APG system
(Stevens, 2001 and onward). Wang et al. (2015) sequenced accessions of few Eastern
Asian species to elucidate the taxonomic status of S. koraiensis Nakai. Attar et al. (2011)
provided a preliminary molecular phylogeny, which was however limited to only 20
Iranian taxa. Navarro Pérez et al. (2013) included 77-108 accessions into a timecalibrated phylogeny. The authors confirmed the monophyly of the genus and
17
postulated its divergence in the Miocene. However, their sampling did not comprise
samples from all distribution areas and proposed subsections; their main focus was the
reconstruction of pollination system shifts in the history of the genus.
2.3.3. Pollination biology and evolution of the staminode
The pollination biology of Scrophularia offers several interesting aspects as well:
Scrophularia flowers are self-compatible but protogynous, thus favoring crosspollination. In part of the species, centrifugal incurvation of the style additionally
separates the sexual organs before the stamens open (see e.g. Shaw, 1962; Ortega
Olivencia and Devesa Alcaraz, 1993a). The flowers present sucrose-rich nectar; the
nectar composition of individual species (with one exception) is largely independent of
their respective pollinator group (Rodríguez Riaño et al., 2014). Ortega Olivencia and
Devesa Alcaraz (1993b), in a study on 24 representatives, found greater nectar and
pollen production in taxa from S. sect. Anastomosantes compared to S. sect.
Tomiophyllum, putatively due to larger corolla and anther sizes in the former. In most
species, the flowers match the characteristics of wasp-flowers; Brodmann et al. (2012)
revealed that they also contain green leaf volatiles in their odour, which are highly
attractive to wasps. It seems intriguing that in many plants, these volatiles are also
indicators of an infestation with herbivores, which constitute suitable prey for wasps.
Consequently, the main pollinators are traditionally considered wasps and
syrphid flies, with further Diptera, bees, and bumblebees complementing the pollinator
spectrum (see Shaw, 1962, Ortega Olivencia and Devesa Alcaraz, 1993b,c; Fateryga,
2011; Valtueña et al., 2013; and references therein). Close pollinator-plant relationships
do not seem to exist, as most pollinators are generalists unable to distinguish among
different Scrophularia species. In some species with large and showy flowers, birds also
act as pollinators (e.g. Ortega Olivencia et al., 2012; see chapter 5.5.). Within the
inflorescences, the main pollinator groups follow a pattern of rather upward than
downward vertical movements, accompanied by horizontal movements whose
proportion is apparently positively correlated to flower size (Valtueña et al., 2013). The
authors conclude that this, together with the fact that dichogamy is not synchronized
throughout the inflorescence, implies that geitonogamy (i.e., self-pollination
accomplished by the transfer of pollen from one flower to the stigma of another flower
on the same plant) is not avoided in Scrophularia.
The importance of its pollinators for Scrophularia is illustrated by results from
Ortega Olivencia and Devesa Alcaraz (1993c), where plants in a study on 22 taxa showed
both reduced fruit-set and seed-set per capsule when pollinators were excluded. An
exception was S. peregrina L., which readily set full fruit following self-pollination
according to Shaw (1962). Altogether, intrafloral self-pollination does not seem to play a
major role in the genus. From the different groups of pollinators, Syrphidae seemed to be
important only in S. sect. Tomiophyllum as noted by Ortega Olivencia and Devesa Alcaraz
(1993c), although these insects visit Anastomosantes flowers as well (Shaw, 1962). The
significance of hoverflies for flowers of S. sect. Tomiophyllum was confirmed by Valtueña
et al. (2013) and also in a study on five Crimean species by Fateryga (2011), who
proposed a shift towards hoverfly pollination in this section in response to the open
landscapes inhabited by its species. The author also speculated on wasp-pollination to
be the ancestral condition in Scrophularia, which was later corroborated by Navarro
Pérez et al. (2013).
Most species of Scrophularia are characterized by an adaxial staminode adnate to
the upper part of the corolla (see chapter 2.3.2. and Fig. 3i). Staminodes, i.e. sterile,
18
modified stamens, usually emerge during an evolutionary reduction of the androecium
(Walker-Larsen and Harder, 2000). Single stamens within a whorl can be modified into
staminodes during the development of zygomorphic (instead of actinomorphic) flowers.
Staminodes thus represent a transitional phase: they can be present as vestigial organs,
which might get lost during subsequent evolution, or can acquire secondary floral
functions and even become essential floral components (Walker-Larsen and Harder,
2000; Ronse De Craene and Smets, 2001). These functions may be attractive (e.g. display
of color, odor), nutritional (e.g. nectar production) or structural (e.g. nectar recipients or
obstacles to selfing or nectar theft) according to Ronse De Craene and Smets (2001) and
connected to pollination in various ways (e.g. also by mediating pollinator contact to the
stigma or anthers). In vestigial, little altered staminodes as in most Scrophularia species,
functions are often difficult to discern. The scale-like distal part of the Scrophularia
staminode obtains various shapes and also may be reduced to a small, awl-shaped
structure like in S. canina L. Rodríguez Riaño et al. (2015b) found that the function of the
staminode is not connected to nectar secretion. Larger staminodes also do not hinder
access to the flower; instead, they act as barriers to reduce the dilution of nectar by the
entry of rainwater (Rodríguez Riaño et al., 2015a). According to López et al. (2016),
larger staminodes act as attraction units to pollinators during multiple visits. However,
they do not improve pollinator contact to the reproductive organs by decreasing the
opening of the corolla or by forcing them into a correct position. Small staminodes do
not seem to fulfil a special role; the function of the exceptionally large staminodes in
some species of S. sect. Tomiophyllum yet remains to be revealed.
2.3.4. Anatomy and phytochemistry
Anatomical studies were conducted by several authors. Scrophularia has
anatropous, unitegmic and tenuinucellate ovules according to investigations in S.
himalensis Royle ex. Benth. by Natesh and Bhandari (1974), who also found two
coexisting types of embryo sac development (polygonum plus allium type) in this
species. Bhandari and Natesh (1985) reported on endosperm development in
Scrophularia. Lersten and Curtis (1997) investigated foliar idioblast occurrence in
Scrophularia and Verbascum; single-celled idioblasts are apparently restricted to these
two genera (Lersten and Curtis, 2001; see chapter 2.3.2.). Within Scrophularia, idioblasts
are more frequent in species of S. sect. Tomiophyllum than S. sect. Anastomosantes,
however they are abundantly present in North American species (Lersten and Curtis,
1997). Makbul and Beyazoğlu (2009) emphasized the taxonomic value both of idioblasts
and the distribution and dimension of sclerenchymatic tissue in cortex and phloem, and
presented stem and leaf sections. Makbul et al. (2006) stated that these anatomical
characters, plus the mean number of stomata, epidermal cell characteristics, and the
diameter of the vascular bundle, might be more important for species identification than
morphological traits.
Typical secondary metabolites found in root and leaf tissues of Scrophularia
include iridoid glycosides, phenylpropanoids, phenolic acids, flavonoids and saponins.
These agents account for the medicinal properties of several species (e.g. antiinflammatory, antibacterial, fungicidal, cardiovascular, or diuretic activities, see de
Santos Galíndez et al., 2002). According to Potter's Herbal Cyclopedia (Williamson,
2003), S. nodosa can be used for wound healing, as a laxative, and is diuretic and antiinflammatory. In Traditional Chinese Medicine (TCM), several species are used for
therapeutic purposes, most notably S. ningpoensis Hemsl. and S. buergeriana Miq., which
are listed in the Encyclopedia of Traditional Chinese Medicines (Zhou et al., 2011) as
19
remedies for various disorders including insomnia and dry eyes. Indications of
pharmaceutic effects were also found in studies on e.g. S. scorodonia L., S. deserti Delile,
S. striata Boiss., S. hypericifolia Wydler, S. orientalis L., or S. oxysepala Boiss. (Diaz et al.,
2004; Stavri et al., 2006; Azadmehr et al., 2013; Kosari-Nasab et al., 2013; Alqasoumi,
2014; Lange et al., 2016; Orangi et al., 2016). In this respect, the ancient reputation of
Scrophularia as a medicinal plant is still valid today.
20
3. Methodology
3.1. Testing for incongruence among phylogenetic trees
When the analysis of two or more molecular markers during phylogenetic
reconstruction yields different trees, the question arises on whether their topologies are
congruent, i.e. support the same phylogenetic hypothesis. Incongruence can result from
a variety of sources (see Discussion, chapter 5.4.), but irrespective of its origin, it can
distort a combined analysis of different markers, especially if datasets lead to wellsupported, but conflicting topologies. Therefore, the first step is to assess if statistically
significant incongruence is present at all. Three methods have been used here (several
more are reviewed in detail in Johnson and Soltis, 1998):
1) Visual inspection for taxa and clades displaying hard incongruence
Two trees are compared and examined for "hard", i.e. well-supported incongruence of
single accessions or entire clades (for example, taxon A receives high support as sister to
taxon B in one tree, but as sister to taxon C in another tree). Congruence is rejected if the
support values for conflicting placements in both trees equal or exceed 70 % bootstrap
support (BS), a widely used threshold established by Mason-Gamer and Kellogg (1996).
Bayesian posterior probabilities (PP) of ≥ 0.95 have also been used; cut-offs may be
decreased in cases where reduced supports, due to high levels of homoplasy or sequence
polymorphism (see chapter 3.3.), are to be expected. By obscuring phylogenetic
relationships, these would prevent taxa from being recognized as hard incongruent,
which is by definition dependent on the support of associated nodes. In this case,
additional examination of phylogenetic networks based on coded polymorphisms (see
chapter 3.4.) is advisable; accessions displaying "soft" incongruence should be further
examined. Identification of hard incongruence taxa was done for all articles of this
dissertation.
2) Testing for incongruence with the Incongruence Length Difference (ILD) test
This statistical test (Farris et al., 1995) evaluates the likeliness of two datasets
supporting the same phylogenetic hypothesis. In principle, a combined analysis of
congruent datasets should result in a tree whose length is equal to the sum of both
lengths of trees yielded by individual analysis of each marker alone. If datasets are
incongruent, combination will result in greater homoplasy and consequently higher tree
length - the null hypothesis is rejected. This is tested by comparing the sum of lengths of
the two single marker trees against that of trees obtained from two datasets which are
made up of randomly chosen characters from both markers (i.e., combined datasets).
The procedure is repeated several times to ensure statistical significance; a p-value of <
0.05 suggests that the data are incongruent. Chloroplast markers thus can be tested
against nuclear markers; if the result is significant, all accessions which putatively cause
the conflict (e.g. already identified hard incongruence taxa) are pruned from the datasets
and the test is repeated. If now congruence is supported, stepwise re-addition of single
taxa helps to assess the individual amount of incongruence introduced by each
respective taxon, and which taxa should be finally excluded from a combined analysis
(see chapter 3.2.). The ILD test was used in Articles I-IV.
3) Templeton's significantly less parsimonious (SLP) test
The SLP test (Templeton, 1983) investigates whether the data of one dataset are in
significant conflict with the topology supported by another dataset. To test this, a
heuristic search with dataset A is constrained by one (or more) topologies representing
relationships supported by dataset B. The trees from this search are compared to those
of an unconstrained heuristic search (or to an associated consensus tree) of dataset A. If
21
the constrained trees are significantly longer (p < 0.05), the null hypothesis is rejected,
i.e., the "strange" topology is in conflict with the data. The SLP test might be used in
addition to the ILD test, as the latter has been criticized for putative weaknesses, e.g. a
high rate of false positives, by Barker and Lutzoni (2002) and Darlu and Lecointre
(2002). The SLP test was applied in Articles II and III.
3.2. Combining incongruent datasets for phylogenetic tree construction
The question whether significantly incongruent datasets should be combined has
been discussed extensively (see Huelsenbeck et al., 1996). Some authors advocate a
concatenation approach which ignores the incongruence altogether (e.g. Gadagkar et al.,
2005), while others have shown that this might produce misleading results (Kubatko
and Degnan, 2007; Weisrock et al., 2012). A widely used alternative is the "conditional
combination approach” (reviewed in Huelsenbeck et al., 1996; Johnson and Soltis, 1998),
where taxa which display significant topological incongruence (see chapter 3.1.), or
more generally introduce conflict into a combined dataset, are excluded prior to the
analysis. Where the incongruence is not too widespread and clearly detectable (see
Articles I and II of this dissertation), this yields reliable and satisfying results.
However, in groups where
reticulation
and/or
incomplete lineage sorting
(ILS) is frequent, pruning of
hard incongruent taxa will
disregard large portions of
the available data. An
elegant solution to this
problem was presented by
Pirie et al. (2008, 2009).
Fig. 5. Exemplary taxon duplication in an alignment with two Their approach allows the
conflicting taxa (4 and 6). The incongruent marker information is inclusion of conflicting data
split, by duplication of the respective taxon into one "plastid-only" into the alignment, by
(cp) and one "nuclear-only" (nr) accession. For more details see Pirie
duplication
of
the
et al. (2008, 2009)
respective taxa into one
"plastid-only" and one "nuclear-only" accession ("taxon duplication approach"; Fig. 5).
This way, analyses will not be impeded by incongruence issues, tree resolution is
improved, and the placement of nuclear and plastid representations in the tree possibly
allows conclusions on the complex evolutionary history of these taxa (Pirie et al., 2008).
While the taxon duplication approach can be very useful for phylogenetic and
biogeographic reconstructions of medium-sized datasets (see Article III of this thesis),
the method becomes increasingly unfeasible for larger samplings in species-rich genera
with many conflicting taxa. In these cases, pruning taxa consequently is no alternative
either; plastid and nuclear data are thus often analyzed separately and then interpreted
individually and compared (as done in Article IV).
3.3. Character and indel coding
When molecular marker datasets are to be analyzed separately, sufficient
resolution in the resulting single marker trees is required for significant interpretation
and conclusions. Unfortunately, some processes which produce among-dataset
22
incongruence (e.g. ILS or hybridization) are often also responsible for lowered supports
and polytomies in the respective phylogenies. In the biparentally inherited nuclear DNA,
markers of which are frequently used for reconstruction, hybridization will result in (at
least) two different copies in the genome of the offspring. Also without any reticulation,
retained polymorphisms from a common ancestor, which persist in a population, can
produce the same patterns, especially in young lineages (where sequence divergence
among species is low anyway). These differences among single copies of a gene lead to
intra-individual site polymorphism in direct sequences (i.e., generated by direct Sanger
sequencing); some examples are illustrated in Fig. 6a. A variety of other processes can
also produce intra-individual variability (see chapter 5.4.); this is especially true for
multi-copy nuclear markers like ITS.
Large
amounts
of
polymorphisms in sequences
hamper
phylogenetic
reconstruction by obscuring
important
relationships,
particularly if they are present
at synapomorphic sites of an
alignment. Many algorithms
used for tree reconstruction
treat the respective sites as
missing data or ambiguous
information,
so
that
unequivocal assignment of the
respective taxa to a certain
phylogenetic group will fail.
This leads to polytomies and
weakly supported nodes and
hinders correct interpretation
of the data (see e.g. Campbell
et al., 1997; Fuertes Aguilar
and Nieto Feliner, 2003;
Fehrer et al., 2009).
In species-poor lineages, the
problem can be solved by
Fig. 6. Sequence polymorphisms and coding methods. Intra- cloning the marker regions of
individual site polymorphism in two exemplary pherograms the
respective accessions
obtained from Scrophularia (a), with corresponding IUPAC (Nieto Feliner and Rosselló,
characters. These ambiguities can be treated as informative, by
2007). In large genera, where
recoding all characters of the alignment as a standard matrix (b);
thereby, each IUPAC code is given a distinct character state. (c), cloning of all taxa cannot be
exemplary coding of plastid indels using the simple indel coding accomplished (for example,
procedure (Simmons and Ochoterena, 2000); the binary matrix is only exemplary cloning was
added to the alignment
performed in Article IV), the
information content drawn from direct sequences can be enhanced by explicitly treating
intra-individual polymorphisms as informative. For example, this can be done by the use
of step matrices during calculations (Potts et al., 2014), which directly model the change
from e.g. nucleotide character "C" to "T" via "Y" ("C" and "T"). Alternatively, single site
variabilities, oligonucleotide motives or the whole alignment can be recoded into a
character matrix, which is then analyzed using step matrices or, more straightforward,
as categorical dataset (Grimm et al., 2007; Potts et al., 2014). For analyses of
infrageneric relationships in Scrophularia (see Article IV), I followed the latter approach:
23
100
200
300
400
500
600
700
800
2a
6
2c
2d
3
5
4
Fig. 7. Indel patterns in a representative sample of 75 Scrophularia accessions. Length type numbers of
indels correspond to those in Table 2 of Article IV. Type 2a and 2d are characteristic for the Polyantha and
Scopolii clades, respectively. However, indels of similar length also occur spontaneously (type 2c). The
longest indels are found in the Orientalis clade and S. rubricaulis Boiss. (types 5 and 6); all 25 analyzed
accessions from the Nodosa and NW / Japan clades feature a characteristic 597-bp indel (type 4)
the complete alignment was recoded as a standard matrix, with each IUPAC code
receiving a distinct number ("0-9" or "?"; Fig. 6b). This way, all nucleotide character
states, simple or ambiguous, are treated as distinct characters; analyses are done using
settings analogous to those for categorical (e.g. morphological) data. This approach does
not discriminate between polymorphisms derived from different processes (which may
be difficult to ascertain), but incorporates all available sites; the amount of additional
information obtained can be considerable depending on the ambiguity present in the
dataset.
Uniparentally inherited markers are usually free from intra-individual
polymorphism. Here, limited resolution on the species level is often caused by a lack of
informative characters in slow-evolving regions. Additional phylogenetic signal may be
contained in insertions-deletions (indel) patterns in the alignment of the ingroup. This
information can be extracted (as done in Articles I-IV) by coding indels as a binary
matrix of present-absent states (1-0) for every accession, which is generated
sequentially for each indel (an example is given in Fig. 6c). This method, known as
"simple indel coding", was presented by Simmons and Ochoterena (2000). The resulting
binary matrix is then added to the alignment and analyzed accordingly; the occurrence
of every indel is thus treated as a single mutation event independent of indel length.
Particular indels may be diagnostic for species groups (as in Scrophularia; Fig. 7) and, if
large enough, might be recognized directly by characteristic length variations of the
amplified PCR products (reduced or increased length of the fragment on the PCR gel,
compared to those of congeners; see Articles II and IV); this constitutes an efficient
distinguishing tool for the respective species groups.
24
3.4. Network methods
To infer the evolutionary history of a group and reveal phylogenetic relationships
among its members, the standard approach is to compute a bifurcating phylogenetic tree
from a given set of sequences. This relies on the tacit assumption that the underlying
processes also follow this bifurcating principle. However, this assumption is often
violated, e.g. during adaptive radiation where multiple new lineages are generated from
one common ancestor; in cases where ancestors and descendants coexist (this would
require labelling internal nodes); or, importantly, when lineages interact with each other
in a "horizontal" way, e.g. by hybridization or introgression (Moulton and Huber, 2009).
Evolutionary mechanisms often are considerably non-treelike, and even if a single
molecular marker supports one bifurcating phylogenetic tree, other genes are likely to
support different phylogenetic hypotheses, due to processes like ILS and other
phenomena which create incongruence as outlined above (Huson and Scornavacca,
2011).
All of these processes are better depicted by a phylogenetic network, rather than
by a bifurcating tree. Thereby, one important type are "split networks". A split
represents a bipartition of a given set of taxa which is based on some kind of information
(e.g. sequence similarity); the split divides the sample into two groups. Generally, each
branch (or edge) in an unrooted phylogenetic tree also represents a split, in a way that
its removal results in two subtrees (groups) supported by the split. A collection of
"compatible" splits makes up a bifurcating tree; if incompatible splits are present, these
can only be visualized in a network (Huson and Bryant, 2006), with each split
represented by a set of parallel edges (for an example see Fig. 8). Split networks can be
created from a variety of data types, including sequences, distances, quartets, or
phylogenetic trees. As they are unrooted, they are not appropriate to explicitly trace a
putative evolutionary history (in contrast to rooted, explicit phylogenetic networks like
hybridization networks); the function of these implicit networks is to visualize
connections and / or incompatibility in a dataset (Huson and Scornavacca, 2011). The
latter is important for exploratory data analysis, a concept advocated by Morrison
(2010). By evaluating the properties of the present data before analyzing them, the
suitability of the intended methods can be estimated and data-inherent pitfalls, which
might lead to erroneous conclusions, can be avoided. For this dissertation, no explicit
networks were constructed to model hybridization and polyploidization, as
incongruence in Scrophularia is also due to other processes (see Discussion, chapter
5.4.). Three types of unrooted phylogenetic networks were used:
1) consensus networks (CN)
If incompatibility / incongruence is present within or among different datasets,
computing a consensus tree from all trees obtained during an analysis will often conceal
part of the results in favor of other - sometimes only slightly better supported relationships. A consensus network (CN; Holland and Moulton, 2003; Holland et al.,
2004) will display all signal above a certain threshold (e.g., "present in 30 % of trees").
Edge lengths of these networks correspond to the split frequency within the sampled
topologies. If trees based on two different markers are used (like in Fig. 8), the
consensus network enables the examination of phylogenetic among-dataset
incongruence, its extent and possible causes (i.e., accessions causing the conflict, which
are usually found close to heavily networked regions). If such conflicting accessions
have already been identified (by the methods described above), these can be excluded
and the network re-generated to see how far the problem is alleviated by pruning taxa.
25
Tree marker 1
1
Tree marker 2
1
2
2
1
6
3
3
4
4
5
5
6
2
1.0
6
3
4
5
Extremely
entangled
relationships, depicted by
high numbers of parallel
edges,
suggest
that
combination of the data might
not be reasonable. (Filtered)
Super Networks can be
constructed in cases where
trees differ with respect to
the
sampled
accessions
(Huson et al., 2004, 2006).
Consensus
networks
or
filtered super networks were
computed in Articles I, III and
IV.
2) Neighbor-Net (NN)
splits graphs
In a nuclear dataset which is
confounded by reticulation
and/or ILS effects, a split
network based on the raw
data can provide important
insights about conflicting
signals which might lead to
problems in phylogenetic tree
reconstruction
(Morrison,
2010). The Neighbor-Net
(Bryant and Moulton, 2004)
is
based
on
pairwise
distances;
information
contained in polymorphic
sequence sites can be
incorporated by basing the
network on polymorphism pdistances (Schliep, 2011; Potts et al., 2014). However, it is important to notice that the
NN, just like the CN, will display all signal contained in the data irrespective of its source
(Bryant and Moulton, 2004; Morrison, 2010), including conflicts based on methodical
artifacts or, in the case of CNs, lack of information. A Neighbor-Net was used to examine
the ITS dataset in Article IV of this dissertation.
3) Haplotype networks
When taxa are too closely related (i.e., of very low sequence divergence) to be resolved
in a phylogenetic tree by the markers used for reconstruction, their relationships can be
displayed in a haplotype network. This type of unrooted phylogenetic network is
different from the split networks discussed above. Here, different haplotypes (i.e.,
sequence "configurations" inherited as a single unit, present in one or more taxa) are
represented by nodes and are joined by edges which denote the difference among the
two connected haplotypes, e.g. the nucleotide position at which they differ (Huson and
Scornavacca, 2011). Missing intermediate states are inferred during calculation and
correspond to assumed unsampled or extinct haplotypes. Haplotype networks can be
Fig. 8. Example for a splits graph: a consensus network
representing compatible and incompatible splits from two trees
yielded by analysis of different markers. The set of parallel edges in
the network colored in green represents the bipartition
{3,4}|{1,2,5,6}; the corresponding branch in the unrooted tree from
marker 1 is marked. The respective split is not compatible with the
red one in the tree from marker 2. The phylogenetic network
illustrates both relationships (using a threshold ≤ 50 % for
displaying splits, here 33 %). Edge lengths are proportional to the
number of trees supporting a split. Drawing adapted from Morrison
(2010)
26
constructed with various methods; the "TCS approach" (Templeton et al., 1992; Clement
et al., 2000), which was used here, is based on statistical parsimony (definition see
Templeton et al., 1992). Normally, plastid sequences (which are collapsed into
haplotypes by the program) at population level are used for these networks; however
they are also helpful when sequence divergence among species is low. Shared
haplotypes across species boundaries in the same geographical region can point
towards introgression/hybridization; common ancestry (but also ancient hybridization
events) might result in haplotypes shared among geographically distant species. Radial
patterns of change from a central haplotype are consistent with radiation processes,
while divergent haplotypes are found in isolated and hence possibly older lineages. As in
split networks, too low diversity of the sequences will result in uncertainties in the
network (there represented by loops). A haplotype network was constructed in Article
III of this dissertation to elucidate the relationships among closely related Scrophularia
species from the Iberian Peninsula and Macaronesia.
27
28
4. Scientific manuscripts
The present dissertation is based on the following four publications:
Scheunert A, Fleischmann A, Olano-Marín C, Bräuchler C,
Heubl G. 2012. Phylogeny of tribe Rhinantheae
(Orobanchaceae) with a focus on biogeography, cytology
and re-examination of generic concepts.
Taxon 61(6): 1269-1285.
Article I
Scheunert A, Heubl G. 2011. Phylogenetic relationships
among New World Scrophularia L. (Scrophulariaceae): new
insights inferred from DNA sequence data.
Plant Systematics and Evolution 291: 69-89.
doi:10.1007/s00606-010-0369-z
Article II
Scheunert A, Heubl G. 2014. Diversification of Scrophularia
(Scrophulariaceae) in the Western Mediterranean and
Macaronesia - Phylogenetic relationships, reticulate
evolution and biogeographic patterns.
Molecular Phylogenetics and Evolution 70: 296-313.
doi: 10.1016/j.ympev.2013.09.023
Article III
Scheunert A, Heubl G. 2017. Against all odds:
reconstructing the evolutionary history of Scrophularia
(Scrophulariaceae) despite high levels of incongruence and
reticulate evolution.
Organisms Diversity and Evolution, Online First.
doi: 10.1007/s13127-016-0316-0
Article IV
The articles are provided in the following chapters.
29
30
4.1. Article I
Phylogeny of tribe Rhinantheae (Orobanchaceae) with a focus on biogeography,
cytology and re-examination of generic concepts.
by Agnes Scheunert, Andreas Fleischmann, Catalina Olano-Marín, Christian Bräuchler &
Günther Heubl
Taxon 61(6): 1269-1285 (2012)
I
The final publication is available on Ingenta Connect at
http://www.ingentaconnect.com/content/iapt/tax/
2012/00000061/00000006/art00008
31
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TAXON 61 (6) • December 2012: 1269–1285
Scheunert & al. • Phylogeny of Rhinantheae
Phylogeny of tribe Rhinantheae (Orobanchaceae) with a focus on
biogeography, cytology and re-examination of generic concepts
Agnes Scheunert,1 Andreas Fleischmann,1 Catalina Olano-Marín,1 Christian Bräuchler1,2 & Günther Heubl1
1 Systematic Botany and Mycology, Ludwig-Maximilians-University (LMU), Menzinger Strasse 67, 80638 Munich, Germany
2 Botanische Staatssammlung München, Menzinger Strasse 67, 80638 Munich, Germany
Author for correspondence: Agnes Scheunert, agnes.scheunert@lrz.uni-muenchen.de
Abstract A molecular systematic approach using DNA sequences of two non-coding chloroplast loci (trnK, rps16) and the
nuclear ITS region was applied to reconstruct phylogenetic relationships within the tribe Rhinantheae (Orobanchaceae). This
tribe includes approximately 19 genera of hemiparasitic plants predominantly occurring in the Old World. An exception is the
genus Bartsia which, 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.
Keywords hemiparasitic plants; nrITS; Orobanchaceae; Rhinantheae; rps16; trnK
Supplementary Material The alignment is available in the Supplementary Data section of the online version of this article
(http://www.ingentaconnect.com/content/iapt/tax).
INTRODUCTION
The cosmopolitan angiosperm family Orobanchaceae
(broomrape family) is a morphologically diverse group of almost 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 Lindenbergia 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 Orobanchaceae 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 nonparasitic 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 imbricate ascending corolla aestivation. This so-called “rhinanthoid
aestivation” is a synapomorphy of all Orobanchaceae, 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
This publication is dedicated to Dr. Markus Bolliger on the occasion of his 60th birthday.
Version of Record (identical to print version).
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TAXON 61 (6) • December 2012: 1269–1285
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 several 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. (previously 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 phylogenybased taxonomic concept for all taxa of Rhinantheae has been
proposed yet, the traditional system of Wettstein (1891) is followed here, with the exception of Pedicularis as a member of
Pedicularideae following Tank & al. (2009).
Morphological characters, generic concepts, and distribution of Rhinantheae. — Apart from petal aestivation and
parasitic habit, Rhinantheae share no other generative or vegetative 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, Bornmuellerantha Rothm. and Tozzia L. (Fischer, 2004). For generic delimitation in Rhinantheae, mainly corolla morphology, but also
palynological characters (pollen size, shape, exine ornamentation) 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 Bolliger, 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 circumscription 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 highest 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).
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Preceding molecular studies on Rhinantheae. — Rather
few phylogenetic studies have addressed generic-level relationships within Rhinantheae exclusively; most of the work has
focused on the phylogenetic framework and evolution of holoparasitism 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 relationships 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 monophyletic 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, Bornmuellerantha, Odontitella, Bartsiella and the African representatives
of Bartsia were not included, thus leaving important questions
unanswered.
The present study is based on an extensive taxonomic
sampling of Rhinantheae to comprise taxa absent in previous
studies. Based on an enlarged dataset, our goals are to test
the previously published phylogenetic hypotheses in a more
comprehensive context, particularly investigating the impact of
narrowly endemic and poorly studied genera (Bartsiella, Bornmuellerantha, Macrosyringion, Nothobartsia, Odontitella) on
phylogenetic reconstruction, and to infer phylogenetic relationships within and between taxa of Bartsia and Odontites to test
existing taxonomic concepts.
MATERIALS AND METHODS
Plant material. — The taxon sampling follows the treatment of Fischer (2004), who recognized Orobanchaceae within
Scrophulariaceae, and covers a representative number of species 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 comprehensive molecular phylogeny of Orobanchaceae by Bennett & Mathews (2006) and the phylogeny of Rhinanthoid
Orobanchaceae by Těšitel & al. (2010). Striga Lour. (tribe
Buchnereae) and Pedicularis (tribe Pedicularideae) were chosen 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 flowers. Nevertheless, it was included in the study so as to increase
the number of South American Bartsia species.
DNA extraction and amplification. — Total genomic DNA
was extracted either from fresh leaf material (three taxa) or
from herbarium specimens (30 taxa) using the NucleoSpin Plant
Version of Record (identical to print version).
TAXON 61 (6) • December 2012: 1269–1285
Scheunert & al. • Phylogeny of Rhinantheae
Fig. 1. Selected species of representative genera of Rhinantheae (Orobanchaceae). A, Melampyrum nemorosum L.; B, Parentucellia latifolia (L.)
Caruel; C, Bartsia alpina L.; D, Bellardia trixago (L.) All.; E, Rhinanthus alectorolophus (Scop.) Pollich; F, Euphrasia officinalis L.; G, Parentucellia viscosa (L.) Caruel; H, Odontites vernus Dumort.; I, Lathraea squamaria L. — Photographs A, E & F, F. Brambach; B–D, G–H, A.
Fleischmann; I, C. Olano-Marín.
Version of Record (identical to print version).
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Scheunert & al. • Phylogeny of Rhinantheae
TAXON 61 (6) • December 2012: 1269–1285
Kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s standard protocol for genomic DNA extraction. An
additional phenol/chloroform extraction step was performed to
remove proteins and potentially interfering secondary metabolites. 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 region, 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 lamialean groups (Schäferhoff & al., 2010; Těšitel & al., 2010).
PCRs were performed with total genomic DNA using Taqpolymerase (Hybaid, AGS, Heidelberg, Germany) and primers 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 profile
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 (MachereyNagel) 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 (G50Superfine, Amersham Biosciences) and were run on an ABI
3730 DNA analyzer (Applied Bio Systems, Foster City, California, 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 software (Edgar, 2004) and adjusted manually using BioEdit v.7.0.5.1 (Hall, 1999); mononucleotide repeats and ambiguously aligned regions were excluded from
further analysis. Before incorporating nuclear and chloroplast
indels in the analyses, their phylogenetic information content
was assessed. This is regarded essential because within the
parasitic lineages of Lamiales (which contain fast-evolving
groups), nuclear indel data in particular are suspected to be
highly homoplasious and thus could distort the inferred results
(Schäferhoff & al., 2010). Ingroup indels were coded according
to the simple indel-coding method (Simmons & Ochoterena,
2000), as implemented in SeqState v.1.4.1 (Müller, 2005) and
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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 categories 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 recommended by Ronquist & al. (2009); for chloroplast and combined
analyses, a mixed dataset was defined (one/two DNA partitions, 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 (Stamatakis & al., 2008) using raxmlGUI 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 potentially different phylogenetic history should be combined has
been the issue of extensive debates (see reviews by Miyamoto
& Fitch, 1995; Queiroz & al., 1995; Huelsenbeck & al., 1996).
In Rhinantheae, several examples of intrageneric reticulate
evolution caused by introgression and hybridization have been
reported, e.g., in Euphrasia, Rhinanthus, Melampyrum, and
Odontites (e.g., Yeo, 1968; Kwak, 1978; Bolliger & al., 1990;
Wesselingh & Van Groenendael, 2005; Liebst, 2008; Těšitel
& al., 2010). As the amount of heterogeneity present on the
intergeneric level cannot be assumed to be neglectible, assessment 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.
Version of Record (identical to print version).
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Scheunert & al. • Phylogeny of Rhinantheae
We applied several methods to assess levels of data heterogeneity: first, phylograms obtained from the chloroplast and
nuclear datasets alone were visually examined and compared
for well-supported discrepancies (“hard incongruence”; MasonGamer & Kellogg, 1996) using a cut-off of 70% ML bootstrap
support. As reticulate relationships were to be expected and support 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 partition 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 successively 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
incongruence 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 contradicting 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 incongruence 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.
RESULTS
Sequencing and alignment. — A total of 98 sequences
were generated for this study, 33 each for the trnK region and
the rps16 intron, and 32 for ITS. As the sequence pherograms
for the ITS marker region provided a clear, unambiguous
signal without any signs of polymorphisms, no cloning was
performed. In a few taxa where sequencing failed, sequences
were obtained from GenBank (http://www.ncbi.nlm.nih.gov)
to complete the taxon sampling: these include Melampyrum
nemorosum L. (ITS), Euphrasia stricta J.P. Wolff ex J.F. Lehm.
(trnK, rps16, ITS), Pedicularis sylvatica L. / P. attollens A. Gray
(trnK, rps16, ITS) and Striga asiatica (trnK, ITS). For Striga,
no rps16 intron sequence was available in GenBank; however,
as the outgroups were intended to be in accordance with those
in Bennett & Mathews (2006) and Těšitel & al. (2010), the
genus was nevertheless used, and rps16 was coded as missing
data for the combined analysis. For the same reason, we used
GenBank sequences for Pedicularis originating from two species (P. attollens for rps16 and ITS and P. sylvatica for trnK,
as this marker sequence was not available for P. attollens). The
combined DNA data matrix of trnK, rps16, and ITS contained
2800 aligned characters. The average sequence length was
1032 basepairs (bp) for trnK, 784 bp for rps16, and 689 bp
for ITS. Detailed information on alignment statistics for all
markers including the proportions of parsimony-informative
characters is provided in Table 1.
Table 1. Alignment characteristics and statistics for ITS, trnK region, rps16 intron, combined chloroplast dataset, and combined dataset. Number
of constant characters, parsimony-informative characters and % parsimony-informative characters refers to non-excluded characters; number of
excluded characters includes peripheral regions of the alignment not suitable for analysis, mononucleotide repeats and regions which could not be
aligned unambiguously; proportion of unknown characters calculated without peripheral regions of the alignment.
ITS
trnK
rps16
Comb. Chloroplast Combined
Number of taxa
36
36
34
36
30
Sequence length
(average)
537–737 bp
(689 bp)
813–1082 bp
(1032 bp)
701–848 bp
(784 bp)
1044–1898 bp
(1772 bp)
1588–2594 bp
(2461 bp)
Aligned length
767 bp
1147 bp
886 bp
2033 bp
2800 bp
Excluded characters
197 bp
108 bp
153 bp
261 bp
458 bp
Constant characters
275 bp
708 bp
561 bp
1269 bp
1544 bp
Parsimony-informative characters
191 bp
133 bp
74 bp
207 bp
398 bp
% parsimony-informative characters
33.51%
12.80%
10.10%
11.68%
16.99%
Unknown characters within alignment
(average)
0–14.63%
(0.96%)
0–22.81%
(1.05%)
0–6.76%
(0.84%)
0–44.20%
(3.40%)
0–35.65%
(2.79%)
Average G + C content
55.24%
34.24%
34.54%
34.39%
40.27%
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TAXON 61 (6) • December 2012: 1269–1285
Chloroplast and ITS analyses. — The combined chloroplast dataset—including coded indels—showed a standard deviation 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 partition, and at 2.602716 for the binary indel data partition. The ITS
dataset, at the end of the Bayes analysis, also had a standard deviation 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; therefore 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
1.00
100
x
93
x
85
1.00
100
Core group of
Rhinantheae
1.00
100
1.00
100
1.00
99
1.00
99
1.00
100
1.00
71
1.00
98 1.00
77
0.88
80
1.00
98
0.78
81
1.00
100 1.00
100
1.00
96
1.00
93
1.00
100
Macrosyringion glutinosum
Bornmuellerantha aucheri
Bornmuellerantha alshehbaziana
Odontites corsicus
Odontites himalayicus
Odontites pyrenaeus
Odontites viscosus
Bartsiella rameauana
Odontites bocconei
Odontites cyprius
IV. Odontites s.l.
1.00
100
Bellardia trixago
Parentucellia viscosa
Parentucellia latifolia
Bartsia mutica
Bartsia sp. Peru
Bartsia canescens
III. Bellardia
1.00
96
II. Hedbergia
1.00
100
Euphrasia stricta
Tozzia alpina
Odontitella virgata
Nothobartsia asperrima
Nothobartsia spicata
Hedbergia abyssinica
Bartsia decurva
Bartsia longiflora
Bartsia longiflora subsp. macrophylla
I. Bartsia s.str.
1.00
88
1.00
100
RRL clade
1.00
100
Rhinantheae
1.00
100
OG
1.00
99
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
Fig. 2. Bayesian consensus tree (cladogram) from the combined chloroplast dataset (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 (corresponding PP values are given, no support in Bayesian inference is denoted by an “x”). PP values obtained from 9002 trees, BS
values obtained from a best-scoring ML tree from 10,000 bootstrap replicates with subsequent maximum likelihood optimization (not shown).
Divergence of the “Rhinantheae” and “core group of Rhinantheae” (as defined in the text) is marked by arrows. Taxa which were excluded in the
combined analysis are highlighted in bold. OG, outgroup; RRL clade, Rhynchocorys-Rhinanthus-Lathraea clade.
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Scheunert & al. • Phylogeny of Rhinantheae
(PP). Melampyrum is revealed as highly supported sister to
an equally supported clade which comprises all remaining ingroup taxa in both analyses; however, support for these two
nodes is weak in the nuclear ML tree (BS: 66 and 64, respectively). 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
Pedicularis spp.
OG
Striga asiatica
Melampyrum nemorosum
Rhynchocorys orientalis
Rhynchocorys kurdica
Rhinanthus alectorolophus
66
RRL clade
0.81
50
0.98
1.00
96
Lathraea squamaria
Rhinantheae
1.00
64
90
Bartsia alpina Germany
Bartsia alpina Italy
Bartsia alpina Finland
Bartsia alpina Canada
I. Bartsia s.str.
0.95
1.00 74
100 0.62
Euphrasia stricta
Tozzia alpina
1.00
0.99
83 0.89
68 1.00
99
1.00
74
Hedbergia abyssinica
Bartsia decurva
Bartsia longiflora
II. Hedbergia
core group of
Rhinantheae
68
Bartsia longiflora subsp. macrophylla
Bellardia trixago
Parentucellia viscosa
1.00
94 0.92
97 0.93
71
0.84
43
1.00
96 1.00
100
0.85
40
Bartsia sp. Peru
Bartsia canescens
Odontitella virgata
Nothobartsia asperrima
Nothobartsia spicata
Macrosyringion glutinosum
Bornmuellerantha aucheri
Bornmuellerantha alshehbaziana
Odontites corsicus
Odontites pyrenaeus
Odontites viscosus
s.l.
1.00
95
Bartsia mutica
IV. Odontites
1.00
83
0.91
61 0.97
0.83
87
35
0.99
62
Parentucellia latifolia
III. Bellardia
1.00
95
Odontites himalayicus
Bartsiella rameauana
1.00
100
Odontites bocconei
Odontites cyprius
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, RhynchocorysRhinanthus-Lathraea clade.
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TAXON 61 (6) • December 2012: 1269–1285
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 Tozzia. 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):
Macrosyringion 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 considered significant following Farris & al., 1995 and Cunningham,
1997), implying conflicting signal within the data.
Sequential exclusion of the taxa showing hard incongruence as defined above (the group of Nothobartsia asperrima / 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, compared 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,
1276
illustrated by sets of parallel edges (branches) which indicate
differing signals within the data (Fig. 4A). Almost no reticulations 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 showing hard incongruence are removed from the network, it takes
on a much more tree-like structure (Fig. 4B), greatly simplifying 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 congruent (P = 0.264) and thus suitable for the combined analyses.
Combined analysis. — A combined analysis was conducted 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 represented 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 accessions 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 Tozzia 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. longiflora subsp. macrophylla (Hedberg) Hedberg), with the latter
two taxa highly supported as sisters (PP: 1.00, BS: 99).
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TAXON 61 (6) • December 2012: 1269–1285
Scheunert & al. • Phylogeny of Rhinantheae
Striga asiatica
Rhinanthus alectorolophus
Euphrasia stricta
Parentucellia
viscosa
Clade III
Bartsia mutica
Bartsia sp. Peru
Bartsia canescens
Melampyrum
nemorosum
Lathraea squamaria
Pedicularis spp.
Rhynchocorys
kurdica
Rhynchocorys
orientalis
A
Bellardia
trixago
Parentucellia latifolia
O. cyprius
Odontites
bocconei
Bartsia alpina Finland
Bartsia alpina Canada
Bartsia alpina Germany
Bartsia alpina Italy
Odontites pyrenaeus
Bartsiella rameauana
Clade I
Tozzia alpina
O. viscosus
Bartsia longiflora
Hedbergia Bartsia Bartsia longiflora subsp. macrophylla
abyssinica decurva
O. himalayicus
O. corsicus
B. aucheri
Bornmuellerantha
alshehbaziana
Clade II
Nothobartsia asperrima
Nothobartsia spicata
Odontitella virgata
Macrosyringion
glutinosum
Clade IV
Rhinanthus alectorolophus
Euphrasia stricta
Striga asiatica
Melampyrum
nemorosum
Lathraea squamaria
Pedicularis spp.
Rhynchocorys
kurdica Rhynchocorys
orientalis
Parentucellia
viscosa
B
Clade III
Bartsia mutica
Bellardia
Bartsia canescens
trixago
Parentucellia latifolia
56.4 29.3
Odontites bocconei
Odontites
O. cyprius
pyrenaeus
53.8 32.1 29.5
Bartsiella
rameauana
69.6
O. viscosus
O. himalayicus
O. corsicus
B. aucheri
Bornmuellerantha
alshehbaziana
39.5
Clade
Bartsia alpina Finland
Bartsia alpina Canada
Bartsia alpina Germany
Bartsia alpina Italy
I
41.6
Tozzia alpina
Hedbergia
abyssinica
Bartsia longiflora
Clade
Bartsia Bartsia longiflora subsp. macrophylla
decurva
II
Clade IV
Fig. . Split consensus networks for the combined chloroplast and ITS data, using a 25% threshold, obtained from the collections of trees produced 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 network based on A) all 36 taxa included in the study, and B) with five taxa displaying high levels of incongruence (Macrosyringion glutinosum,
Bartsia sp. Peru, Nothobartsia asperrima, Nothobartsia spicata and Odontitella virgata, shown in bold italics in A), removed using the “exclude
selected taxa” command (see text). The tree from the Bayesian analysis with all markers combined and based on the reduced sampling presented
in B) is shown in Fig. 5. Clades I–IV are shaded grey: I, Bartsia s.str.; II, Hedbergia; III, Bellardia; IV, Odontites s.l. — O., Odontites; B., Bornmuellerantha.
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Scheunert & al. • Phylogeny of Rhinantheae
TAXON 61 (6) • December 2012: 1269–1285
Striga asiatica
OG
Pedicularis spp.
Melampyrum nemorosum
1.00
99
Rhinantheae
1.00
100
1.00
100
Rhinanthus alectorolophus
0.96
77
Bartsia alpina Germany
0.61
95
Bartsia alpina Finland
1.00
core group of
Rhinantheae
Rhynchocorys kurdica
Lathraea squamaria
Bartsia alpina Italy
I. Bartsia s.str.
1.00
95
Rhynchocorys orientalis
RRL clade
1.00
100
1.00
100
Bartsia alpina Canada
Euphrasia stricta
95
Tozzia alpina
Hedbergia abyssinica
1.00
100
Bartsia decurva
1.00
99
1.00
96
Bartsia longiflora
II. Hedbergia
1.00
97
Bartsia longiflora subsp. macrophylla
Bellardia trixago
Parentucellia latifolia
1.00
100 1.00
96
0.98
75
1.00
100
1.00
99
Bartsia canescens
Bartsia mutica
Bornmuellerantha aucheri
Bornmuellerantha alshehbaziana
Odontites corsicus
Odontites himalayicus
Odontites pyrenaeus
1.00
91
0.80
50
1.00
100
Odontites viscosus
s.l.
1.00
82
IV. Odontites
1.00
100
1.00
100
III. Bellardia
Parentucellia viscosa
1.00
100
Bartsiella rameauana
Odontites bocconei
Odontites cyprius
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.
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Scheunert & al. • Phylogeny of Rhinantheae
The sister relationship between clade III and IV is highly
supported in the Bayesian analysis and receives moderate support in the ML analysis (PP: 0.98, BS: 75).
Clade III (“Bellardia”) comprises the two included species of Neotropical Bartsia, two accessions of Parentucellia
Viv., and Bellardia trixago (L.) All. The monotypic Bellardia 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 relationship 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 Odontites being part of two subclades which receive maximum to
moderate support: one subclade (PP: 1.00, BS: 82) is composed of four Odontites species and the monotypic Bartsiella
(Bartsiella rameauana (Emb.) Bolliger) nested within it, being 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 subclade (PP: 1.00, BS: 100) consists of the sister species O. corsicus G. Don and O. himalayicus Pennell, and of the monophyletic Bornmuellerantha, comprising B. aucheri (Boiss.) Rothm.
and the recently described B. alshehbaziana (Dönmez & Mutlu,
2010). Given the positions of Bornmuellerantha and Bartsiella,
Odontites is rendered paraphyletic in this analysis.
DISCUSSION
Biogeography of Rhinantheae. — Těšitel & al. (2010)
conducted a dispersal-vicariance analysis and identified
temperate western Eurasia as the origin of the Rhinanthoid
Orobanchaceae, in accordance with the Laurasian origin north
of the Tethyan Sea as assumed by Wolfe & al. (2005) and the
Eurasian origin inferred for the cosmopolitan hemiparasitic
Euphrasia by Gussarova & al. (2008). Most genera of the core
group of Rhinantheae sampled here have a distinct center of
diversity in the Mediterranean area. An exception are the frutescent perennial Afromontane species of Bartsia sect. Longiflorae 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 lineage of
Rhinantheae, based on its distinctive corolla morphology. However, 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 pattern, the core Rhinantheae agree with some other typical elements 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 Bellardia 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 Argentina, and seem to descend from a Mediterranean lineage
represented by the extant Parentucellia latifolia, which is
widespread across the Mediterranean area and beyond, ranging 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 arrived 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 especially 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 Odontites (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 unambiguously dissociated from the remaining members of the genus 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 (equalling 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 chromosome 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 suggests 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-flowered, hemiparasitic, frutescent Afromontane species of Bartsia (Bartsia sect. Longiflorae), B. decurva and B. longiflora (including B. longiflora subsp. macrophylla; Hedberg & al., 1979; Molau, 1990) are associated with
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TAXON 61 (6) • December 2012: 1269–1285
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 palynology provide strong evidence for this clade to form a natural 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 exclusively characterized by a rotate corolla symmetry (Hedberg
& al., 1980; Molau, 1988). The unique actinomorphic symmetry
of the flowers observed in Hedbergia (Molau, 1988) might 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 chromosome count of 2n = app. 28 for B. decurva (as B. macrocalyx
R.E. Fr.) was reported by Hedberg (Hedberg, 1957; Hedberg
& Hedberg, 1977; Hedberg & al., 1979), and no count for Hedbergia 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 Parentucellia 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 racemose inflorescences of the members of the Bellardia clade are
predominantly multilateral (partially unilateral in the Neotropical 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 Parentucellia. 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 position 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. Considering morphological, molecular and biogeographical evidence,
including Parentucellia in Bellardia seems reasonable; in doing
so, the generic name Bellardia All. has nomenclatural priority
over the younger name Parentucellia Viv. As the New World
1280
species of Bartsia s.l. are nested within Parentucellia and the
Bellardia clade, these species also should be included in Bellardia. The cytological data available for the group give some
additional support, as both Bellardia and Parentucellia, as well
as New World Bartsia share a chromosome base number of
x = 12 (although this is a common base number in Rhinantheae):
Bellardia trixago is a diploid with 2n = 24 (Speta, 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 internal 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 observed 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, applying 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 regarding 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. Furthermore, support values for a clade IV including Macrosyringion
are low (PP: 0.78, BS: 81) in the chloroplast phylogeny, raising the question whether the genus should be included into a
more broadly defined Odontites. Chromosome numbers cannot 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 ambiguous as well: the prolonged corolla tubes, which could possibly
represent a synapomorphy for the genus, were considered not
significant enough to support it as distinct (Rothmaler, 1943).
The shortly bifid corolla lip, however, additionally used by
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Scheunert & al. • Phylogeny of Rhinantheae
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 genus 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 Nothobartsia has frequently changed in the past, as the plants unite morphological characters of both Bartsia and Odontites (Molau,
1990; Bolliger & Molau, 1992). Both taxa were originally described as Bartsia (e.g., Wettstein, 1891) before a separate 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 incongruence in the single-marker reconstructions and almost
exclusively accounts for the highly networked central part 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 similar 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 Macrosyringion. 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 (Rothmaler, 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 Rhinantheae, 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 chloroplast 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 species of Nothobartsia, which are intermediate between Bartsia
and Odontites s.l. (Bolliger & Molau, 1992). Equally, the annual Odontitella shares characters with both Odontites s.l. and
Bartsia s.l. (Rothmaler, 1943; Bolliger, 1996), the latter suggesting a possible relationship to the Hedbergia clade.
NEW GENERIC CIRCUMSCRIPTIONS
As evident from our molecular phylogenetic reconstructions 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, requires 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 septentrionale 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 Norweg. 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. Fleischm. & Heubl,
comb. nov. ≡ Bartsia decurva Hochst. ex Benth. in Candolle, Prodr. 10: 545. 1846 – Holotype: [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 nomenclatural 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 latifolia 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 Parentucellia (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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TAXON 61 (6) • December 2012: 1269–1285
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 exemplarily 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 – Lectotype (designated by Molau in Opera Bot. 102:
76. 1990): Peru, Lima, without date, Dombey s.n. (P; isotype: 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: Turkey, Antalya, Gazipaşa, 1760 m, 23 Sep 2006, Dönmez
& Mutlu AAD 14036 (HUB; isotypes: E, INU, M!, MO).
CONCLUSION
The Rhinantheae as studied here are characterized by some
obvious topological incongruences between the plastid and nuclear datasets. Nothobartsia constitutes the most conspicuous example of reticulation in the evolutionary history of Rhinantheae,
explaining part of the incongruent patterns observed. Hybridization 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 circumscription of Bartsia, Bellardia, and Hedbergia, and corroborate
a broader circumscription of Odontites. Further 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 several 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.
1282
ACKNOWLEDGEMENTS
The authors would like to thank Ulf Molau and Markus Bolliger
for helpful correspondence; the curators of the Munich Herbarium
(M), especially Franz Schuhwerk for providing important material
for this study; Ali A. Dönmez and Birol Mutlu for making available
material from the newly described Bornmuellerantha alshehbaziana;
Eberhard Fischer for providing material of African Bartsia; Daniel
Pinto Carrasco for his supporting correspondence; and two anonymous
reviewers for helpful comments on the manuscript. Tanja Ernst is
acknowledged for technical assistance.
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Appendix. List of taxa used in the phylogenetic analyses with voucher information. Previously published sequences of ITS, trnK and rps16 are provided
with reference citations. Key to references: (1): Těšitel & al., 2010; (2): Schäferhoff & al., 2010; (3): Tank & Olmstead, 2008; (4): Müller & al., 2004; (5):
Morawetz & Wolfe, 2009; (6): Young & al., 1999.
Taxon name; region of origin, coll. date, collector, coll. no. (voucher location); acc. no. ITS; acc. no. trnK; acc. no. rps16
Bartsia alpina L.; Germany, 2000, Förther, H., 10816 (M); JF900502; JF900567; JF900535; – Bartsia alpina L.; Canada, 1965, Doppelbaur, H., 203 (M)
JF900505; JF900570; JF900538; – Bartsia alpina L.; Finland, 1971, Federley, B., s.n.; JF900504; JF900569; JF900537; – Bartsia alpina L.; Italy, 1972, Lippert, 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); JF900511; JX629749; JF900543; – Bartsia longiflora Hochst. ex Benth.;
Kenya, 1978, Grau, J., 1899 (M); JF900510; JF900575; JX629747; – Bartsia longiflora subsp. macrophylla (Hedberg) Hedberg; Rwanda, 2010, Fischer &
Thiel, 20415 (private herb. E. Fischer); JF900519; JF900583; JF900551; – Bartsia mutica (Kunth) Benth.; Argentina, 2008, Bräuchler, C., 5170 (M); JF900517;
JF900581; JF900549; – Bartsia sp.; Peru, 2001, Henning, T. & Schneider, C., 18 (M); JF900516; JF900580; JF900548; – Bartsiella rameauana (Emb.) Bolliger;
Morocco, 1951, Rauh, W., 393 (M); JF900523; JF900587; JF900555; – Bellardia trixago (L.) All.; Spain, 2010, Schuhwerk, F., 27 (M); JF900513; JF900577;
JF900545; – Bornmuellerantha alshehbaziana Dönmez & Mutlu; Turkey, 2006, Dönmez, A. & Mutlu, B., 14036 (HUB); JF900522; JF900586; JF900554; –
Bornmuellerantha aucheri (Boiss.) Rothm.; Iran, 2001, Podlech, D. & Zarre, Sh., 55243 (M); JF900521; JF900585; JF900553; – Euphrasia stricta J.P. Wolff
ex J.F. Lehm.; Czech Republic, 2007, Svobodova, S., 5091 (CBFS); FJ790051(1); FJ790111(1); n/a; – Euphrasia stricta J.P. Wolff ex J.F. Lehm.; n/a, n/a, Borsch,
T., 3785 (BONN) n/a; n/a; FN794093(2); – Hedbergia abyssinica (Benth.) Molau; Ethiopia, 1973, Ash, J.W., 2054 (M); JF900509; JF900574; JF900542; – Lathraea squamaria L.; Germany, 2010, Olano-Marín, C., 1 (M); JF900500; JF900565; JF900533; – Macrosyringion glutinosum (M. Bieb.) Rothm.; France, 1997,
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Appendix. Continued.
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); JF900507; JF900572; 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; – Odontites viscosus (L.) Clairv.; France, 1985, Perrin, F., 12515 (M); JF900524; JF900588; JF900556; – Parentucellia latifolia (L.) Caruel; 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; – Tozzia
alpina L.; Austria, 1998, Panzer, R., s.n. (M); JF900512; JF900576; JF900544.
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4.2. Article II
Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae):
new insights inferred from DNA sequence data.
by Agnes Scheunert & Günther Heubl
Plant Systematics and Evolution 291: 69-89 (2011)
II
The final publication is available at Springer via
http://dx.doi.org/10.1007/s00606-010-0369-z
51
52
Plant Syst Evol (2011) 291:69–89
DOI 10.1007/s00606-010-0369-z
ORIGINAL ARTICLE
Phylogenetic relationships among New World Scrophularia L.
(Scrophulariaceae): new insights inferred from DNA sequence
data
Agnes Scheunert • Günther Heubl
Received: 27 April 2010 / Accepted: 4 October 2010 / Published online: 28 October 2010
Ó Springer-Verlag 2010
Abstract The genus Scrophularia L. (Scrophulariaceae)
comprises 200–300 species, of which approximately 19 are
distributed in North America and the Greater Antilles. To
investigate phylogenetic and biogeographic relationships of
the New World species, two intergenic spacers (trnQ-rps16
and psbA-trnH) of chloroplast DNA and nuclear ribosomal
ITS were sequenced. Phylogenetic analyses revealed three
distinct New World clades that correspond to their geographical distribution and are corroborated by morphological characters. Phylogenetic inference indicates the
eastern American S. marilandica L. as sister to all Antillean
species; for colonization of the Caribbean archipelago, a
late Miocene dispersal event from the North American
mainland is assumed. There is evidence for a hybrid origin
of the most widespread North American species, S. lanceolata Pursh. The results further suggest that S. nodosa L. is
sister to all New World and three Japanese species of
Scrophularia. The latter form an Eastern Asian–Eastern
North American (EA-ENA) disjunction with six New
World species. We propose an eastern Asian origin for the
New World taxa of Scrophularia. Divergence times estimated using a relaxed molecular clock model suggest one or
more Miocene migration events from eastern Asia to
the New World via the Bering Land Bridge followed by
diversification in North America.
Keywords Scrophularia North America Greater
Antilles Molecular phylogeny trnQ-rps16 intergenic
spacer psbA-trnH intergenic spacer
A. Scheunert (&) G. Heubl
Department Biology I, Ludwig-Maximilians-University Munich,
Menzinger Str. 67, 80638 Munich, Germany
e-mail: agnes.scheunert@gmx.net
Introduction
Scrophularia L. (Scrophulariaceae), commonly known as
figwort, is a widespread genus of mainly holarctic distribution (Hong 1983) and comprises about 200 (Fischer
2004) to more than 300 species (Willis 1973). Its origin is
assumed to be the Himalayan region (Stiefelhagen 1910)
while its primary diversity center is found in Iran and
adjacent areas, where 64 species occur (Grau 1981). Fiftyseven species are reported for Turkey (Lall and Mill 1978)
and nearly as many for Russia/Middle Asia, and 36 species
are listed for China (Shu 1998). Species numbers decrease
in Europe [only six species in Central Europe according to
Hartl (1965)], but another secondary diversity center is
found on the Iberian Peninsula with 22 species (Ortega
Olivencia 2009). The number of species of Scrophularia is
considerably lower in the New World: about 18–20 species
are known from North America and the Caribbean (Greater
Antilles) (USDA 1982; Liogier 1994).
The Scrophulariaceae, as traditionally defined by morphological characters, is not supported by molecular analyses. Phylogenetic studies based on DNA sequencing have
radically changed their circumscription since reconstructions indicated that the family does not represent a monophyletic entity. Olmstead and Reeves (1995) identified two
monophyletic clades named Scroph I and Scroph II, the
former including Buddleja L., Selago L., Verbascum L.,
and the type genus Scrophularia. The clade Scroph I was
named Scrophulariaceae s.str. by Olmstead et al. (2001),
while Scroph II is equivalent to Plantaginaceae. Subsequent molecular phylogenetic studies on the delimitation
of the Scrophulariaceae (e.g., Oxelman et al. 1999, 2005;
Rahmanzadeh et al. 2004; Albach et al. 2005) have generally confirmed the pattern revealed by Olmstead and
Reeves (1995) and Olmstead et al. (2001).
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70
Despite the large number of infrafamilial studies on
Scrophulariaceae (e.g., Albach and Chase 2001; Kornhall
et al. 2001; Gebrehewit et al. 2000), there is a considerable
lack of knowledge concerning phylogenetic relationships
within the familiar type genus Scrophularia. Major revisions of the genus were provided by Wydler (1828), Bentham (1846), and Boissier (1879). The most comprehensive
treatment of the genus was done by Stiefelhagen (1910).
He arranged 145 species into two sections and five subsections, using leaf anastomosis, petal length, shape of the
corolla tube, and life form as distinguishing features. As no
extensive studies have been conducted on the genus since
then, a systematic revision using methods from molecular
biology is of great interest. The present study focusing on
the New World taxa of Scrophularia is a first step in a
series of treatments for major geographical areas towards
the goal of a comprehensive revision of the genus.
Stiefelhagen (1910), in his treatment of the genus,
argued that all New World species described at that time
(except S. macrantha Greene ex Stiefelhagen, a distinctive
species native to New Mexico) were conspecific with
S. nodosa L. Actually, the widespread species do share a
very similar morphology. However, this assumption was
rejected by Pennell (1935; 1947). In his treatment of the
eastern (Pennell 1935) and far western (Pennell 1947)
North American species, he recognized two species for
eastern North America (S. marilandica L. and S. lanceolata
Pursh) and five species for the westernmost area (S. villosa
Pennell, S. atrata Pennell, S. californica Cham. & Schltdl.,
S. multiflora Pennell, S. lanceolata). Regional studies were
published for Arizona and New Mexico (Tidestrom and
Kittell 1941), Arizona (Kearney and Peebles 1951), California (Munz and Keck 1959), and Nevada (Edwin 1959).
A comprehensive study on western North American
Scrophularia was provided by Shaw (1962), but no phylogenetic study based on molecular data of New World
Scrophularia is available to date.
In the West Indies, Scrophularia has undergone an
extensive diversification that corresponds to the generally
high plant diversity in the Caribbean (Myers et al. 2000):
while 11–13 species (depending on the synonymy
employed) occur on the large North American mainland,
the islands of the Greater Antilles host 7 or 8 species (7 of
which are endemic to the Dominican Republic) characterized by notable morphological differences. As only a
small number of species of Scrophularia have expanded
into tropical regions, the understanding of their relationships to holarctic representatives is of special interest.
In this paper, we present a phylogenetic analysis of New
World Scrophularia based on sequence data from nuclear
internal transcribed spacer regions (ITS 1 and 2) and
chloroplast trnQ-rps16 and psbA-trnH intergenic spacers.
The particular objectives were to (1) test the monophyly of
123
A. Scheunert, G. Heubl
the New World taxa and analyze phylogenetic relationships
among them, especially regarding the origin of the West
Indian taxa, (2) examine Stiefelhagen’s taxonomic concept
of reducing almost all North American species of Scrophularia into the synonymy of S. nodosa, (3) elucidate
patterns of cytogenetic evolution based on the molecular
phylogeny, (4) identify biogeographic diversity patterns
within this alliance, and (5) gain insight into the colonization pathways leading from the assumed Eurasian origin
of the genus to the New World.
This contribution is part of a more comprehensive study
in preparation on the evolution of Scrophularia.
Materials and methods
Plant material
Leaf material for DNA extraction was removed from herbarium specimens in A, GH, M, MSB, UTEP, W, and WU.
In three cases, material was taken from cultivated plants in
the greenhouses of the Munich Botanical Garden. The
sampling strategy was to include all species of Scrophularia occurring in North America and in the Caribbean as
far as possible. Scrophularia multiflora and S. serrata
Rydb. were sampled to gain more information about their
status and taxonomic rank, which have been discussed for
some time. No material could be obtained from S. pluriflora Urb. & Ekman, S. tuerckheimii Urb., and S. bahorucana Zanoni, three local endemics of Hispaniola. Several
eastern Asian species were added to the sampling because
preliminary molecular analyses revealed close relationships between nearctic and eastern Asian species of Scrophularia (results not shown). Furthermore, to assess the
question of a close relationship of S. nodosa to the New
World species of Scrophularia [or even conspecifity, as
proposed by Stiefelhagen (1910)], we analyzed three
accessions of S. nodosa, collected from Germany, Armenia, and the USA. One rather widespread Eurasian species
(S. umbrosa Dumort.) completed the sampling. Verbascum
nigrum (Scrophulariaceae) was chosen as an outgroup
because Verbascum was found to be sister to the Scrophularia alliance by Olmstead and Reeves (1995) and
Olmstead et al. (2001). Additionally, two more distant
outgroup species, Hemimeris centrodes Hiern (Scrophulariaceae) and Russelia verticillata Kunth (Plantaginaceae), were sampled. Altogether, the analysis included 32
taxa: 12 species occurring in mainland North America, 5
from the Greater Antilles, 8 from eastern Asia, 1 Eurasian
species, 3 accessions of S. nodosa, and 3 outgroup taxa.
Table 1 lists all taxa and summarizes additional information such as voucher specimen data and GenBank accession
numbers.
Source
Date
Collector
Coll. no.
Herbarium
GB (T-R)
GB (ITS)
GB (P-T)
Russelia verticillata Kunth
Costa Rica, Guanacaste
Prov.
13.10.1990
P. Döbbeler
3795
M
–
HQ130062
–
Hemimeris centrodes Hiern
South Africa, Cape
Prov.
04.09.1976
P. Goldblatt
4033
M
HQ130033
HQ130063
–
Verbascum nigrum L.
Germany, Bavaria
18.06.1998
H. Wunder
nn
M
HQ130034
HQ130064
HQ130094
Scrophularia atrata Pennell
USA, California, Santa
Barbara Co.
30.04.1958
H. M. Pollard
nn
W 1960/21086
HQ130051
HQ130081
HQ130111
Scrophularia buergeriana
Miq.
Greenhouse cultivated
plantsa
Cult. since 2008
Material from Kor. Nat.
Arb.b, cultivated by
A. Scheunert
Cult. no 001/1-1
MSB
HQ130040
HQ130070
HQ130100
Scrophularia californica
Cham. & Schltdl.
USA, California, Santa
Cruz Co.
04.05.1976
A. L. and
H. N. Moldenke
30898
M
HQ130049
HQ130079
HQ130109
Scrophularia desertorum
(Munz) R. J. Shaw (S.
californica Cham. &
Schltdl. var. desertorum
Munz)
USA, California, Mono
Co.
28.05.1959
P. H. Raven
14265
GH
HQ130057
HQ130087
HQ130117
Scrophularia densifolia
Urb. & Ekman
Scrophularia domingensis
Urb.
Dominican Republic
10.12.1969
A. H. Liogier
17213
GH
–
HQ130093
–
Dominican Republic
28.02.1929
E. L. Ekman
H11711
GH
HQ130059
HQ130089
–
Scrophularia duplicatoserrata Makino
Japan, Shizuoka Pref.
09.11.2002
T. Sugawara
2110903
A
HQ130046
HQ130076
HQ130106
Scrophularia eggersii Urb.
Dominican Republic
30.05.1964
B. Augusto
1499
A
HQ130060
HQ130090
HQ130119
Scrophularia grayana
Maxim. ex Kom.
Japan, Northern
Honshu, Iwate Pref.
09.07.1986
H. Tohda, T. Nemoto,
Y. Endo, H. Hoshi
1155
A
HQ130044
HQ130074
HQ130104
Scrophularia kakudensis
Franch.
Greenhouse cultivated
plants
Cult. since 28.07.08
Material from Bot.
Gard. Tübingen,
Germany, cultivated
by A. Scheunert
2008/1036, cult.
no 002/1-1
MSB
HQ130045
HQ130075
HQ130105
Scrophularia koraiensis
Nakai
Greenhouse cultivated
plants
Cult. since 28.07.08
Material from Kor. Nat.
Arb., cultivated by
A. Scheunert
Cult. no 003/1-1
MSB
HQ130039
HQ130069
HQ130099
Scrophularia laevis
Wooton & Standl.
USA, New Mexico,
Dona Ana Co.
06.07.1993
R. D. Worthington
22161
M
HQ130047
HQ130077
HQ130107
Scrophularia lanceolata
Pursh
USA, Massachusetts,
Hampshire Co.
28.06.1977
H. E. Ahles
83075
M
HQ130050
HQ130080
HQ130110
Scrophularia marilandica
L.
nn
1990
V. Bates
10313
Harvardc
HQ130055
HQ130085
HQ130115
Scrophularia macrantha
Greene ex Stiefelhagen
USA, New Mexico,
Grant Co.
02.09.1996
C. E. Freeman
129
UTEP
HQ130122
HQ130092
HQ130121
71
123
Taxon
Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae)
Table 1 Taxa included in the present study, with voucher information on country of origin, collector/collector’s number, date of collection, herbarium, and GenBank accession numbers
72
123
Table 1 continued
Taxon
Source
Date
Collector
Coll. no.
Herbarium
GB (T-R)
GB (ITS)
GB (P-T)
Scrophularia micrantha
Desv. ex Ham.
Dominican Republic
07/1910
H. v. Türckheim
3064
WU
HQ130058
HQ130088
HQ130118
Scrophularia minutiflora
Pennell
Dominican Republic
14.08.1968
A. H. Liogier
12075
GH
HQ130061
HQ130091
HQ130120
Scrophularia montana
Wooton
USA, New Mexico, San
Miguel Co.
19.08.1984
S. R. Hill
15272
GH
HQ130052
HQ130082
HQ130112
Scrophularia multiflora
Pennell
USA, California, Kern
Co.
25.04.1951
W. J. Dress
3171
M
HQ130048
HQ130078
HQ130108
Scrophularia musashiensis
Bonati
Japan, Toyama Pref.
26.06.1991
J. Jutila, H. Fujino,
M. Yoshizoki
473
GH
HQ130043
HQ130073
HQ130103
Scrophularia ningpoensis
Hemsl.
Japan, Toyama Pref.;
garden culture
03.10.1991
J. Jutila
769
GH
HQ130041
HQ130071
HQ130101
Scrophularia nodosa L.
(Germany)
Germany, Bavaria
29.06.1999
D. Podlech
nn
MSB 116671
HQ130037
HQ130067
HQ130097
Scrophularia nodosa L.
(Armenia)
Armenia, Lori Prov.
01.07.2003
G. Fayvush,
K. Tamanyan,
H. Ter-Voskanian,
E. Vitek
03-0549
MSB 123419
HQ130038
HQ130068
HQ130098
Scrophularia nodosa L.
(USA)
Scrophularia parviflora
Wooton & Standl.
USA, Massachusetts,
Franklin Co.
USA, Arizona, Santa
Rita Mountains
23.08.1977
H. E. Ahles
84733
M
HQ130036
HQ130066
HQ130096
25.08.1932
V. Douglas
1588?
GH
HQ130056
HQ130086
HQ130116
Scrophularia serrata Rydb.
USA, Idaho, Idaho Co.
13.07.1937
F. W. Pennell,
L. Constance
20885
GH
HQ130053
HQ130083
HQ130113
Scrophularia umbrosa
Dumort.
Scrophularia villosa
Pennell
Iran, Chaharmahal va
Bakhtiyari Prov.
USA, California, L.A.
Co.
17.07.2003
M. R. Parishani
14232
M
HQ130035
HQ130065
HQ130095
10.05.1962
P. H. Raven
17711
GH
HQ130054
HQ130084
HQ130114
Scrophularia yoshimurae T.
Yamaz.
Taiwan, Nantou Hsien
07.10.1992
C.-C. Liao
718
A
HQ130042
HQ130072
HQ130102
Pref. Prefecture, Prov. province, Co. county, GB Genbank accession number, T-R trnQ-rps16 intergenic spacer, P-T psbA-trnH intergenic spacer
Plants grown in the greenhouses of the Botanical Garden Munich, Germany
a
Korean National Arboretum, Gwangwhanum Forest, Seoul, Rep. of Korea
c
Harvard University, herbarium not known
A. Scheunert, G. Heubl
b
Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae)
DNA extraction, amplification, and sequencing
Total DNA was extracted from dried leaf material using the
NucleoSpin Plant Kit (Macherey-Nagel). Protocols followed those provided by the manufacturer, except for an
additional extraction step with phenol/chloroform to
remove potentially interfering secondary compounds. The
extracted DNA was resuspended in 50 ll elution buffer
(10 mM Tris-HCl), and a standard amount of 1 ll of the
solution was used for amplification (higher amounts up to
3 ll in cases where PCR yielded insufficient amounts of
product).
Three noncoding regions from nuclear DNA (ITS) and
chloroplast DNA (trnQ-rps16 and psbA-trnH intergenic
spacers) were chosen for phylogenetic analysis. The
markers were amplified from total DNA using Taq-polymerase (AGS), or Phusion-polymerase (New England
Biolabs) whenever Taq-polymerase did not provide satisfactory results. For ITS, we used the universal primers
ITS1 and ITS4 (and in five difficult cases ITS2 and ITS3)
described by White et al. (1990), alongside aITS1 and
aITS4 designed for angiosperms by Meimberg (2002). In
four cases, additional primers designed exclusively for
73
problematic taxa were necessary, but these were used only
in sequencing reactions, not for PCR (for exact position
and additional information about all primers, see Fig. 1a–
c). The trnQ-rps16 intergenic spacer fragment was amplified using primers 1 (trnQ-F) and E (rps16-1R) described
by Calviño and Downie (2007). These primers, originally
designed for Apiaceae, were here shown to work for
Scrophulariaceae as well. Two internal primer sets designed
for the trnQ-rps16 spacer fragment were used for sequencing
(see Fig. 1a). The chloroplast psbA-trnH intergenic spacer
was amplified with primers psbA (forward) and trnH2R
(GUG) (reverse) as described by Shaw and Small (2004).
PCR reactions were performed in volumes of 50 ll
(or rarely 100 ll) containing a dNTP solution of 2.5 mM,
Taq-polymerase with 1 U/ll, primer solutions with a concentration of 100 pmol/ll, and differing amounts of
unquantified genomic DNA. When necessary, an alternative preparation containing 0.05% bovine serum albumin
(BSA) and 100% dimethyl sulfoxide (DMSO) was used for
ITS. The 109 Thermo Pol (TP) reaction buffer used for
‘‘standard’’ PCR with Taq polymerase consisted of
100 mM KCl, 100 mM (NH4)2SO4, 200 mM Tris-HCl,
20 mM MgSO4, 10% Triton X-100 at a pH of 8.8 (25°C).
LSC
IRb
IRa
SSC
(a)
1 (trnQ-F)
SPF/SPR2
trnQ-rps16 intergenic
E (rps16-1R)
SPR/SPF2
spacer
DELETION
trnQ gene
(b)
rps16 gene
psbA (F)
trnH2R (R)
psbA-trnH intergenic spacer
psbA gene
(c)
aITS1-F
trnH gene
ITS1-F
18S rDNA
ITS2-R/3-F
ITS 1
ITS IF/IR
5,8S rDNA
Fig. 1a–c Schematic illustration of marker regions in Scrophularia
used for this study, with relative positions of primers. F indicates
forward, R indicates reverse primers. The trnQ-rps16 and psbA-trnH
intergenic spacers are part of the large single copy (LSC) unit of the
plastome (overview above marker diagrams). a Map of the trnQrps16 intergenic spacer region of Scrophularia showing the deletion
within the spacer; position within the LSC is indicated by a black
circle. Primers 1 (trnQ-F) and E (rps16-1R) according to Calviño and
Downie (2007). Primer sequences designed for this study, written 50
to 30 : SPF: GAC AAC TGT TCA GTC TAT CTG, SPR: CAC GTT
TGA TCT TCA TAG G, SPF2: CCT ATG AAG ATC AAA CGT G,
ITS4-R
ITS 2
aITS4-R
26S rDNA
SPR2: CAG ATA GAC TGA ACA GTT GTC. b Map of the psbAtrnH intergenic spacer region of Scrophularia; position within the
LSC indicated by a black square. Primers psbA (F) and trnH2R
(R) according to Shaw and Small (2004). c Map of the ITS region of
Scrophularia with relative positions of the internal transcribed spacers
1 and 2. ITS1-F, ITS2-R, ITS3-F, and ITS4-R according to White
et al. (1990); aITS1-F and aITS4-R according to Meimberg (2002).
Primer sequences designed for this study, written 50 to 30 : ITS-IF:
AAT CCC GTG AAC CAT CGA GTT, ITS-IR: AAC TCG ATG
GTT CAC GGG ATT
123
74
A. Scheunert, G. Heubl
The modified TP buffer for BSA/DMSO ITS-PCR consisted of 50 mM KCl, 200 mM (NH4)2SO4, 750 mM TrisHCl, 15 mM MgSO4. The 59 HF reaction buffer for
Phusion PCR (Finnzymes) contained 7.5 mM MgCl2.
Detailed amounts of the components for each PCR reaction
are given in Table 2.
A thermocycler type T-Personal 48 (Biometra), type
Primus 96 plus (MWG-Biotech), or type 2720 (Applied
Biosystems) was used for amplification. ITS/trnQ-rps16
spacer amplification programs started with a 5 min initial
denaturation step at 94°C; followed by 40 cycles of 30 s/1
min denaturation (94°C), 30 s annealing (54°C), and 1 min
15 s/2 min extension (72°C); ending with a final extension
step of 10 min (72°C). The psbA-trnH spacer program was
identical to that of ITS except for a lower annealing temperature (48°C). Successful PCR reactions were either
purified with NucleoSpinÒ Extract II-Kit (Macherey-Nagel)
following the manufacturer’s instructions or were reduced
to 25 ll and then purified in 4 ll units with 0.025 ll exonuclease I and 0.25 ll shrimp alkaline phosphatase (Sap) in
a 5 ll preparation with 0.0725 ll 109 TP buffer (ExoSap
purification). Purification using columns proved particularly useful with ITS as ExoSap seemed to work inefficiently on ITS fragments for unknown reasons.
Cycle Sequencing was carried out using the BigDyeÒ
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems)
in a final volume of 20 ll. Runs were performed on an ABI
3730 48 capillary sequencer (Applied Biosystems). In all
cases, markers were sequenced bidirectionally using the same
primers as in PCR reactions, sometimes supplemented by
sequences from the internal sequencing primers, to achieve
maximum reliability of the results. Bidirectional sequencing
proved essential for the psbA-trnH intergenic spacer because
sequencing terminated regularly and from both ends at two
nucleotide repeat regions in the central part of the sequence.
Alignment, indel coding, and phylogenetic
reconstruction
All sequences generated in this study were assembled,
edited, and aligned manually using BioEdit 7.0.5.1 (Hall
Table 2 Components and
corresponding amounts of
chemicals used for PCR
reactions conducted for this
study
123
Primer (each) (ll)
1999). The alignment is available from the corresponding
author upon request. Ambiguously aligned characters and
mononucleotide repeat units were excluded from further
analyses. The beginning and end of the alignments where
not all of the taxa provided complete data were also
excluded. For Bayesian and parsimony analyses, informative indels (i.e., indels presumably containing phylogenetic
information) resulting from the alignment were coded
using the simple indel coding algorithm proposed by
Simmons and Ochoterena (2000), which is implemented in
SeqState (Müller 2005). A present/absent indel matrix
(coded as 1/0) was added at the end of the alignment. The
three markers were analyzed in a single combined dataset
as well as in one ITS and one combined chloroplast dataset.
In accordance with Bull et al. (1993), the combined
matrix was tested for incongruence between nuclear and
chloroplast markers. To assess cases of ‘‘hard incongruence’’ (Mason-Gamer and Kellogg 1996) between the two
datasets, two maximum parsimony bootstrapping analyses,
one for each dataset, were conducted (using the same settings as for the bootstrap analysis of the combined dataset)
and the resulting trees visually examined for well-supported contradicting placement of taxa, using a cutoff of
70% bootstrap support following Mason-Gamer and Kellogg (1996).
In addition, two statistical tests were used: the incongruence length difference (ILD) test as a suitable first step
in detecting incongruences (Cunningham 1997; Hipp et al.
2004), and Templeton’s significantly less parsimonious test
(SLPt; Johnson and Soltis 1998; Templeton 1983) to
compensate for putative weaknesses of the ILD test pointed
out by Barker and Lutzoni (2002). The ILD test, called the
partition homogeneity test (PHT) in PAUP* 4.0b10
(Swofford 2003), computed 1,000 replicates with MAXTREES option set to 100 and was executed on the combined dataset, excluding coded indels, and after removing
constant characters from the matrix. The PHT was conducted on the combined chloroplast dataset as well, to test
the two chloroplast markers against each other. For the SLP
test (implemented in PAUP* 4.0b10 as the nonparametric
pairwise test), we performed separate runs for the ITS and
Standard PCR
(all markers)
BSA/DMSO PCR
(ITS only)
Phusion PCR (ITS
and trnQ-rps16 spacer)
0.125
0.1
0.25
Reaction buffer (ll)
5.0 (109 Thermo Pol)
5.0 (modified)
10 (59 HF)
dNTPs (ll)
2.5
4.0
4.0
Polymerase (ll)
1.0 (Taq)
1.0 (Taq)
0.25 (Phusion)
BSA/DMSO (ll)
–
0.5/2.5
–
Unquantified DNA (ll)
1.0 (-3.0)
1.0 (-3.0)
1.0 (-3.0)
Total (ll)
50.0
50.0
50.0
Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae)
the chloroplast dataset. A 70% consensus tree from an
unconstrained heuristic search was tested against the trees
obtained from a constrained heuristic search (level of significance P = 0.05; the P value reported represents the
maximum found in the respective test). As the constraint,
we used an artificial tree reflecting the topology suggested
by the other marker/dataset, but with only the outgroup and
contradicting nodes resolved (topologies correspond to the
70% consensus trees obtained when assessing cases of hard
incongruence, see respective paragraph in the ‘‘Results’’
section). The heuristic searches were run with the same
settings as all maximum parsimony (MP) calculations in
this study (see below).
Phylogenetic reconstruction analyses were performed
with a Bayesian inference (BI), an MP, and a maximum
likelihood (ML) approach. MrBayes 3.1.2 (Ronquist and
Huelsenbeck 2003) was used for BI calculations using the
metropolis-coupled Markov Chain Monte Carlo (MCMC)
algorithm. A mixed dataset was defined for MrBayes
[DNA characters in part 1, binary characters (coded indels)
in part 2]; for the DNA partition, the analyses assumed the
general time reversible (GTR) model (Tavaré 1986) with
rates differing across sites following a gamma distribution,
and allowing a proportion of invariant sites; for the coded
indel characters, settings as recommended in the MrBayes
3.1 manual were employed. MrBayes was then executed
with two runs and four chains (hot chains with the default
temperature t = 0.2) for 2 million generations, sampling
every 100th generation. In all cases, average standard
deviation of split frequencies fell below 0.01 long before 2
million generations were computed. A burn-in of 1/4 of the
samples was discarded, and the remaining trees were
summarized in an 80% majority rule consensus tree.
MrBayes settings were identical for all analyses of combined and individual ITS and cpDNA datasets.
Maximum parsimony analyses were performed with
the combined (ITS and cpDNA) dataset including coded
indels using PAUP* 4.0b10 (Swofford 2003) with the
following parameters: all characters unordered and
equally weighted, coded indel characters not treated as
separate data partition but added at the end of the alignment; heuristic search with random sequence addition,
TBR branch swapping, 50 random-addition-sequence
replicates, and MAXTREES option set to 1,000. Bootstrapping was done using the same settings and computing 5,000 bootstrap replicates (summarized in a 50%
bootstrap majority rule consensus tree as a cladogram and
a phylogram).
Maximum likelihood analysis was carried out with
RAxML v. 7.0.4 (Stamatakis et al. 2005, 2008) as implemented in the CIPRES (Cyberinfrastructure for Phylogenetic Research) portal v. 1.14 (Miller et al. 2009), using
75
only the combined dataset without coded indels. A bootstrap run with 5,000 replicates and subsequent maximum
likelihood optimization was performed with Russelia verticillata as the outgroup and no constraint tree defined.
Rapid bootstrap analysis used the CAT model; for the final
ML search, rate heterogeneity was modeled using a gamma
distribution and allowing a proportion of invariant sites,
which is estimated in the course of the run (model as in
Bayesian analysis).
Dating of divergence times
A BI approach was used to estimate divergence times of
the Scrophularia lineages examined in this study. The
algorithm implemented in BEAST v. 1.5.2 (Drummond
and Rambaut 2007) employs the MCMC to co-estimate
topology, substitution rates, and node ages (Drummond
et al. 2002) and was executed on the combined dataset.
A NEXUS file analogous to the one used for MP analyses
(but without coded indels) was used for calculations in
BEAST, but to avoid a basal polytomy, Hemimeris centrodes was excluded from the analysis; its unresolved
position between Russelia verticillata and Verbascum
nigrum would have made correct assignment of calibration points problematic. As in all analyses with the
combined dataset, S. lanceolata and S. serrata were
excluded as well so as to avoid incongruence problems
during topology estimation. Rate variation among sites
was modeled using the same substitution model as in
MrBayes and PAUP (GTR model) to ensure comparability of the results. We employed a relaxed molecular
clock model (Drummond et al. 2006) relying on uncorrelated rates drawn from a log-normal distribution, and a
Yule tree prior for speciation. To further support the
results of the phylogenetic analyses, tree topology was
co-estimated during the BEAST runs for comparison
purposes. The clock was calibrated using the assumed
divergence time between Plantaginaceae and Scrophulariaceae as proposed by Bremer et al. (2004); see the
‘‘Discussion’’ section for further details. The value of
76 mya, referred to as ‘‘Plantaginaceae stem age’’ by the
authors, was assigned to node 1 of Fig. 6 (value indicated
by brackets) with a normal-distributed prior and a standard deviation of 0.5 my. Two independent MCMC runs
were performed with 15,000,000 generations each, with
every 1,000th generation sampled. A burn-in of 10% per
run was discarded after assessing convergence with Tracer v. 1.4.1. Input data and results were edited and processed with the respective programs included in the
BEAST program package. The resulting maximum clade
credibility tree displaying mean heights was edited using
FigTree v. 1.2.3.
123
76
A. Scheunert, G. Heubl
Results
Amplification
The length of the trnQ-rps16 intergenic spacer fragment
differed considerably across the examined species. Within
the alignment of 1,362 basepairs (bp), some species
showed a 480 bp deletion that drastically shortened
sequence length (see also Table 3). This deletion can easily
be recognized in PCR products (Fig. 2) and is shared by all
North American as well as Japanese taxa and S. nodosa.
Likewise, S. umbrosa is characterized by a 262 bp deletion.
Sequence divergence and alignment
The combined data matrix of the internal transcribed spacer
(ITS) and the trnQ-rps16 and psbA-trnH intergenic spacers
comprised a total of 2,940 aligned characters. Detailed
information about alignment characteristics and statistics
of MP analysis is given in Table 3. Sequence length of the
trnQ-rps16 intergenic spacer was 657 bp (with deletion)
and 1,061 bp (complete) on average; ITS sequences had an
average length of 583 bp, psbA-trnH sequences were
493 bp long.
Whenever possible, taxa were analyzed using sequence
information from all three markers; in some cases, where
sequencing of single markers failed, the taxa were included
and scored as missing data for the respective markers: for
Russelia verticillata, no chloroplast sequences could be
obtained; Hemimeris centrodes provided trnQ-rps16 spacer
sequences only. No psbA-trnH sequences could be
Fig. 2 Gel electrophoresis image of trnQ-rps16 intergenic spacer PCR
products from different species of Scrophularia. Mainland Asian
species show the full sequence length (3 S. ningpoensis, 4 S. yoshimurae). North American mainland species (5 S. montana, 6 S. serrata, 7
S. villosa, 9 S. marilandica), West Indian species (8 S. minutiflora), and
Japanese species (1 S. musashiensis, 2 S. duplicato-serrata) show
reduced length due to a 480 bp deletion. A taxon with a smaller deletion
(such as in S. umbrosa) is indicated by an arrow
generated from two of the West Indian taxa (S. densifolia
Urb. & Ekman, S. domingensis Urb.); trnQ-rps16 and ITS
sequence information of these taxa was included nevertheless so as to increase the sampling with respect to the
West Indian species.
Phylogenetic reconstruction
The combined chloroplast dataset showed no significant
heterogeneity in the PHT (P = 0.132). Consequently, both
chloroplast markers were combined for all remaining
analyses in favor of a more robust phylogenetic hypothesis.
Table 3 Alignment characteristics and statistics of maximum parsimony (MP) analysis for trnQ-rps16 intergenic spacer, psbA-trnH intergenic
spacer, ITS, and combined dataset
Combined
trnQ-rps16 spacer
ITS
psbA-trnH spacer
No. of taxa
32a
30
32
28
Sequence length (bp) (mean)
572–2,287
(1,714.59)
349–756 (656.87);
992–1,176 (1,061.33)
395–862 (583.34)
435–554 (493.29)
Aligned length (bp)
Excluded characters (bp)
2,940
688
1,362
136
910
346
668
206
Constant characters (bp)
1,834
1,048
388
391
Parsimony-uninformative characters (bp)
294
143
100
53
Parsimony-informative characters (bp)
164
49
88
32
Parsimony-informative characters (%)
7.16
3.95
15.28
6.72
No. of coded indels
40
14
12
14
Unknown characters within sequences (bp) (mean)
3–841 (43.5)
0–837 (35.13)
0–54 (7.69)
0–16 (3.29)
Average G-C content (%)
37.99
28.69
59.67
23.03
Number of constant characters, parsimony-(un)informative characters, and % parsimony-informative characters refer to nonexcluded characters.
G-C content is without outgroup taxa. Sequence length is given separately for species with/without the 480 bp deletion (see text)
a
Number of taxa includes S. lanceolata and S. serrata; see text for details
123
Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae)
77
1.00
0.92
0.93
1.00
1.00
1.00
0.87
0.87
0.99
0.97
1.00
0.92
0.96
0.59
0.62
1.00
0.96
0.99
0.85
0.56
0.60
0.87
0.57
0.69
0.96
1.00
0.98
0.96
0.76
0.73
1.00
1.00
1.00
1.00
0.98
1.00
1.00
0.99
0.91
0.97
1.00
0.99
0.92
0.92
1.00
0.61
0.66
0.68
0.70
0.84
0.87
1.00
1.00
1.00
1.00
1.00
0.93
1.00
1.00
1.00
S. buergeriana
S. ningpoensis
S. yoshimurae
S. nodosa USA
S. nodosa Germ.
S. kakudensis
S. nodosa Armenia
S. musashiensis
S. duplicato-serrata
S. grayana
S. marilandica
S. micrantha
S. minutiflora
S. domingensis
S. eggersii
S. densifolia
S. multiflora
S. californica
S. atrata
S. desertorum
S. villosa
S. laevis
S. macrantha
S. montana
S. parviflora
Eastern
Asia
S. nodosa
Japan
3: Eastern N Am.
1.00
0.95
0.92
0.99
0.99
1.00
2: California
1.00
North
America
1: New Mexico
1.00
1.00
Russelia verticillata
Hemimeris cent.
Outgroup
Verbascum nigrum
S. umbrosa
Eurasia
S. koraiensis
Fig. 3 Bayesian consensus tree (cladogram), from a combined
dataset (ITS and trnQ-rps16 and psbA-trnH intergenic spacers)
excluding S. lanceolata and S. serrata. Geographic distribution and
names are given for each clade. Black dots on terminal branches
indicate presence of the 480 bp deletion in the trnQ-rps16 sequence
of the respective taxon; the position where the deletion was
introduced is marked by an arrow. Posterior probabilities (PP)
exceeding 80% are given above each node, corresponding bootstrap
support (BS) values from a MP 50% majority rule consensus tree (not
shown) are indicated below. Maximum likelihood (ML) bootstrap
support values of the respective nodes are plotted beneath the BS
values. PP values were obtained from 30,002 trees, BS values from
5,000 bootstrap replicates with 1,000 saved trees per replicate, and
ML values from 5,000 bootstrap replicates with subsequent maximum
likelihood optimization. Asterisk indicates the position of an
additional node supported by MP analysis only (79% BS), and
therefore not shown in this figure, assigning the Asia clade as sister to
the Japan-New World (J-NW) clade and the S. nodosa clade (see also
the ‘‘Discussion’’ section). Triangle indicates the position of an
additional, yet insufficiently supported node (therefore not shown in
this figure) in Bayesian (62% PP), BEAST (74% PP), and ML (54%
ML bootstrap support) analyses, assigning the New Mexico clade as
sister to the remaining New World clades (see the ‘‘Discussion’’
section). Hemimeris cent. Hemimeris centrodes, Germ Germany
For the nuclear (ITS) and combined chloroplast (trnQrps16 and psbA-trnH spacer) dataset, the PHT displayed
significant heterogeneity (P = 0.004). A series of PHT
tests (results not shown), tentatively removing single species from the dataset, revealed S. lanceolata and S. serrata
to be responsible for the conflict. When these taxa were
excluded from the combined dataset, the PHT found the
remaining data to be highly congruent (P = 0.704). Visual
inspection of the majority rule consensus trees obtained
from MP bootstrapping analyses of the nuclear and chloroplast datasets (not shown) supports the results of the
PHT: the only instance of a hard incongruence sensu
Mason-Gamer and Kellogg (1996), i.e., differences in
topology supported by 70% BS or more, involves the
placement of S. lanceolata and S. serrata. In the
chloroplast gene tree, the two taxa are in one clade with
S. marilandica; in the ITS tree, they are sister to S. laevis
Wooton & Standl., S. macrantha, and S. montana Wooton.
We also examined possible incongruences between the
datasets using Templeton’s significantly less parsimonious
(SLP) test. When performed on the ITS dataset, the test
was significant (pmax = 0.0045), which means that the
nuclear tree (70% consensus from the unconstrained
search) is a significantly better fit to these data than the
rival chloroplast topologies (trees obtained in the constrained search). The reciprocal test also rejected the null
hypothesis (pmax = 0.0018), suggesting the rival ITS
topology is significantly noncongruent as compared to the
unconstrained dataset. The number of character sites
available for the test was adequate in both tests
123
78
Fig. 4 The 50% majority rule
consensus tree (phylogram)
from the PAUP* maximum
parsimony bootstrap analysis,
from a combined dataset (ITS
and trnQ-rps16 and psbA-trnH
intergenic spacers) excluding
S. lanceolata and S. serrata.
The consensus was generated
from 5,000 bootstrap replicates
with 1,000 saved trees per
replicate. Numbers above
branches indicate mutational
changes of the respective
branch, dashed branches are not
shown with correct lengths, but
were shortened
A. Scheunert, G. Heubl
55
Russelia verticillata
91
Hemimeris centrodes
47
34
Verbascum nigrum
64
6
7
S. koraiensis
10
11
S. buergeriana
10
4
38
S. umbrosa
S. ningpoensis
9
S. yoshimurae
2
5
11
11
1
S. nodosa USA
S. nodosa Germany
0 S. kakudensis
14
2
7
10
mutational
changes
2
14
S. nodosa Armenia
S. musashiensis
S. duplicato-serrata
S. grayana
5
9
S. marilandica
11
0 S. micrantha
3
14
8
5
4
1
3
1
4
2
7
3
8
S. minutiflora
4
2
S. domingensis
S. eggersii
S. densifolia
S. multiflora
S. californica
S. atrata
S. desertorum
S. villosa
11
10
11
15
16
(N = 13–15/18–20), assuring the reliability of the results.
Trees obtained from the constrained search in the ITS test
were 12 steps longer than the unconstrained 70% consensus; constrained trees in the chloroplast test had 17 steps in
excess.
Taking into account the results of the statistical analyses, a combined nuclear/chloroplast dataset was used for
phylogenetic analyses, but S. lanceolata and S. serrata
were excluded and examined in two separate analyses
(nuclear and chloroplast markers). The Bayesian consensus
tree from the combined analysis is shown in Fig. 3, each
node displaying its posterior probability (PP) values.
Maximum parsimony analysis of the combined dataset
resulted in three most parsimonious trees. The bootstrap
(BS) 50% majority rule consensus tree (length 620 steps,
CI = 0.82, RI = 0.75) yielded the same topology as the
Bayesian consensus tree, yet with much weaker support
values in five cases, whereas one additional node was
generated. The BS support values were attached to their
corresponding nodes in the BI cladogram in Fig. 3. The MP
50% majority rule consensus including branch lengths is
shown in Fig. 4.
Maximum likelihood optimization of the combined
dataset resulted in a final optimization likelihood of
123
2
5
S. laevis
S. macrantha
S. montana
S. parviflora
-6,180.96, with the alpha parameter being 0.566429. The
proportion of invariant sites was 0.16, the best-scoring ML
tree had a length of 0.340060 and corresponded to the
topology of the BI and MP analyses. ML bootstrap support
values were attached to their respective nodes (below the
maximum parsimony BS values) in the Bayesian cladogram in Fig. 3. Support values are highly similar to those
of the MP analysis, with six values being weakly supported
compared to the Bayesian consensus (five of them similarly
weak in MP analysis).
All New World species of Scrophularia form a strongly
(92% PP) to weakly (61% BS / 66% ML) supported clade.
Apart from three New World subclades related to geographical distribution, the clade includes the three Japanese
taxa and is referred to as the Japan-New World clade
(J-NW clade). The Japanese taxa form a grade towards the
New World Eastern North America (ENA) clade and
indicate an intercontinental disjunction (though weakly
supported): S. grayana Maxim. ex Kom. is linked somewhat more closely to the eastern North American species
(87% PP / 57% BS / 69% ML), while support for the
whole Japan–Eastern North America clade (J-ENA clade)
including S. musashiensis Bonati and S. duplicato-serrata
Makino remains very weak (85% PP / 56% BS / 60% ML).
Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae)
cpDNA
1.00
5
1.00
1.00
0.98
4
1.00
0.99
1.00
1.00
0.98
1.00
0.77
1.00
1.00
0.87
0.96
1.00
1.00
1.00
Russelia verticillata
Hemimeris centrodes
Verbascum nigrum
S. umbrosa
S. koraiensis
S. buergeriana
S. ningpoensis
S. yoshimurae
S. nodosa USA
S. nodosa Germany
S. kakudensis
S. nodosa Armenia
S. musashiensis
S. duplicato-serrata
S. grayana
S. lanceolata
S. serrata
S. marilandica
S. micrantha
S. minutiflora
S. domingensis
S. eggersii
S. densifolia 3
S. multiflora
S. californica
S. atrata
S. desertorum
2
S. villosa
S. laevis
S. macrantha
S. montana
S. lanceolata
S. serrata
S. parviflora 1
79
ITS
5
1.00
1.00
4
0.95
0.93
0.94
0.70
0.70
1.00
1.00
1.00
1.00
0.94
0.96
1.00
1.00
0.98
1.00
0.90
Fig. 5 Bayesian consensus trees (cladograms) from a combined
chloroplast dataset (trnQ-rps16 and psbA-trnH intergenic spacer) (left
side) compared to the ITS dataset (right side). Posterior probabilities
(obtained from 30,002 trees) exceeding or equaling 70% are given
above each node. New World clades 1–3 are indicated by boxes.
Scrophularia lanceolata and S. serrata are shown within boldly lined
boxes at their differing positions within each tree. 1 New Mexico
clade, 2 California clade, 3 ENA clade, 4 S. nodosa clade, 5 Asia
clade
Apart from that, three strongly supported North American subclades can be distinguished: a ‘‘New Mexico
clade’’ (clade 1) with S. laevis and S. macrantha grouped
together with high support (100% PP / BS / ML); S.
montana is sister to this subclade (100% PP / 93% BS /
100% ML). Scrophularia parviflora Wooton & Standl.,
which is indicated as sister to all the rest of the New
Mexico clade, is confined to New Mexico and Arizona,
while the other members of the clade are exclusively distributed in New Mexico. The whole clade receives strong
support in all analyses (100% PP / BS / ML). A well
supported ‘‘California clade’’ (clade 2; 100% PP / 84% BS /
87% ML) shows S. villosa as sister to four Californian
species that remain within an unresolved polytomy. Apart
from California, S. californica occurs in British Columbia
and Canada, and S. desertorum (Munz) R. J. Shaw is also
found in Nevada. Scrophularia atrata and S. villosa are
only very locally distributed. The ENA clade (clade 3)
contains a highly supported subclade (100% PP / 98% BS /
100% ML) of five species endemic to several islands of the
Greater Antilles (‘‘West Indies clade’’; distribution map in
Fig. 7). These again are divided into two subclades, one
with three species (S. densifolia, S. domingensis, S. eggersii
Urb.) restricted to Hispaniola, the other with two more
widespread taxa (S. micrantha Desv. ex Ham. and S.
minutiflora Pennell). Scrophularia marilandica receives
high support as sister to that group (100% PP / 98% BS /
96% ML).
Bayesian inference (96% PP), MP (76% BS), and ML
(73% ML bootstrap support) analyses show high support
for a sister relationship of all New World taxa (including
the Japanese subgroup) to the widely distributed S. nodosa.
All S. nodosa accessions included in the dataset form a
strongly (100% PP / 99% BS / 97% ML) supported clade,
with S. kakudensis Franch., a species distributed in eastern
China, Korea, and Japan, nested within.
The remaining Asian species (S. koraiensis Nakai,
S. buergeriana Miq., S. ningpoensis Hemsl., and
S. yoshimurae T. Yamaz.) are placed in a single clade
receiving high support (100% PP / 99% BS / 100% ML).
This ‘‘Asia clade’’ is indicated within an unresolved
polytomy at the base of the ingroup in BI and ML analysis.
123
80
A. Scheunert, G. Heubl
6
46,86
9
8
3
10
28,56
13
*
12
18,17 14
7
15
15,84
12,34
24,16
17
16
19
8,52 18
21
11
20
22,27
8,32
23
22
14,57
75,0
70,0
60,0
50,0
40,0
30,0
20,0
10,0
24
CLADE 3
4
2
CLADE 2
5
1
CLADE 1
[75,99]
Russelia vert.
Verbascum nigrum
S. umbrosa
S. koraiensis
S. buergeriana
S. ningpoensis
S. yoshimurae
S. nodosa USA
S. nodosa Ger.
S. kakudensis
S. nodosa Arm.
S. musashiensis
S. duplicato-serr.
S. grayana
S. marilandica
S. micrantha
S. minutiflora
S. domingensis
S. eggersii
S. densifolia
S. multiflora
S. californica
S. atrata
S. desertorum
S. villosa
S. laevis
S. macrantha
S. montana
S. parviflora
0,0
Fig. 6 Maximum clade credibility tree (chronogram with time scale)
estimated by BEAST from a combined dataset (ITS and trnQ-rps16
and psbA-trnH intergenic spacers) excluding Hemimeris centrodes, S.
lanceolata, and S. serrata. Nodes receive posterior probability values
exceeding 80% (PP values not shown); one exception with 0.68/
0.69% PP is marked by an asterisk (see text). A time constraint of
76 mya (75–76.98 mya) was employed at node 1 for the
Plantaginaceae stem age according to Bremer et al. (2004). Mean
age and 95% higher posterior density values (HPD) for each node are
given in Table 4. Divergence time estimates of important groups are
indicated below the node numbers. Values are in millions of years
before present (mya); value in square brackets represents the
constrained age of the calibration node. Russelia vert. Russelia
verticillata, Ger. Germany, S. duplicato-serr. S. duplicato-serrata
Table 4 Divergence time estimates calculated by BEAST (maximum
clade credibility tree shown in Fig. 6)
and is therefore not reliable, it was excluded from Fig. 3
(position marked by an asterisk). Scrophularia umbrosa,
which is widely distributed but does not occur in the New
World, is linked to none of the clades and appears as sister
to the rest of the Scrophularia taxa sampled.
To further reveal the phylogenetic positions of the
excluded S. lanceolata and S. serrata, two separate BI
analyses were performed, one from ITS and one from the
combined chloroplast dataset (trnQ-rps16 and psbA-trnH).
The resulting consensus trees are compared in Fig. 5.
The basal branches of the combined chloroplast cladogram strongly correspond to the trnQ-rps16 gene tree (not
shown), while the combination of both chloroplast markers
increases support for the terminal clades. The topology is
highly similar to that of the ITS tree, but with better resolution in the basal nodes. A paraphyletic grade leading
from the Japanese taxa to the ENA clade is not supported in
any case, but all Japanese taxa (ITS) or at least S. grayana
(chloroplast tree) are positioned in an unresolved polytomy
with the ENA clade. Like the Asia clade and S. nodosa
clade, the three New World clades are highly supported by
both markers (90–100% PP), however, the ENA clade does
not include S. marilandica in the chloroplast tree. The
placement of S. lanceolata and S. serrata in the two trees is
Node
Mean age
(mya)
95% HPD
Node
Mean age
(mya)
95% HPD
1
75.99
75.00–76.98
13
4.20
0.4–10.2
2
46.86
25.83–72.59
14
15.84
7.57–26.11
3
28.56
13.97–46.5
15
12.34
5.64–20.71
4
12.41
4.68–22.5
16
8.52
3.63–14.75
5
6
6.82
7.99
1.88–13.25
2.22–15.69
17
18
1.03
5.66
0.01–2.8
1.86–10.32
7
24.16
11.51–39.15
19
2.39
0.37–5.12
8
10.44
3.11–19.89
20
8.32
2.09–17.24
9
3.03
0.39–7.08
21
4.00
0.94–8.44
10
1.08
0.02–2.9
22
14.57
6.22–25.2
11
22.27
10.9–36.35
23
8.34
2.88–15.34
12
18.17
8.68–29.95
24
3.57
0.65–7.61
Mean ages in million years before present (mya) with corresponding
95% higher posterior density values (HPD) are given for each node.
Node numbers correspond to labels in Fig. 6
However, MP analysis places it as sister group to S. nodosa
and the J-NW clade with moderately high support (79%
BS). As the node collapses in Bayesian and ML analyses
123
Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae)
Fig. 7 Distribution of
Scrophularia in the Greater
Antilles. Symbols: filled dot
S. minutiflora, blank dot
S. micrantha (probably
synonymous with
S. minutiflora), triangle
S. domingensis, square
S. eggersii, star S. densifolia.
The position of the symbols
does not necessarily correspond
to actual distribution areas.
Present distribution areas of
S. marilandica in Florida
(USDA NRCS National Plant
Data Center 2010) are marked
by crosses
81
Florida
Bahamas
Mexico/
Yucatan
Cuba
Belize
Puerto Rico
Jamaica
Honduras
500 km
Hispaniola
Lesser
Antilles
Panama
Venezuela
substantially different. In the ITS analysis, these taxa are
part of the New Mexico clade. The combined chloroplast
phylogeny places them in a clade with S. marilandica
(causing the latter to split away from the ENA clade). Both
placements are highly supported (98%/100% PP) and point
out the ambivalent status of the most widespread species of
Scrophularia in North America.
Divergence time estimation
The maximum clade credibility tree from the BEAST
analysis is shown in Fig. 6 alongside a time scale and
divergence times. It revealed the same topology as the
cladogram inferred by MrBayes (some nodes with slightly
lower/higher posterior probabilites; results not shown).
One node supported with [80% PP in the Bayesian cladogram received only 68/69% support in the BEAST analysis (marked by an asterisk in Fig. 6), but was maintained
nevertheless so as to allow interpretation of the tree on the
basis of the MrBayes cladogram. The mean number of
substitutions per site per million years across the tree was
estimated to be 7.9296 9 10-4 (standard deviation: 8.8268
9 10-6). The standard deviation r of the uncorrelated lognormal relaxed clock was 0.598, which identifies moderate
rate heterogeneity among branches; a strict clock is
therefore rejected by the data.
In the absence of Scrophularia fossils and due to the
limited fossil record of Scrophulariaceae and Lamiales, the
clock was calibrated using time estimates from a study with
better fossil backup (Bremer et al. 2004). Of course, results
obtained by using approximations as a basis have to be
treated with caution; see the ‘‘Discussion’’ section for a
methodical review of that approach. The derived stem age
of the ingroup (separation from its sister group) is
46.86 mya (Fig. 6, node 2). According to our results, the
most recent common ancestor of New World and Japanese
Scrophularia arose at the Oligocene-Miocene boundary
(24.16 mya; Fig. 6, node 7). The split into the three North
American clades occurred in the early Miocene (ca.
22.27 mya, node 11), with subsequent diversification
within the New World clades themselves starting from
14.57 mya (late Miocene, New Mexico clade 1) to
8.32 mya (early Pliocene, California clade 2). The beginning of species diversification in the Antilles is dated to the
early Pliocene as well (node 16). All estimated mean ages/
divergence times and corresponding 95% higher posterior
densities (HPD) are shown in Table 4.
Discussion
Diversification and character evolution within New
World Scrophularia
Analyses of a combined dataset using nuclear ITS and
plastid trnQ-rps16 and psbA-trnH revealed three highly
supported North American clades (Fig. 3). It is evident that
tree topology is correlated with geographical distribution.
The clades are confined to California, New Mexico, and
eastern North America including the West Indies (i.e.,
ENA clade), respectively. Three Japanese taxa form a
grade leading towards the ENA clade, representing a
(weakly supported) Japan–Eastern North America species
alliance (J-ENA clade).
123
82
Within the California clade, S. villosa is sister to all
other taxa. This perennial, shrubby taxon, which can reach
up to 3.5 m in height, apparently diverged early within the
Californian lineage. Its restricted distribution on three
Californian and Mexican islands (Santa Catalina, San
Clemente, and Guadalupe) supports this assumption. Its
densely villose inflorescence with slender white glandular
hairs even covering the sepals is unique within the clade.
Other features are shared with S. atrata, a very local species restricted to two Californian counties: a dark maroon
to almost blackish urceolate corolla with a constricted
orifice, and a lanceolate-oblong or (in the case of S. villosa)
rudimental and awn-like staminode. Although S. atrata is
placed in an unresolved polytomy with the other Californian taxa, a close relationship to S. villosa is apparent from
a morphological viewpoint. The remaining species of the
clade have a more or less bicolored corolla with a red or
brownish dorsal upper lip and a pale to greenish lower lip,
and a clavate to obovoid brownish staminode. In contrast to
S. atrata and S. villosa, which are not found above 500 m,
these species occur up to 1,000 m (S. californica) and
3,000 m (S. desertorum) above sea level.
Scrophularia californica, the most widespread species
within the California clade, is native to coastal California.
In the inland regions and in southern California, it is
replaced by S. multiflora. Various names and taxonomic
ranks have been assigned to this taxon. The name was
introduced by Pennell (1947) as a nomen novum for
S. californica var. floribunda Greene (described in Greene
1894) because of the earlier S. floribunda Boiss. & Bal.
(described in Boissier 1856). Scrophularia californica var.
floribunda had already been elevated to species rank as
S. floribunda (Greene) A. Heller (1906), mainly because of
its distinctive geographical distribution. Shaw (1962)
modified its taxonomic status again by treating it as
S. californica subsp. floribunda (Greene) Shaw. We agree
with Pennell’s (1947) taxonomic concept and accept it as a
distinct species because of the smaller, shorter maroon
corolla and the more paniculately branched inflorescences.
Scrophularia desertorum (Munz) Shaw occurs on dry
mountain slopes farther inland and extends into western
Nevada. It was described as a variety of S. californica by
Munz (1958), but elevated to species rank by Shaw (1962)
because of its ecological characteristics.
The second distinct New World clade comprises four
species largely confined to New Mexico. Shared morphological features within this group include a slender conical
capsule and ovate to broadly lanceolate, decurrent leaves.
Scrophularia parviflora, distributed in southern New
Mexico and central and southeastern Arizona, is sister to
the rest of the clade. While its stem is densely puberulent
and the flowers possess a spatulate light brownish staminode, the other species have glabrous stems (S. montana in
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A. Scheunert, G. Heubl
the lower parts only) and rounded staminodes. Scrophularia laevis and S. montana are restricted to the mountains
of central New Mexico, with S. laevis distributed even
more locally in the southern parts of that area. Both species
are highly similar, but triangular-lanceolate, acute sepals
set S. laevis apart from S. montana with its ovate, obtuse
sepals. A conspecific status as proposed by Shaw (1962) is
not supported by molecular analyses. Sister to S. laevis is S.
macrantha, an outstanding species that has large, showy
red corollas of 13–22 mm length, with erect lobes and a
slightly constricted orifice; the flowers are almost entirely
covered with glandular trichomes. Pollination by hummingbirds has been proven for this species by Lightfoot
and Sivinski (1994), which is unique within North American Scrophularia and probably the whole genus. Scrophularia macrantha is extremely rare and known only from
two counties of southwestern New Mexico. Shaw (1962)
mentions some localities of S. montana in this region as
well.
Biogeography of the Caribbean species of Scrophularia
Like the North American mainland clades, the Antillean
species form a highly supported lineage [West Indies clade,
see Eastern North America (ENA) clade in Fig. 3, but
without S. marilandica]. A distribution map of all West
Indian species is shown in Fig. 7. Putative morphological
synapomorphies of the West Indies clade are as follows:
lanceolate to linear, acute sepals, ovate to globular capsules, a filiform or rudimentary staminode, and a characteristic blackening of leaves and stems upon drying. The
species of the first subclade are most likely synonymous: S.
micrantha Desv. ex Ham. was described in Hamilton
(1825) and is mentioned in Urban (1898, 1903). Later, it
appears as synonym to S. minutiflora (see, e.g., Britton and
Wilson 1925), a taxonomic decision accepted today (Liogier 1994). The long shared branch in the phylogram
(Fig. 4) with low resolution of the terminal taxa corroborates this view and is additionally supported by the almost
identical morphological features. Scrophularia minutiflora
is rather widespread across the West Indies and occurs on
the islands of Cuba, Jamaica, Puerto Rico, and Hispaniola.
The other subclade comprising S. densifolia, S. domingensis, and S. eggersii is restricted to Hispaniola.
Scrophularia marilandica, which is widely distributed
in the eastern parts of North America and eastern Canada,
is sister to the species of the Greater Antilles with high
support in all analyses. While there are no distinctive
shared morphological characters, the phylogenetic results
get support from biogeographic reconstructions of the
Antillean region. According to current knowledge, the
Greater Antilles originated as a submerged volcanic arc
between North and South America (Pindell and Barrett
Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae)
1990) during the lower Cretaceous (130–110 mya) and
subsequently drifted eastwards on the Caribbean Plate
(Donovan and Jackson 1994). The landmasses are thought
to have been subaerial only since the mid-Eocene (45 mya;
Iturralde-Vinent and MacPhee 1999), although this topic is
still being discussed. With the continued drifting, Cuba
(attaching to the North American plate) separated from NHispaniola and proto-Puerto Rico (on the Caribbean plate)
during the Oligocene (Hedges 1996; Iturralde-Vinent and
MacPhee 1999) or the early Miocene (Graham 2003a,
2003b); eventually, all three islands were fixed to the North
American plate. The separation of Puerto Rico from Hispaniola probably dates to the early Miocene as well (Graham 2003a).
Various colonization patterns have been proposed for
several Antillean species, including tertiary vicariance due
to separation of islands (Negrón-Ortiz and Watson 2003),
dispersal (also repeatedly, leading to reticulate patterns)
from North or South America (Fritsch 2003), combinations
of vicariance and dispersal (McDowell et al. 2003), taxa
originating from the early Tertiary boreotropical flora
(Lavin et al. 2001), or from in situ adaptive radiation
(Graham 2003b). Hedges (2006) states that dispersal is
likely to be the key factor in Antillean colonization by
terrestrial vertebrates, often followed by adaptive radiation.
This also applies to Scrophularia with seven species
occurring in the West Indies, while only one species
inhabits the eastern North American mainland. Furthermore, the divergence time between S. marilandica and the
Antillean species was dated to ca. 12 mya (late Miocene)
in the relaxed molecular clock analysis (Fig. 6), a time
when all Greater Antillean islands were already separated
from each other. This excludes two other main colonization
patterns being discussed: proto-Antillean vicariance (Rosen
1975) and the Aves Land Bridge model (Iturralde-Vinent
and MacPhee 1999). Colonization of the Greater Antilles
should thus have occurred via long distance dispersal. The
inferred divergence time for the Antillean species is
somewhat later, but still in concordance with the results
provided by Hedges et al. (1992), who studied albumin
evolution in West Indian terrestrial vertebrates to date
divergences between West Indian and Central/South
American taxa and found that most splits date to the midCenozoic (Eocene to Miocene).
Scrophularia marilandica occurs in large parts of eastern North America today, for example in the northern parts
of Florida as well (see Fig. 7), whereas no species of
Scrophularia have ever been reported from tropical Central
or South America. For this reason, the evidence is strong
that the common ancestor of the West Indian lineage
occurred in Florida and colonized the Antilles from there,
dispersing to Cuba. This was probably facilitated by the
fact that the wet forests of western Cuba are remarkably
83
similar to the vegetation of Florida, and that presumably,
Cuba was fully emergent in its present position by the late
Miocene, 19–12 mya (Iturralde-Vinent and MacPhee 1999;
Lewis and Draper 1990). Migration pathways to Hispaniola, Puerto Rico, and Jamaica remain speculative; Hispaniola may have been colonized before the early Pliocene,
when the Hispaniola endemic lineage diverged from the
more widespread S. minutiflora (Fig. 6). Jamaica most
likely was not colonized before that time as well because it
remained inundated from the latest Eocene until ca.
10 mya (Lewis and Draper 1990) and was never connected
to a land mass after that time (Buskirk 1985).
While dispersal in principle is the usual mechanism of
Antillean colonization and was found in many other
Antillean and New World taxa [e.g., Poitea (Fabaceae;
Lavin 1993), Styrax sect. Valvatae (Styracaceae; Fritsch
2003), Cuphea (Lythraceae; Graham 2003b), Erithalis
(Rubiaceae; Negrón-Ortiz and Watson 2003)], the modes
of diaspore dispersal are diverse and in this case cannot be
fully elucidated. However, as the Florida coastal region is
influenced by the northeast trade winds that come across
the Atlantic, incidental dispersal over the relatively short
distance (Florida–Cuba: 160 km) is likely (Graham 2003a,
b). Between the islands, the distances involved are even
shorter (Cuba–Haiti: 72 km, Dominican Republic–Puerto
Rico: 96 km) and there are various possibilities for overcoming these distances: hurricanes with unusual yet proven
west to east tracks are mentioned in Hedges (2006), and
Powell (1999) discussed strong easterly surface winds
associated with westerly hurricanes. In addition, seeds
could have been transported via local water currents
(Hedges et al. 1992); the transport of plant seeds in soil
stuck to drifting vegetation was described by Renner
(2004), and this mode of dispersal has already been documented for West Indian vertebrates (Censky et al. 1998;
Knapp 2000). Altogether, species diversification subsequent to colonization from the North American mainland
and dispersal within the Antilles presumably accounts for
the current distribution of Scrophularia in the Caribbean.
The colonization of the Greater Antilles is accompanied
by morphological changes. The large reddish brown
corollas of S. marilandica do not suggest a close relationship to S. minutiflora with its small, white corollas, but the
latter are typical features for much of the flora of the
Antilles and especially Cuba. According to Borhidi (1996),
plants with very small flowers, so-called micranthia (cf. S.
micrantha!), are adapted to pollination by minute, endemic
insects. Again, the Hispaniola clade differs from S. minutiflora in possessing ovate-lanceolate leaves with crenate to
serrate margins (in contrast to triangular-ovate, coarsely
serrate leaves) and large yellow corollas (up to 12 mm).
The species are perennial herbs or subshrubs and are
adapted to coniferous forests of higher elevations (up to
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84
3,100 m). This as well is in contrast to the annual life cycle
of S. minutiflora, which inhabits shaded banks and humid
forests between 800 and 2,000 m elevation. The appearance of woody lifeforms derived from herbaceous ancestors in the course of island colonization [so-called insular
woodiness as coined by Carlquist (1974)] has been discussed for the pantropic giant lobelias (Campanulaceae) by
Knox et al. (1993) and for Echium (Boraginaceae) and
Pericallis (Asteraceae) on the Canary Islands (Böhle et al.
1996; Panero et al. 1999).
The origin of Scrophularia lanceolata
Scrophularia lanceolata is the most widespread species
within New World Scrophularia. It is found throughout the
United States except in a few southern states and extends to
southern Canada as well, while all other American species
(except S. marilandica) have a restricted distribution.
Scrophularia lanceolata in all analyses forms a highly
supported clade with S. serrata, a species which is known
from only Idaho and Colorado and is generally regarded as
synonymous with S. lanceolata. The only distinguishing
feature is the color of the staminode, which is yellowishgreen in S. lanceolata and purple in S. serrata. But as
purple staminodes can also be found in individuals of S.
lanceolata (D. E. Boufford, pers. comm.), different staminode color may not be sufficient for assigning species rank
to S. serrata.
In all tree topologies, both species are closely linked.
When the nuclear and chloroplast datasets are analyzed
separately (Fig. 5), the trnQ-rps16/psbA-trnH subset
groups the two taxa with S. marilandica, while the ITS
phylogeny shows them within the New Mexico clade.
Consequently, a different evolution of the markers concerned is apparent, which is best explained by a hybridization event involved in the origin of S. lanceolata. Under
this assumption, crossing of S. marilandica with an
ancestral species from the New Mexico clade (possibly
resembling S. parviflora) is most likely. Successful crossings between North American Scrophularia species have
already been conducted by Shaw (1962), who found that
most North American species will cross fairly easily, also
due to pollination by non-species-exclusive insects such as
wasps and syrphid flies (Trelease 1881; Robertson 1891).
In S. marilandica and S. parviflora, similar habitats and
overlapping flowering times additionally facilitate
hybridogenesis.
Numerous morphological characters shared between S.
lanceolata and its putative parental species support a
hybrid origin hypothesis. Morphologically, S. lanceolata is
remarkably similar to S. marilandica; the two species can
be distinguished only by leaf margin shape (more finely
serrate in S. marilandica), sepal shape (somewhat acute in
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A. Scheunert, G. Heubl
S. lanceolata, while broadly ovate in S. marilandica), and
color of the staminode (yellow-greenish vs. purple in
S. marilandica); however, none of these features is reliable
given the great variability within the species. For example,
S. lanceolata occasionally develops purple staminodes
(see above) and Pennell (1935) reported an aberrant form
(S. marilandica var. viridis) with green staminodes. The
striking similarity has already led to confusion regarding
species delimitation in the past. Pennell (1935), in his
treatment of the eastern temperate Scrophulariaceae,
transferred 10 species into synonymy with S. lanceolata
and S. marilandica. On the other side, morphological
features shared between S. lanceolata and S. parviflora
include similar color patterns of the corolla (greenish with
a shade of red-brown on the upper lip in S. lanceolata,
green with reddish lobes in S. parviflora) and the spatulate
staminode, which is unique within the New Mexico clade.
The slender ovoid capsules in S. lanceolata are intermediate between the conical capsules in S. parviflora and the
globular ones in S. marilandica, while capsules and flowers
in S. lanceolata are larger than in S. marilandica. The
combination of advantageous characters acquired through
hybridization might have been the key factor enabling this
unique species of Scrophularia to colonize almost the
whole North American continent.
Phylogenetic position of New World Scrophularia
within the genus
Scrophularia nodosa, which is widely distributed in Eurasia and the New World, is sister to the J-NW clade with
high to moderate support in all analyses. Therefore, conspecificity of S. nodosa and the New World species as
proposed by Stiefelhagen (1910) must be rejected based on
our molecular analyses. However, close relationships
between the taxa are apparent as S. nodosa (in addition to
the three Japanese species) is the only known Old World
species characterized by the large trnQ-rps16 deletion
typical for New World species of Scrophularia.
Preliminary studies conducted on Scrophularia placed S.
nodosa and a set of New World taxa in one highly supported clade with several eastern Asian species (results not
shown). An Asian origin of the North American taxa is
suggested by several of our results. First, the affiliation of
three Japanese taxa (possessing the deletion in trnQ-rps16)
to the ENA clade (although weakly supported) indicates a
link between eastern Asia and North America. Intercontinental biogeographic relationships of that kind have been
observed in many plant genera and correspond to a general
Eastern Asian–Eastern North American (EA–ENA) disjunct pattern (see Xiang et al. 2000), which has been
extensively discussed (e.g., Boufford and Spongberg 1983;
Li 1952; Boufford 1998; Wen 1999). Sometimes, eastern
Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae)
Asian and North American taxa form sister clades, as in
Torreya (Taxaceae; Li et al. 2001) and Aesculus (Sapindaceae; Xiang et al. 1998); several other examples are
mentioned in Soltis et al. (2001). In this study, Japanese
species are even linked to one of the New World clades and
thus are part of the clade containing all North American
taxa. Asian plant species that are more closely related to
their New World relatives than to species of their own
continent were discussed by Li et al. (2003). This pattern of
relationships often involves Japanese taxa and was found,
e.g., in Hamamelis (Hamamelidaceae) by Wen and Shi
(1999) and Li et al. (2000); the latter state that the same
relationships are found in several other groups (see also
Kim and Jansen 1998).
Second, Scrophularia nodosa, which is sister to all
North American taxa, has been shown in this study for the
first time to have an eastern Asian species nested within.
Scrophularia kakudensis was put into synonymy with S.
nodosa by Stiefelhagen (1910) and has the same chromosome number as S. nodosa (2n = 36; Nishikawa 1985;
Scherer 1939). Consequently, it can be assumed that S.
nodosa itself, an ancestor, or a close relative occurs in
eastern Asia, which also supports an Asian origin hypothesis. Third, further evidence comes from an additional node
in the MP analysis, placing the eastern Asian species as
sister to S. nodosa and the J-NW clade (Fig. 3, position of
the node marked by an asterisk). Although this node collapses in ML and Bayesian analyses and was therefore not
included in the figure, it receives a bootstrap support value
of 79% in MP, which indicates a weak signal for the proposed relationship. Altogether, there is strong evidence that
the J-NW clade arose from a S. nodosa-like Asian ancestor
characterized by a large deletion in the trnQ-rps16 marker.
Chromosome evolution
Regarding phylogenetic reconstructions, there is evidence
that Asian Scrophularia species were involved in the formation of the Japan-New World clade. Regarding reported
karyological data, all members of the three New World
clades are high polyploids with 2n = 86–96 (e.g., Shaw
1962). It has been suspected that all species endemic to the
New World have 2n = 96, with the lower counts being
attributable to problems in identifying very small chromosomes. An exception occurs in S. montana, which
possesses 2n = 70–76 chromosomes (Shaw 1962). Polyploidy is also documented for the Japanese species of
the J-NW clade: S. grayana has 2n = 94 chromosomes
(Kamada et al. 2007). No counts are available for S. musashiensis or S. duplicato-serrata, but it is likely that the
whole J-NW clade is characterized by high polyploidy in
contrast to S. nodosa with a chromosome number of
2n = 36. Assuming a polyploid ancestor that gave rise to
85
the J-NW clade, interspecific hybridization and autopolyploidization with S. nodosa or its ancestor involved is
conceivable. Although no direct evidence is available at
present, this hypothesis gains support from the existence of
polyploid Scrophularia species and a remarkable variation
of chromosome numbers in eastern Asia [from 2n = 24 in
S. dentata Royle ex Benth. (Mehra and Vasudevan 1972)
and 2n = 30 in S. buergeriana (Lee 1967), to 2n = 50–56
in S. incisa Weinm. (Vaarama and Hiirsalmi 1967) and
2n = 90 in S. ningpoensis (Ma et al. 1984)].
Phylogenetic dating
Estimating divergence times within families of Lamiales is
extremely difficult due to the limited fossil record. There
are two studies on a larger scale using reference fossils as
calibration points to estimate clade ages: Bremer et al.
(2004) dated families of asterids, Wikström et al. (2001)
timed angiosperm cladogenesis. The stem and crown ages
of Lamiales have been estimated by these authors at 106/
97 mya (mid Cretaceous) and 77–81/71–74 mya (late
Cretaceous), respectively.
To compensate for missing fossils, authors dealing with
families belonging to Lamiales have used inferred ages
from the studies mentioned above for calibration of their
phylogenies, e.g., Phrymaceae (Nie et al. 2006) and also
Scrophulariaceae (Datson et al. 2008). Nie et al. (2006)
additionally dated divergence times by calibration with
three reference fossils from Bignoniaceae and Oleaceae.
This approach is likely to produce more accurate results
because calibration points are closer to the examined
family. However, calibration based on these fossils was not
possible in our study due to problems in aligning distant
outgroups when using markers variable enough to resolve
structures on an infrageneric level. From families other
than Scrophulariaceae, only Russelia verticillata (Plantaginaceae) could be included successfully into the sampling.
Calculations of divergence times were also complicated by
the incomplete sampling within the genus. As outlined by
Linder and Hardy (2004), the potential absence of basal
lineages can lead to younger dates obtained for the start of
radiation. Thus, all divergence times provided in this study
must be regarded as an initial attempt to establish a chronological framework for the radiation of lineages within
Scrophularia.
To calibrate the present phylogeny, we decided to follow Bremer et al. (2004). Their results are probably more
reliable than those provided by Wikström et al. (2001), as
six chloroplast regions (compared to three) and six reference fossils (compared to one) were used. Furthermore, the
obtained divergence times for Lamiales (106 mya) was
recently corroborated by Janssens et al. (2009), assessing
the age at 104 ± 8.2 mya. As for the group concerned in
123
86
this study, Bremer et al. (2004) date the separation of
Scrophulariaceae from the Plantaginaceae at ca. 76 mya.
Many asterids appear in the fossil record of the late Cretaceous (Magallon et al. 1999), and the values fit the
minimum age of Scrophulariaceae based on their appearance in the fossil record (late Eocene; Magallon et al.
1999). This ‘‘Plantaginaceae stem age’’ of 76 mya was
used as calibration point. Although Plantaginaceae and
Scrophulariaceae are not sister groups in the phylogeny
provided by Bremer et al. (2004) but form a grade leading
towards the Lamiales crown group families (such as Verbenaceae, Acanthaceae, Bignoniaceae), a study on
Scrophulariaceae has already been conducted successfully
including only Plantaginaceae and a few related Lamiales
stem group taxa (Datson et al. 2008). With calibration
applied, the resulting divergence time of the ingroup
(46.86 mya) is optimally congruent with Bremer et al.
(2004) who estimate Selago (which is less closely related
to Scrophularia) to have split away 48 mya. Datson et al.
(2008), using calibration points according to Wikström
et al. (2001), specify this divergence at 39.5 mya. The split
between Verbascum and Scrophularia as inferred by Datson et al. (2008) dates to ca. 24.3 mya, compared to
46.86 mya in the present study.
Intercontinental disjunction
According to Stiefelhagen (1910), the origin and native
range of Scrophularia is assumed to be in Asia, more
precisely, in the Himalayan region. Southwestern China is
one of the biologically richest temperate regions in the
world today (Wu 1988; Sun 2002), and it is hypothesized
that the uplift of the Himalaya-Tibetan plateau (which
started 40–50 mya) and subsequent increase in geological
complexity in that region (resulting in a diverse mixture of
habitats) has contributed to the process of diversification
(Wu 1988; Nie et al. 2005). As the sampling of the present
study does not allow any implications regarding the center
of origin, we follow Stiefelhagen’s (1910) assumption in
that point. Relaxed molecular clock estimates suggest the
divergence of Scrophularia from its sister taxon at ca.
47 mya. From its center of origin, it would then have
spread to eastern Asia, to Europe, and to North America
(Stiefelhagen 1910).
Based on an Asian origin of New World Scrophularia,
the observed relationships point towards ancient transBeringian migrations. As outlined by Tiffney (1985), the
Beringian Land Bridge (BLB) allowed migrations from the
early Tertiary until the late Eocene, and also in the second
half of the Tertiary (until 5.5–4.8 mya) for cool-temperate
taxa (Marinovich and Gladenov 1999; Tiffney and Manchester 2001). In contrast, migration via the North Atlantic
Land Bridge (NALB) seems unlikely because of the
123
A. Scheunert, G. Heubl
closure of this pathway at the Eocene-Oligocene boundary
(Tiffney 1985), which is considerably earlier than the
earliest possible migration of Scrophularia to the New
World (early Miocene, between nodes 7 and 11 in Fig. 6)
as determined by relaxed molecular clock analyses. Thus,
migration to the New World from Asia through Beringia
has to be assumed for Scrophularia, a hypothesis which
was doubted but not ruled out by Hong (1983). Other
Northern Hemisphere genera are also known to have dispersed using that route: Scheen et al. (2004) found that
North American species of Cerastium (Caryophyllaceae)
originated as a result of Asian taxa migrating across the
BLB prior to its breakup. An ancestral species of Aralia
(Araliaceae) was hypothesized by Wen et al. (1998) to
have migrated into North America via Beringia and
diversified into the present-day species. Asian Aralia species occur in China, Japan, Korea, and the Himalayas,
while the North American species are found in California,
New Mexico, Arizona, and eastern North America, a distribution that is identical to that of Scrophularia.
Although relationships among the three New World
clades are not resolved in the tree topologies, a rough
scenario for the colonization of the North American continent can be outlined. Assuming a common ancestor in
Asia, the most parsimonious explanation for the current
distribution pattern involves one or two migration
events across the BLB in the Miocene (between 22.27
and 8.32 mya), with subsequent diversification into the
Californian and New Mexican lineages (clades 1 and 2).
Independently, another lineage underwent a radiation
in Japan before likewise colonizing the New World in the
late Miocene (between 15.84 and 12.34 mya) and giving
rise to the eastern North American/Caribbean lineage
(clade 3).
An additional node in BI, BEAST, and ML analyses
placed the New Mexico clade sister to the other New
World clades, but support values were too low for the node
to be reliable (62 / 74% PP and 54% ML bootstrap support), so it was not included in Fig. 3 (position marked by a
triangle). However, if this weak signal reflects the true
relationships, another, less likely scenario has to be considered, where one single migration event across the BLB
led to the colonization of western North America. From
there, a recolonization of Japan via the BLB with subsequent return to the New World would have created the
EA-ENA disjunction present in the phylogeny, and eventually the West Indies clade.
However, as long as no reliable evidence supports any
of the possibilities, we tend to favor the most parsimonious
explanation. Apart from that, a molecular phylogenetic
study focusing on Scrophularia on a worldwide scale is in
preparation and should provide answers to the remaining
questions.
Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae)
Acknowledgments We acknowledge Prof. Dr. Dave Boufford for
his valuable introduction to the North American species of Scrophularia, his kind support of this study, and his helpful comments on
the manuscript. Furthermore, we thank the herbaria and curators of A,
GH, M, MSB, UTEP, W, and WU for permitting the examination of
their specimens and for help in obtaining leaf material. The Botanical
Garden Tübingen (Germany) is acknowledged for providing seeds, as
well as Till Hägele and the Korean National Arboretum Seoul (Rep.
of Korea) for plant material. We thank Florian Turini for help with the
data analyses, Tanja Ernst for laboratory assistance, and the whole lab
group of Prof. Heubl for helpful discussions and comments on the
manuscript. This study was supported by the Universität Bayern e.V.
by means of the Bayerisches Eliteförderungsgesetz (BayEFG).
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4.3. Article III
Diversification of Scrophularia (Scrophulariaceae) in the Western Mediterranean
and Macaronesia - Phylogenetic relationships, reticulate evolution and
biogeographic patterns.
by Agnes Scheunert & Günther Heubl
Molecular Phylogenetics and Evolution 70: 296-313 (2014)
III
The final publication is available on Science Direct via
http://dx.doi.org/10.1016/j.ympev.2013.09.023
75
76
Molecular Phylogenetics and Evolution 70 (2014) 296–313
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Diversification of Scrophularia (Scrophulariaceae) in the Western
Mediterranean and Macaronesia – Phylogenetic relationships,
reticulate evolution and biogeographic patterns
Agnes Scheunert ⇑, Günther Heubl
Systematic Botany and Mycology, Department Biology I, Ludwig-Maximilians-University, GeoBio Center LMU, Menzinger Strasse 67, 80638 Munich, Germany
a r t i c l e
i n f o
Article history:
Received 14 May 2013
Revised 2 August 2013
Accepted 25 September 2013
Available online 4 October 2013
Keywords:
Biogeography
Iberian Peninsula
Incongruence
Polyploidy
Scrophularia
Taxon duplication
a b s t r a c t
The flora of the Mediterranean region and Macaronesia is characterized by high levels of species diversity
and endemism. We examined phylogenetic relationships of Scrophularia within one of its secondary centers of diversity located in the Iberian Peninsula and adjacent Macaronesia. In total, 65 ingroup accessions
from 45 species, representing an almost complete sampling of the region, were analyzed using sequences
from the internal transcribed spacer region (ITS) and the plastid trnQ-rps16 intergenic spacer. Phylogenetic relationships were inferred using Bayesian inference, maximum likelihood and statistical parsimony networking. Incongruence between datasets was assessed with statistical tests and displayed by
split networks. Biogeographic inferences incorporating information from both markers (despite low resolution in some parts of the trees) and all incongruent taxa were accomplished with a novel combination
of methods, using trees generated with the taxon duplication approach as input for Bayesian binary
MCMC (BBM) analysis as implemented in RASP.
Nuclear and chloroplast markers support a clade which comprises the majority of Iberian and Macaronesian species and consists of three subclades. Analyses of the substantial incongruence observed among
markers indicate reticulate evolution and suggest that Scrophularia species diversity in this region is largely attributable to hybridization; a combination of both polyploidy and dysploidy in the karyotypic evolution of Western Mediterranean Scrophularia taxa is proposed. Our results provide support for an ancient
hybridization event between two widespread lineages, which resulted in an allopolyploid ancestor of the
Iberian – Macaronesian group with 2n = 58 chromosomes. The ancestor then diverged into the three main
lineages present in the Iberian Peninsula, Northern Africa and Macaronesia today. Subsequent interspecific hybridizations at different ploidy levels additionally generated new species. Presumably, hybridization and diversification within the genus in the Western Mediterranean have not been restricted to one
particular event, but occurred repeatedly. It can be assumed that the topographical complexity found in
the Iberian Peninsula has promoted diversification and hybrid speciation processes in Scrophularia, and
that isolation in glacial refugia has preserved recent and ancient lineages. For the Macaronesian taxa, biogeographic analyses support several origins, by colonizations from at least four distinct lineages.
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
The Mediterranean basin is recognized as a global biodiversity
hotspot and harbors app. 25,000 vascular plant species (Comes,
2004; Médail and Myers, 2004; Myers et al., 2000). As defined by
Médail and Quézel (1997), important areas of plant endemism
and floristic richness in the Western Mediterranean and Macaronesia are the High and Middle Atlas mountains, the Baetic – Rifan
complex, the Maritime and Ligurian Alps, the Tyrrhenian Islands,
and the Canary Islands and Madeira. One of two main centers of
⇑ Corresponding author.
E-mail address: agnes.scheunert@gmx.net (A. Scheunert).
1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ympev.2013.09.023
biodiversity in the Mediterranean basin is found in its western part
and includes the Iberian Peninsula and Morocco (Médail and
Quézel, 1997). It is assumed that patterns of plant speciation in
the Mediterranean Basin have been shaped by climatic shifts and
geological events, like the Betic Crisis and Messinian Salinity Crisis
(Garcia-Castellanos et al., 2009; Krijgsman et al., 1999; Lonergan
and White, 1997), the onset of the Mediterranean climate rhythm
(Suc, 1984; Thompson, 2005), and the Quaternary glaciations (Suc,
1984). Additionally, its topographical complexity was suggested to
promote diversification and speciation processes. Fragmentation
and contraction of distribution ranges have triggered the isolation
of populations during glacial periods, thereby causing allopatric
speciation. Following inter- and postglacial range expansion,
A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
hybrid zones developed where different genetic lineages came into
contact (Blanco-Pastor et al., 2012).
The genus Scrophularia L. (Scrophulariaceae), belonging to the
order Lamiales and closely related to Verbascum L. (Datson et al.,
2008; Olmstead et al., 2001), includes app. 270 extant species
(Ortega Olivencia, 2009). Representatives of the mainly holarctic
genus occur in both the Old and New World; the primary center
of diversity is located in the Irano – Turanian region including
the Caucasus and Central Asia (Gorschkova, 1997; Grau, 1981; Lall
and Mill, 1978). One of two secondary diversity centers is found in
the Iberian Peninsula and adjacent Macaronesia with 28 species,
more than half of which are endemic (Dalgaard, 1979; Ortega Olivencia, 2009). The last taxonomic treatment of the genus was done
by Stiefelhagen (1910), who classified the genus according to two
sections: section Anastomosantes Stiefelhagen (= section Scrophularia), with two subsections Vernales Stiefelhagen and Scorodoniae
(Benth.) Stiefelhagen (= subsection Scrophularia), and section Tomiophyllum Bentham, comprising subsections Farinosae Stiefelhagen,
Orientales Stiefelhagen, and Lucidae Stiefelhagen (which largely
corresponds to sect. Canina G.Don). Leaf venation, petal
length, shape of the corolla tube, and life form were used as
distinguishing characters. In a revision of the genus for the Iberian
Peninsula, Ortega Olivencia and Devesa Alcaraz (1993a)
confirmed the assignment of the respective species to sections
Anastomosantes / Scrophularia (20 species) and Canina G.Don (three
species); S. tanacetifolia was moved to section Scrophularia by the
authors.
Species of Scrophularia are annual, biennial or perennial herbs,
subshrubs or small shrubs with pinnate to undivided leaves of
various forms. The inflorescence is a thyrse with cymose, often
dichasial or monochasial partial inflorescences. The flowers are
characterized by a more or less equally 5-lobed calyx, a mostly
zygomorphic, distinctly 2-lipped corolla and typically a rudimentary fifth stamen of various shapes at the base of the upper lip.
The fruit is a capsule with septicidal – septifragous dehiscence
(Fischer, 2004). Representatives of the genus inhabit regions from
coasts and lowlands to high plateaus and alpine regions, where
the majority of species is found. Preferred habitats include mountain slopes and rock crevices, but also forests, scrubs and grassland as well as roadsides or disturbed areas. Species occurring
in moist habitats (e.g. on river banks) are mainly restricted to section Anastomosantes, while section Tomiophyllum also contains
more xerophytic elements; however, real desert plants are rare.
Karyological studies on taxa of the genus were confined to particular geographic areas (Carlbom, 1969, 1964; Dalgaard, 1979;
Grau, 1976; Ortega Olivencia and Devesa Alcaraz, 1990; Shaw,
1962) or merely reported chromosome numbers (Vaarama and
Hiirsalmi, 1967), which range from 2n = 22 (e.g. in S. divaricata Ledeb.; Vaarama and Leikas, 1970) to 2n = 96 (e.g. in S. desertorum
(Munz) R.J. Shaw; Shaw, 1962). Many taxa from the Iberian Peninsula and Macaronesia are characterized by 2n = 58 (Grau, 1976);
and most of the species occurring in the New World are high
polyploids with 2n = 70–96 (Shaw, 1962). Shaw (1962) and
Carlbom (1969) postulated that allopolyploid evolution increased
species diversity and variability within Scrophularia. Indeed,
hybridization is frequent in the genus (natural hybrids have been
erroneously described as distinct species by Menezes, 1908,
1903), and is additionally supported by flower morphology and
pollinator preferences. While some of the few large-flowered Scrophularia (S. sambucifolia L., S. grandiflora DC. and S. trifoliata L. from
the Western Mediterranean, and S. calliantha Webb. & Berthel.
from Gran Canaria) were shown to possess a mixed pollination
syndrome between insects and passerine birds (and even juvenile
lizards in S. calliantha; Ortega-Olivencia et al., 2012), wasp pollination was recently revealed to be the ancestral condition within the
genus (Navarro-Pérez et al., 2013). Wasps (Vespidae) are
297
considered the main pollinators (Faegri and van der Pijl, 1979;
Ortega Olivencia and Devesa Alcaraz, 1993b; Wilson, 1878); other
insects like bees (Valtueña et al., 2013) and syrphid flies (Knuth,
1909; Ortega Olivencia and Devesa Alcaraz, 1993b; Robertson,
1891) complete the pollinator spectrum, which thus mostly consists of generalist pollinators unable to distinguish between different species of Scrophularia. In addition, the mostly protogynous
flowers are often self-compatible (although rarely self-pollinating;
Ortega Olivencia and Devesa Alcaraz, 1993b, 1993c; Shaw, 1962;
Trelease, 1881; Valtueña et al., 2013), and reproductive barriers
among related species tend to be weak as well. Artificial cross-pollination experiments have shown the potential for hybridization:
successful crossings were performed by Shaw (1962) and Carlbom
(1964) on North American, Dalgaard (1979) on Macaronesian, and
Goddijn and Goethart (1913) and Grau (1976) on some European
species. Altogether, the above-mentioned ecological and morphological factors make interspecific hybridization, in combination
with karyotype evolution including polyploidy and dysploidy, very
likely to have played an important role in the diversification and
speciation history of Scrophularia.
In the current work, which is part of an extensive molecular
phylogenetic study of the genus, we specifically address the following questions: (1) What are the phylogenetic relationships
among Scrophularia taxa in the Iberian Peninsula and in Macaronesia? (2) Is the high species diversity observed in the Iberian Peninsula the result of hybridization events or due to other factors? (3)
Which parental taxa were involved in the origin of the polyploid
Iberian species? Which event gave rise to the unusual chromosome
number 2n = 58 found in many taxa? (4) Which historical biogeographic processes have affected present distribution patterns of
Scrophularia in the Iberian Peninsula? Are the species occurring
on the Canary Islands and Madeira monophyletic (implying a single colonization event), or were there multiple colonizations?
To achieve this goal, we analyzed the plastid trnQ-rps16 intergenic spacer, which has been successfully employed in a recent
study on New World Scrophularia (Scheunert and Heubl, 2011),
and the nuclear ribosomal internal transcribed spacer (ITS) region,
which continues to be widely used for phylogenetic analyses on
the interspecific level (Nieto Feliner and Rosselló, 2007).
2. Material and methods
2.1. Plant material, DNA extraction, sequencing
The taxon sampling strategy was designed to span the distribution range of Scrophularia in the Western Mediterranean (especially the Iberian Peninsula), Macaronesia and Northern Africa.
The present study comprises all 22 species of the Iberian Peninsula
according to Ortega Olivencia (2009), 27 out of 28 species occurring in the Western Mediterranean as a whole (e.g. Cartier, 1975;
Ortega Olivencia, 2009; Zángheri, 1976; S. heterophylla was not
sampled but in the region is confined to Istria only), 17 out of 21
species occurring in Northern Africa (Ibn Tattou, 2007; Qaiser,
1982; Quézel and Santa, 1963; Täckholm, 1974; the four absent
species are either narrow endemics with close association to sampled species, or putative synonyms), and all of the nine species
from Macaronesia (Dalgaard, 1979). Additionally, four species from
other diversity centers and the putative center of origin (‘‘Asian
species’’) were sampled; these include three Southern Asian/Eastern Asian taxa (S. urticifolia Wall., S. ningpoensis Hemsl., S. yoshimurae T.Yamaz.) and one species distributed in Southwestern Asia,
Turkey and the Caucasus (S. amplexicaulis Benth.). Altogether, the
sampling thus consists of 45 ingroup species. For a rough estimate
of the degree of intraspecific genetic variation, 13 species were
sampled with two or more accessions (see Table 1), laying
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A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
Table 1
Analyzed taxa with voucher information on collectors, localities and collection years, herbaria, and GenBank accession numbers. Abbreviated identifiers for individual accessions as
used in the text are given after the respective locality (in quotes). Reference citations of previously published sequences: (1), Vargas et al. (2009); voucher information for the respective
sequence as given there; (2), Kornhall and Bremer (2004); (3), Scheunert and Heubl (2011). CULT., plants grown in the greenhouses of the Botanical Garden Munich, Germany – the
original locality and the supplier (in brackets) are given where known. Herb., Herbarium; acc. no., accession number; Bot. Gard., Botanical Garden, n/a, information not available.
Taxon
Year
Locality (country, province/district)
Collector
Collector
no.
Herb.
Acc. no.
trnQ-rps16
Acc. no. ITS
Antirrhinum majus L.
Russelia verticillata Kunth
Hemimeris centrodes Hiern
Nemesia cheiranthus E.Mey. ex
Benth.
Selago corymbosa L.
Verbascum arcturus L.
Verbascum nigrum L.
S. amplexicaulis Benth.
S. auriculata L.
S. auriculata ‘‘balbisii Hornem.’’
S. alpestris J.Gay ex Benth.
S. arguta Sol.
S. arguta Sol.
1999
1990
1976
1974
Spain, Cordoba
Costa Rica, Guanacaste
South Africa, Cape
South Africa, Cape
M. Nydegger
P. Döbbeler
P. Goldblatt
P. Goldblatt
36531
3795
4033
2534
MSB
M
M
M
KF447311
–
HQ1300333
KF447312
FJ4876151
HQ1300623
HQ1300633
KF447249
1962
1998
1977
1993
2008
1998
1905
2010
Vlok
D. Phitos
H. Wunder
K.H. Rechinger
H. Förther
A. Scheunert
D. Podlech
C.J. Pitard
A. Scheunert
2514
603
S
M
M
M
M
MSB
MSB
M
MSB
–
KF447313
HQ1300343
KF447315
KF447247
KF447248
KF447332
KF447368
KF447367
AJ5506032
KF447250
HQ1300643
KF447252
KF447287
KF447291
KF447269
KF447308
KF447307
S. arguta Sol.
S. bourgaeana Lange
1995
1994
South Africa, Beaufort West
Crete, Chanía
Germany, Bavaria
Iran, Tehran
Morocco, Tétouan
CULT., orig.: Spain, Cantabria (J. Grau)
France, Pyrénées-Atlantiques
Fuerteventura, Puerto del Rosario
CULT., orig.: Lanzarote, Teguise (M.
Erben)
Morocco, Tiznit
Spain, Salamanca
D. Podlech
M. Martinez Ortega
MSB
MA
KF447366
KF447333
KF447306
KF447270
S. calliantha Webb. & Berthel.
S. canina ssp. canina L.
S. canina ssp. ramosissima
(Loisel.) P.Fourn.
2011
1995
1976
CULT., orig.: Gran Canaria, n/a
Morocco, Tiznit
Sardinia, Oristano
A. Scheunert
D. Podlech
U. Hecker
52494
(MA
631819)
010/1-1
52525
I 774
(Hec.
1560)
MSB
MSB
MJG
KF447362
KF447320
KF447323
KF447302
KF447257
KF447260
S. crithmifolia Boiss.
1983
Spain, Malaga
M
KF447321
KF447258
S.
S.
S.
S.
S.
1991
1933
1973
1988
2010
Egypt, Sinai Peninsula
Morocco, Rif-Atlas
Spain, Cadiz
La Palma, Fuencaliente; ‘‘pal’’
CULT., orig.: Tenerife, Monte del
Cuchillo; ‘‘ten’’
CULT., orig.: n/a (Bot. Gard.
Erlangen 218/2007)
CULT., orig.: n/a (Bot. Gard. Madrid
262-80); ‘‘na’’
Spain, Zamora; ‘‘za’’
E. Bayer, J. Grau & G. López
González
D. Podlech
Sennen (& Mauricio)
H. Merxmüller & W. Gleißner
M. Nydegger
A. Scheunert
49719a
8461
29073
25415
011/1-1
MSB
W
M
M
MSB
KF447325
KF447351
KF447322
KF447360
KF447361
KF447262
KF447289
KF447259
KF447300
KF447301
A. Scheunert
004/1-1
MSB
KF447348
KF447285
A. Scheunert
016/1-1
MSB
KF447370
KF447278
MA
KF447342
KF447279
A. Scheunert
(MA
510365)
009/1-1
MSB
KF447364
KF447304
A. Scheunert
013/1-1
MSB
KF447365
KF447305
A. Scheunert
014/1-1
MSB
KF447363
KF447303
W. Lippert
M. Nydegger
M.A. Carrasco, S. Castroviejo &
M. Velayos
K.H. Rechinger
A. Scheunert
25095
33671
13801SC
M
MSB
MA
KF447353
KF447337
KF447336
KF447292
KF447274
KF447273
10358
007/1-1
M
MSB
KF447329
KF447369
KF447266
KF447309
12546
7444
M
M
W
KF447319
KF447350
KF447334
KF447256
KF447288
KF447271
769
03-0549
GH
MSB
HQ1300413
HQ1300383
HQ1300713
HQ1300683
MSB
HQ1300373
HQ1300673
MA
KF447343
KF447280
M
M
MSB
KF447317
KF447335
KF447354
KF447254
KF447272
KF447293
deserti Delile
eriocalyx Emb. & Maire
frutescens L.
glabrata Aiton
glabrata Aiton
57228
7104
005/1-1
55135
015/1-1
S. grandiflora DC.
2010
S. herminii Hoffmanns. & Link
2009
S. herminii Hoffmanns. & Link
1987
S. hirta Lowe
2010
S. hirta Lowe
2010
S. hirta Lowe
2010
S. hispida Desf.
S. laxiflora Lange
S. laxiflora Lange
1989
1995
1996
S. libanotica Boiss.
S. lowei Dalgaard
1957
2010
S. lucida L.
S. lyrata Willd.
S. macrorrhyncha (Humbert,
Litard. & Maire) Ibn Tattou
S. ningpoensis Hemsl.
S. nodosa L.
1967
1971
2006
Iraq, Sulaymaniyyah (Kurdistan)
CULT., orig.: Madeira, n/a (Bot.
Gard. Madeira 61/2009)
Greece, Attica
Crete, Chanía
Morocco, n/a
1991
2003
CULT., orig.: n/a
Armenia, Lori
S. nodosa L.
1999
Germany, Bavaria
J. Jutila
G. Fayvush, K. Tamanyan, H. TerVoskanian & E. Vitek
D. Podlech
S. oxyrhyncha Coincy
1995
Spain, Badajoz
J.L. Perez Chiscano
S. peregrina L.
S. pyrenaica Benth.
S. racemosa Lowe
1987
1971
2011
E. & M. Mayer
H. Merxmüller & B. Zollitsch
A. Scheunert
S. reuteri Daveau
S. sambucifolia L.
S. scopolii var. scopolii Hoppe ex
Pers.
S. scopolii var. grandidentata
1974
1996
1967
Croatia, Dalmatia
France, Pyrénées-Atlantiques
CULT., orig.: Madeira, n/a (Bot.
Gard. Madeira 47/2009)
CULT., orig.: Spain, Avila (J. Grau)
Spain, Cadiz
CULT., orig.: Poland, Małopolskie
(Bot. Gard. Wroclaw)
Italy, L’Aquila
(MSB
116671)
(MA
560760)
12049
27178
006/1-1
J. Grau
D. Podlech
J. Grau
Sc-143
54066
Sc-63
M
M
M
KF447344
KF447352
KF447330
KF447281
KF447290
KF447267
H. Merxmüller & J. Grau
20788
M
KF447331
KF447268
1965
CULT., orig.: Madeira, n/a; ‘‘mad.
(1)’’
CULT., orig.: Madeira, n/a; ‘‘mad.
(2)’’
CULT., orig.: Madeira, Pico de Ruivo
(M. Erben); ‘‘mad. (r)’’
Morocco, Tadla-Azilal
Spain, Cadiz, Algeciras; ‘‘alg’’
Spain, Cadiz, Los Barrios; ‘‘bar’’
J. Grau
G. & W. Sauer
M. Staudinger
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A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
Table 1 (continued)
Taxon
Year
Locality (country, province/district)
Collector
Collector
no.
Herb.
Acc. no.
trnQ-rps16
Acc. no. ITS
(Ten.) Boiss.
S. scorodonia L.
S. scorodonia L.
1986
2011
H. Hertel
A. Scheunert
33369
017/1-1
M
MSB
KF447338
KF447339
KF447275
KF447276
S. smithii ssp. smithii Hornem.
2010
A. Scheunert
018/1-1
MSB
KF447355
KF447295
S. smithii ssp. smithii Hornem.
S. smithii ssp. smithii Hornem.
S. smithii ssp. langeana (Bolle)
Dalgaard
S. smithii ssp. langeana (Bolle)
Dalgaard
S. smithii ssp. langeana (Bolle)
Dalgaard
S. sublyrata Brot.
S. syriaca Benth.
S. syriaca Benth.
S. syriaca ‘‘hypericifolia Wydler’’
S. tanacetifolia Willd.
S. tenuipes Coss. & Durieu
2010
1971
2010
Madeira, Funchal; ‘‘mad’’
CULT., orig.: Tenerife, Anaga
mountains (M. Erben); ‘‘ten’’
CULT., orig.: Tenerife, Chamorga
(M. Erben); ‘‘cha’’
CULT., orig.: Tenerife, n/a; ‘‘ten. (1)’’
Tenerife, Taganana; ‘‘tag’’
CULT., orig.: Tenerife, Aguamansa;
‘‘ag’’
Tenerife, Los Erjos; ‘‘erj’’
A. Scheunert
019/1-1
A. Scheunert
008/1-1
MSB
M
MSB
KF447310
KF447356
KF447357
KF447294
KF447296
KF447297
M
KF447358
KF447298
CULT., orig.: Tenerife, Los Silos;
‘‘sil’’
Portugal, Estremadura
Israel, Negev; ‘‘isr’’
Tunisia, Gafsa; ‘‘tun’’
Iraq, Al Ramadi
Spain, Valencia
Algeria, Skikda
A. Scheunert
012/1-1
MSB
KF447359
KF447299
4564
M
M
MSB
M
M
M
KF447345
KF447326
KF447324
KF447327
KF447340
KF447318
KF447282
KF447263
KF447261
KF447264
KF447371
KF447255
S. trifoliata L.
S. umbrosa Dumort.
S. urticifolia Wall.
1977
2003
Corsica, Cap Corse
Iran, Chaharmahal & Bakhtiyari
n/a; voucher specimen: LP0908740
E. Bayón & R. Vogt
K. Tielbörger
D. Podlech
K.H. Rechinger
A. Aguilella & I. Mateu
A. Dubuis, H. Maurel & R.
Rhamoun
H. Merxmüller & W. Lippert
M.R. Parishani
JL
KF447349
HQ1300353
KF447314
KF447286
HQ1300653
KF447251
S. valdesii Ortega Oliv. & Devesa
1982
Spain, Salamanca
J.L. Fernández Alonso
M
M
HU/
HZU
MA
KF447341
KF447277
S. vernalis ssp. clausii (Boiss. &
Buhse) Grau
S. viciosoi Ortega Oliv. & Devesa
1974
Iran, Azerbaijan
W. Rechinger & J. Renz
M
KF447316
KF447253
2002
Spain, Malaga, Antequera; ‘‘ant’’
B. Cabezudo
MA
KF447346
KF447283
S. viciosoi Ortega Oliv. & Devesa
S. xanthoglossa Boiss.
S. yoshimurae T.Yamaz.
1973
1992
1992
Spain, Malaga, El Torcal; ‘‘tor’’
Israel, Negev
Taiwan, Nantou Hsien
H. Merxmüller & W. Gleißner
K. Tielbörger
C.-C. Liao
M
M
A
KF447347
KF447328
HQ1300423
KF447284
KF447265
HQ1300723
2007
2010
1986
1992
1980
1957
1983
1986
particular emphasis on Macaronesian taxa. Samples do not represent the whole distribution range of the respective species as this
would have gone beyond the scope of this study; therefore, as no
accessions from the Azores (S. auriculata) and the Cape Verdes (S.
arguta) were available, biogeographic conclusions regarding Macaronesia were restricted to the Canary Islands and Madeira. Seven
outgroup species were chosen from the Scrophulariaceae (Verbascum nigrum L., Verbascum arcturus L., Selago corymbosa L., Hemimeris centrodes Hiern, Nemesia cheiranthus E.Mey. ex Benth.) and
Plantaginaceae (Russelia verticillata Kunth, Antirrhinum majus L.)
based on results by Olmstead et al. (2001), Datson et al. (2008)
and Scheunert and Heubl (2011). Information on voucher specimens as well as accession numbers is provided in Table 1. Chromosome numbers for sampled ingroup taxa were obtained from the
database of Index to Plant Chromosome Numbers (IPCN; http://
www.tropicos.org/Project/IPCN; last accessed on 10.05.2013) and
from the literature, especially Grau (1976) and Ortega Olivencia
and Devesa Alcaraz (1990).
Leaf material for DNA sequencing was obtained from herbarium
specimens (55 accessions from collections in A, GH, HU/HZU, M,
MA, MJG, MSB, nd W), and from plants cultivated in the greenhouses of the Botanical Garden in Munich (16 accessions, vouchers
deposited in MSB; See Table 1). Seeds for cultivation were acquired
from seed banks, economic providers or collected during field
trips; to avoid confounding effects of uncontrolled hybridization
in the greenhouses, plants were grown in isolation; furthermore,
generally no F1 plants were sampled for this study. Specimens
were checked for correct species identification whenever possible.
One non-coding chloroplast (cp) region (the trnQ-rps16 intergenic spacer) and one nuclear ribosomal (nr) region (the internal
transcribed spacer region, ITS) were chosen for phylogenetic
W. Nezadal
34195
9514
15531
18440
31405
14232
17
(MA
519560)
49744
(MA
789425)
29144
718
analyses. Total genomic DNA was extracted from dried leaf material using the NucleoSpin Plant Kit (Macherey–Nagel, Düren, Germany) following the manufacturer’s standard protocol, while
applying an additional phenol/chloroform extraction step to remove proteins and potentially interfering secondary compounds.
PCR reactions as well as subsequent purifications and sequencing
reactions were performed according to the procedure described
in Scheunert and Heubl (2011; no ExoSap purification), using the
following primers: for ITS, primers ITS1 and ITS4 (White et al.,
1990), supported by ITS2 and ITS3 (White et al., 1990), aITS1 and
aITS4 (Bräuchler et al., 2004) and ITSIR (Scheunert and Heubl,
2011) in problematic cases; for trnQ-rps16, primers 1 (trnQ-F)
and E (rps16-1R) (Calviño and Downie, 2007), and SPF, SPR, SPF2
and SPR2 (Scheunert and Heubl, 2011). Primers were used for
amplification and sequencing, except for aITS1 and aITS4 (PCR
only), and SPF2 and SPR2 (sequencing only). Markers were sequenced bidirectionally in cases where the quality of single sequences proved insufficient.
2.2. Phylogenetic analyses
Alignments were generated with MAFFT v.6 (Katoh and Toh,
2008; Katoh et al., 2002) using the slow iterative refinement FFTnS-I algorithm, 1PAM/j = 2 as scoring matrix, a gap opening penalty of 1.5 and an offset value of 0.0. All alignments were refined
manually using BioEdit v.7.1.11 (Hall, 1999); mononucleotide repeats and ambiguously aligned regions were excluded from further
analyses. ITS sequences were checked for potential pseudogenes
(Bailey et al., 2003; Hershkovitz and Zimmer, 1996; Jobes and
Thien, 1997; Liu and Schardl, 1994). In order to assess their phylogenetic information content before incorporating them into the
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A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
final dataset, nuclear and chloroplast indels were tentatively added
or removed from the single marker phylogenetic analyses and the
results compared. Ingroup indels were coded as binary states (discarding excluded alignment regions) using the simple indel coding
method by Simmons and Ochoterena (2000) as implemented in
SeqState v.1.4.1 (Müller, 2005).
The two markers were first analyzed separately using both a
Bayesian inference (BI) and maximum likelihood (ML) approach.
MrModelTest v.2.3 (Nylander, 2004) suggested the GTR + C substitution model as best fit to the data, with a proportion of invariant characters for the nuclear matrix only (Akaike information criterion). For
Bayesian analyses including indel data, a mixed dataset was defined,
using the model settings recommended in Ronquist et al. (2009) for
the binary partition. Bayes runs were performed with MrBayes v.3.2
for 64 bit systems (Ronquist et al., 2012), using one cold and three
heated Markov Chain Monte Carlo (MCMC) chains with temperature
t = 0.10 for ITS and t = 0.05 for trnQ-rps16. For each of two independent runs per marker, 10 106 generations were completed, sampling every 2000th generation. The first 10% trees of each run were
discarded as burn-in and the remaining 9002 trees used for computation of the majority-rule consensus tree.
ML analyses were performed with RAxML v.7.2.8 (Stamatakis
et al., 2008) using raxmlGUI v.0.95 (Silvestro and Michalak,
2012). Pairs of accessions with completely identical sequences
were represented by only one sequence in the analysis; the removed accession was then manually added to the final tree. A rapid
bootstrap run with 10,000 replicates was followed by an ML
optimization, defining Antirrhinum as outgroup and using the same
models as in Bayesian analyses for DNA data partitions, and
BINGAMMA for the binary indel partition. Each analysis yielded
one fully resolved best-scoring ML tree.
To obtain more detailed information about clades lacking internal resolution in Bayesian and ML analyses, levels of nucleotide
divergence among sequences (uncorrected (‘‘p’’) distances and
numbers of total character differences) were determined separately for each partition using the ‘‘pairwise distance’’ option in
PAUP v.4.0b10 (Swofford, 2003). Calculations were performed
based on the sequence data taken into account for Bayesian and
ML analyses. In addition, the number of plastid haplotypes and
their relationships were inferred for a subset of 36 accessions (corresponding to the 23 species of the ‘‘IPM’’ clade, definition see Section 3.2.) by generating a statistical parsimony network
(Templeton et al., 1992), using the median joining algorithm with
subsequent MP calculation as implemented in TCS v.1.21 (Clement
et al., 2000). Generally, gaps were regarded as missing data, while
coded indels used in Bayesian and ML analyses were added to the
sequence matrix. The number of mutations among haplotypes was
calculated with a maximum parsimony connection limit of 95%
(=14 steps), using equal weights and setting epsilon to zero.
accessions or clades were then subjected to the Incongruence
Length Difference (ILD) test (Farris et al., 1995) implemented in
PAUP as Partition Homogeneity Test; accessions within incongruent clades were additionally tested alone. Applying an approach
also used by van der Niet and Linder (2008), all sequences were
first pruned from the dataset and then re-added and tested separately. Significant accessions were excluded; from those yielding
insignificant results (p > 0.05), a combination of as many as possible was re-included into the dataset. All excluded accessions were
then duplicated for the ancestral area reconstruction (see Section 2.5); these are referred to as ‘‘conflicting taxa/accessions’’ in
the text. Tests were run with 1000 replicates, maxtrees set to
100 and heuristic searches with 50 random addition sequence replicates. Constant characters were removed from the data matrix
prior to the test (following Cunningham, 1997; Lee, 2001).
As the ILD test has been shown to have certain weaknesses, e.g. a
high false positive rate (Barker and Lutzoni, 2002; Darlu and
Lecointre, 2002), all hard incongruence accessions were additionally subjected to Templeton’s significantly less parsimonious test
(SLP test; Templeton, 1983; ‘‘nonparametric pairwise test’’ in
PAUP); ILD-significant accessions or clades were only allowed for
exclusion/duplication if the SLP test was significant for at least
one of the two datasets. Furthermore, in order to assess whether
partly (i.e., in one of the markers) unresolved or weakly supported
positions represented insufficient information or a distinct phylogenetic hypothesis, several of such groups/species (especially present in the chloroplast tree) were also tested. These included the
Macaronesia clade (sister to the Auriculata clade in ITS only), the
Asian species (well supported as sister to S. scopolii and the IPM
clade in ITS only), and S. grandiflora, S. trifoliata and S. tanacetifolia
within the IPM clade (part of the Auriculata/Scorodonia clade in
ITS only). Constraint topologies were simplified from clades in the
Bayesian consensus tree of the other marker; the clades are indicated by the respective constraint numbers in Fig. 1 (exact constraint topologies are provided in Supplementary Fig. S1).
Searches were done with maxtrees set to 5000, 50 replicates with
five trees held at each cycle of the stepwise addition procedure,
and the number of trees retained in each random-addition sequence replicate limited to 100. Significance was set at <0.05 following Templeton (1983).
Finally, to illustrate incongruities between the individual gene
trees (Holland et al., 2004), a tree-based filtered super network
(FSN; algorithm by Huson et al., 2006) was constructed based on
1501 trees from the posterior distribution of the first run of each
single marker Bayesian analysis. Calculations were performed with
SplitsTree v.4.12.3 (Huson and Bryant, 2006), applying the equal
angle splits transformation and the Convex Hull algorithm, with
no filtering of selected splits. Construction of the FSN was done
using default settings, with edge weights displayed as ‘‘tree size
weighted mean’’, and with the minimum number of trees set to
751 (corresponding to a 25% trees threshold any split must be present in to be displayed in the FSN).
2.4. Identification and testing of incongruence
2.5. Ancestral area reconstruction
Patterns of phylogenetic incongruence were explored using several methods as suggested by Hipp et al. (2004). First, the phylogenies yielded from single marker analyses were visually examined
and compared for incongruent placements of individual accessions
or whole clades; congruence was rejected if support values for the
contradictory placements exceeded or equalled 70% bootstrap support (BS) (‘‘hard incongruence’’, Mason-Gamer and Kellogg, 1996),
a cutoff which has successfully been used in several studies (e.g.,
Maureira-Butler et al., 2008; Moline et al., 2007; Scheunert et al.,
2012). A Bayesian posterior probability (PP) of P0.95 was additionally defined as sign of hard incongruence. The respective
Recently developed models for ancestral state inference are able
to account for the uncertainty present in phylogenetic reconstructions. This is particularly important in topologies with polytomies
and weakly supported nodes (Ronquist, 2004). For biogeographic
analyses in this study, we used the Bayesian Binary Markov Chain
Monte Carlo (MCMC) algorithm as implemented in RASP v.2.0b (Yu
et al., 2011), which takes trees from the posterior distribution of a
Bayesian analysis as input and infers ancestral distributions using a
full hierarchical Bayesian approach. Another essential requirement
for obtaining reliable results in biogeographic reconstructions is
that all available data are included; to enable simultaneous analy-
2.3. Sequence divergence and statistical parsimony network analysis
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A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
OUTGROUP
„Arguta“
0.83
0.98 80
0.99 86
96
0.81
5
0.99 79
79
„Nodosa“
0.99
85
1.00
57
3
0.95
61
1.00
94
0.96
83
0.64
75 1.00
100
0.95
90
1.00
93
„Canina“
1.00
98
8
0.96
46
0.98
70
„Auriculata“
9
6
4
10
0.58
78
0.96
89
1.00
96
0.99
64
7
„Macaronesia“
x
100
0.89
49
x
93
1.00
81
0.92
80
„IPM“
„Scorodonia“
1.00
87
11
OUTGROUP
S. arguta Lanzarote
S. arguta Fuertev.
S. ningpoensis
S. arguta Morocco
S. yoshimurae
S. lowei*
S. amplexicaulis
S. urticifolia
S. nodosa Germany
S. nodosa Armenia
S. nodosa Germany
S. alpestris
S. nodosa Armenia
S. bourgaeana*
S. bourgaeana*
S. vernalis ssp. clausii
S. vernalis ssp. clausii
S. peregrina
S. alpestris
S. scopolii
S. tenuipes
S. scopolii var. grandid.
S. lucida
S. umbrosa
S. crithmifolia*
S. peregrina
S. frutescens
S. tenuipes
S. canina
S. arguta Lanzarote
S. canina ssp. ramosissima
S. arguta Fuertev.
S. syriaca Israel
S. arguta Morocco
S. syriaca Tunisia
S. lowei*
S. syriaca „hypericifolia“
S. deserti
S. libanotica
S. syriaca Israel
S. deserti
S. syriaca „hypericifolia“
S. xanthoglossa
S. xanthoglossa
S. ningpoensis
S. syriaca Tunisia
S. yoshimurae
S. libanotica
S. urticifolia
S. lucida
S. amplexicaulis
S. crithmifolia*
S. scopolii
S. frutescens
S. scopolii var. grandid.
S. canina
S. pyrenaica
S. canina ssp. ramosissima
S. umbrosa
S. pyrenaica
S. macrorrhyncha
S. trifoliata
S. grandiflora*
2
S. macrorrhyncha
S. viciosoi* Antequera
S. grandiflora*
S. viciosoi* El Torcal
S. herminii* n/a
S. auriculata „balbisii“
S. herminii* Zamora
S. hispida
S. oxyrhyncha*
1
S. racemosa*
S. sublyrata*
S. sambucifolia
S. reuteri*
S. eriocalyx
S. valdesii*
S. auriculata
S. auriculata „balbisii“
S. lyrata
S. hispida
S. trifoliata
S. racemosa*
S. s. ssp. smithii* Tag.
S. sambucifolia
S. s. ssp. smithii* Ten. (1)
S. eriocalyx
S. s. ssp. smithii* Cha.
S. auriculata
S. glabrata* Ten.
S. lyrata
S. s. ssp. langeana* Ag.
S. s. ssp. smithii* Tag.
S. s. ssp. langeana* Erj.
S. s. ssp. smithii* Ten. (1)
S. s. ssp. langeana* Sil.
S. s. ssp. smithii* Cha.
S. glabrata* Pal.
S. s. ssp. langeana* Ag.
S. calliantha*
S. s. ssp. langeana* Erj.
S. hirta* Mad. (r)
S. s. ssp. langeana* Sil.
S. hirta* Mad. (1)
S. glabrata* Pal.
S. hirta* Mad. (2)
S. glabrata* Ten.
S. laxiflora Alg.
S. calliantha*
S. laxiflora Bar.
S. hirta* Mad. (r)
S. scorodonia Mad.
S. hirta* Mad. (1)
S. scorodonia Ten.
S. hirta* Mad. (2)
S. herminii* n/a
S. viciosoi* Antequera
S. herminii* Zamora
S. viciosoi* El Torcal
S. oxyrhyncha*
2
S. laxiflora Alg.
1
S. sublyrata*
S. laxiflora Bar.
S. reuteri*
S. scorodonia Mad.
S. scorodonia Ten.
S. valdesii*
S. tanacetifolia*
S. tanacetifolia*
ITS
1.00
97
8
0.98
90 0.95
85
„Nodosa“
4 5
0.96
67 1.00
100
1.00
100
6
1.00
81
1.00 „Arguta“
99 1.00
99
0.99
66 0.97
„Canina 1“
70 1.00
91 0.93
53
0.96 „Canina
97 1.00
94
2“
1.00
97 3
10
9
0.66
80
0.98
67
7
1.00
99
„Auriculata“
0.93
50
0.89
66
„IPM“
1.00
76
„Macaronesia“
1.00
86 0.98
62
„Scorodonia“
11
trnQ-rps16
Fig. 1. Bayesian majority-rule consensus trees (cladograms) from the nuclear internal transcribed spacer (ITS) and the plastid trnQ-rps16 intergenic spacer, with outgroup
taxa reduced to a single branch. Posterior probabilities (PP) are given above each node; results from Maximum Likelihood (ML) analyses were plotted onto the cladograms,
with bootstrap support values (BS) given below each node. Branches supported by at least 50% BS in the best-scoring ML trees but lacking in the Bayes consensus trees are
represented by dashed lines. Both Bayesian and ML support values are given only if support at the node equals or exceeds either 0.85 PP or 75% BS (‘‘x’’ denotes cases where
the node was not present in the fully resolved tree of the respective analysis). Major clades are indicated by boxes, divergence of the IPM clade and the Canina clade is marked
by arrows. Conflicting taxa (definition see Section 2.4.) are highlighted in bold. Solid dots (Iberian Peninsula) and triangles (Macaronesia) on terminal branches indicate the
distribution of the respective (sub)species; endemic taxa are additionally marked by asterisks. Circled numbers refer to constraint topologies used in the SLP test. Fuertev.,
Fuerteventura; grandid., grandidentata; n/a, information not available; other abbreviations according to Table 1.
sis of all relevant data in a combined dataset containing several
cases of hard incongruence and potential hybrid taxa, we
constructed a combined trnQ-rps16/ITS data matrix following the
‘‘taxon duplication approach’’ by Pirie et al. (2009; 2008; also
applied in e.g. Linder et al., 2013). All conflicting accessions
(definition see Section 2.4.) were included twice, once as trnQrps16-only-sequence (with nuclear characters coded as missing),
and once as ITS-only-sequence. The modified matrix which thus
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A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
comprised 93 sequences was used for a ‘‘duplicated analysis’’ using
MrBayes with the same settings as for single marker analyses, except for the temperature being set to t = 0.0001 to permit conversion of the chains without having to enforce topological
constraints. A ML analysis (using settings from the chloroplast
marker calculations) was done for comparison purposes.
Distributions of species were assigned to 15 areas, subspecies
were given their own respective distribution; to avoid erroneous
inferences due to human influence, only native occurrences were
taken into account. Single accessions from the same species were
coded with the distribution of the respective species rather than
their individual origin, to avoid mistakes caused by taxa not
sampled across their whole distribution range. An exception is S.
auriculata ‘‘balbisii’’ (see Table 1); as the distribution of S. balbisii
Hornem. is difficult to infer (due to naming confusions and conspecificity with S. auriculata L. according to Ortega Olivencia,
2009), but cannot necessarily be assumed to be identical to that
of S. auriculata L. given different phylogenetic positions in the ITS
tree (Fig. 1), this accession was coded with its geographical origin.
The chosen areas are mainly areas of endemism (here defined as a
geographic region inhabited by two or more species displaying
congruent distributions; Harold and Moor, 1994) based on present-day natural distributions of Scrophularia taxa; some of those
were further subdivided according to palaeogeographic or climatic
characteristics. In detail, areas were defined as follows: A, Azores;
B, Canary Islands (including Cape Verde Islands); C, Madeira; D,
Western Mediterranean (from Portugal to Italy including Sicily);
E, Eastern Mediterranean (from Slovenia and Croatia to Crete and
Cyprus including the Balkan Peninsula); F, Western North Africa
(Morocco to Tunisia); G, Eastern Africa (Libya to Somalia); H, W-/
N-/C-Europe (from the British Isles to Norway and Austria, excluding France); I, E-Europe and Western (‘‘European’’) Russia (from
Czech Republic eastwards, Baltic States, Russia as far as Ural and
Pechora rivers); J, Lebanon/Syria/Israel s.l.; K, Southwestern Asia
(Arabian Peninsula, Iran and Iraq); L, Turkey and the Caucasus
(including the Talysh Mountains); M, Southern/Southeastern Asia
(India to Myanmar, including Afghanistan and Pakistan); N, Central
Asia and Siberia (Kazakhstan and southwards, Southern Siberia
(Russia), northwards as far as species of the genus occur); O, Eastern Asia, Mongolia and ‘‘Russian Far East’’ (China to Japan and Taiwan, southeasternmost Russia from Sakhalin to the Zeya River).
The outgroup taxa, as well as the virtual outgroup, were assigned a wide distribution (i.e., occurring in all defined areas),
which matches the real distribution of Verbascum, the closest relative of Scrophularia. Ancestral area distributions were estimated for
ingroup nodes only. The maximum number of ancestral areas inferred at each node was constrained as recommended by Ronquist
(1997): assuming that ancestral ranges were similar to those of
present-day descendants (Sanmartín, 2003), ‘‘maxareas’’ was set
to five as the majority of species (39 out of 45) now occur in no
more than five areas. Five independent runs of the Bayesian Binary
MCMC were conducted with one million generations each, sampling every 100th tree and discarding 1/5th of the trees as burnin. State frequencies were estimated (F81 model) with a Dirichlet
distribution of 1.0, and among-site rate variation was modeled
across a gamma distribution as suggested by MrModeltest. An
additional run with identical settings but maxareas set to two
yielded congruent results.
3. Results
3.1. Sequence variation
Between Scrophularia and outgroup, p distances from chloroplast DNA sequences were smallest between Verbascum nigrum
and S. nodosa L. from Germany (0.04074) and largest between
Hemimeris and S. hispida Desf. (0.16982). In the nuclear dataset,
values ranged from 0.05060 between Verbascum nigrum and S. arguta Sol. from Morocco, to 0.19403 between Antirrhinum and S. nodosa from Armenia. Among ingroup taxa, chloroplast and nuclear
DNA distances varied from 0.00000 to 0.03515 in trnQ-rps16 (between S. vernalis L. and S. hispida), and from 0.00000 to 0.05602
in ITS (between S. syriaca Benth. from Tunisia and S. tanacetifolia
Willd.). Completely identical sequences in trnQ-rps16 as well as
ITS were found in several cases, especially within the Auriculata,
Scorodonia and Macaronesia clades (definitions see Section 3.2).
All uncorrected distances and total pairwise character differences
are provided in Supplementary Table S1.
3.2. Phylogenetic analyses
ITS sequences showed no length changes in conserved parts
indicative of pseudogenes, and G + C contents (see Table 2) were
similar to those previously published for the genus (Scheunert
and Heubl, 2011), so all sequences were regarded as derived from
functional copies. Altogether, the sampling covers 72 accessions, of
which new sequences were generated for 71 accessions; the ITS sequences of two outgroup taxa (Antirrhinum, Selago) were obtained
from GenBank (NCBI). As sequencing of the trnQ-rps16 intergenic
spacer failed for Russelia and Selago, and no sequence was available
in GenBank, the species were coded as missing for the respective
marker. The aligned trnQ-rps16 matrix thus consisted of 70 accessions and 1345 characters and the ITS matrix of 72 accessions and
617 characters. Detailed information on average lengths of sequences and further alignment characteristics including parsimony
– informative characters is given in Table 2.
Thirty-four ingroup indels were coded for the trnQ-rps16 dataset and 17 for the ITS dataset. Analyzing the trnQ-rps16 dataset in
MrBayes with and without indels showed that support values increased in 12 cases (decrease in six cases) when using indels, and
that four new nodes were supported (results not shown). Using indels with the ITS dataset (results not shown) generally did not alter
support values severely; however, disregarding them resulted in
one additional ingroup node and three considerably increased support values. Consequently, indels were only coded for the trnQrps16 dataset in the final calculations.
MrBayes runs on the single marker datasets had reached convergence after 10,000,000 generations (standard deviation of split
Table 2
Sequence and alignment characteristics, and statistics from maximum likelihood (ML)
analysis for trnQ-rps16 intergenic spacer and ITS. Percentage of parsimony –
informative characters referable to non-excluded characters; lengths and G + C
content calculated based on the sequences as present in the alignment without any
exclusions (aligned length). Sequence divergence values based on calculation of p
distances (dissimilarity distances). alpha, the alpha value of the gamma shape
parameter as inferred by ML calculations; SD, sequence divergence; avg., average; bp,
basepairs; No., number.
trnQ-rps16
No. of taxa (including outgroups) 70
Sequence length (avg.)
622–1126 bp
(1022 bp)
Aligned length
1345 bp
Non-excluded characters
1306 bp
Parsimony-informative
133 bp (10.18%)
characters
Average G + C content
26.65%
Min – max SD Outgroup–
4.07–16.98%
Ingroup
Min – max SD Ingroup
0.00–3.52%
ML tree score
4512.522
ML tree length
0.626
Alpha
1.173
ITS
72
506–587 bp
(556 bp)
617 bp
591 bp
129 bp (21.83%)
61.40%
5.06–19.40%
0.00–5.60%
3554.120
2.145
0.371
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A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
frequencies 0.004 and 0.003, respectively). Log-likelihood curves,
acceptance rates, chain swap frequencies and potential scale
reduction factors suggested effective mixing and stationarity of
the chains. The majority-rule consensus trees for the single marker
datasets are shown in Fig. 1. Information about likelihoods and tree
lengths in ML analyses is given in Table 2; the alpha parameter was
estimated at 1.173 for the chloroplast DNA partition and 17.947 for
the chloroplast binary indel data partition, and at 0.371 for the nuclear dataset, while the proportion of invariant characters was
0.135. Results obtained by single marker ML analyses were consistent with those from Bayesian phylogenetic inference, so the
Bayesian majority consensus trees are provided with both Bayesian
posterior probabilities (above) as well as ML bootstrap supports
(below) where either PP P 0.85 or BS P 75. Nodes supported in
ML but absent in the majority-rule BI tree were added, applying
a 50% BS threshold for displaying branches.
Large parts of the chloroplast and nuclear tree topologies are
incongruent; however, there is support for a clade (cp PP: 0.93,
BS: 50/nr PP: 1.00, BS: 81) containing the majority of the Iberian
as well as the Macaronesian species (‘‘Iberian Peninsula – Macaronesia’’ = ‘‘IPM’’ clade, Fig. 1). Within the IPM clade, three major
subclades can be distinguished: one includes S. scorodonia L.
(‘‘Scorodonia’’ clade) and is highly to weakly supported by both
analyses (cp PP: 0.98, BS: 62/nr PP: 1.00, BS: 87). Another comprises several species alongside S. auriculata (‘‘Auriculata’’ clade)
and receives high support (cp PP: 1.00, BS: 99/nr PP: 1.00, BS:
96). However, while these two clades as a whole are supported
by cp and nr analyses, their composition is slightly different among
the datasets: three taxa remain unresolved in trnQ-rps16 (S. tanacetifolia, S. grandiflora, S. trifoliata) which are part of the Auriculata or
Scorodonia clades in ITS. A third subclade forming a largely unresolved polytomy contains all but one of the Macaronesian perennial endemics (‘‘Macaronesia’’ clade; does not contain the
Madeiran S. racemosa Lowe) and is sufficiently supported by the
chloroplast tree only (PP: 1.00, BS: 76). Relationships within as
well as among the Scorodonia, Auriculata and Macaronesia clades
are only poorly resolved; the Auriculata clade is subtended by an
exceptionally long branch in both analyses, while the Scorodonia
clade features a long branch in ITS only (see Supplementary
Fig. S2). Altogether, the IPM clade comprises 68% of all Iberian/
Macaronesian species and 13 of the 16 species endemic to the
two regions. Apart from the IPM clade, two smaller clades were
identified by both analyses: a ‘‘Nodosa’’ clade containing the two
accessions of the holarctic S. nodosa as well as the Iberian endemic
S. bourgaeana Lange (cp PP: 0.95, BS: 85/nr PP: 0.99, BS: 79), and an
‘‘Arguta’’ clade consisting of the three accessions of the mainly
Northern African and Southwestern Asian S. arguta and the Madeiran endemic S. lowei Dalgaard (cp PP: 1.00, BS: 99/nr PP: 0.99, BS:
96). From the species included with more than one accession, only
S. nodosa is revealed as monophyletic in both analyses (cp PP: 0.98,
BS: 90/nr PP: 0.81, BS: 79).
3.3. Phylogenetic Incongruence
While several major clades identified in the nuclear phylogeny
are also present in the cp tree topology, large degrees of incongruence are indicated between the two markers, on the level of single
accessions as well as whole clades regarding their composition and
relationships. Focusing only on cases of well-supported, hard
incongruence (P70% BS/P0.95 PP as defined above), this is true
for instance for the species of the ‘‘Canina group’’ (S. canina L.
and other species from subsection Lucidae Stiefelhagen) and S. lucida L.: while in the chloroplast phylogeny they are part of two separate clades with moderate support (Fig. 1, cp tree, ‘‘Canina 1’’, PP:
0.93, BS: 53; ‘‘Canina 2’’, PP: 1.00, BS: 94) and S. lucida remains
unresolved, they are merged into one well supported clade in the
nuclear tree (Fig. 1, nr tree, ‘‘Canina’’, PP: 0.96, BS: 83), with S. lucida highly supported as sister, and with no trace of subclades corresponding to the clades of the cp phylogeny. Another example is the
IPM clade, which is sister to S. scopolii Hoppe ex Pers. in the ITS
phylogeny (Fig. 1, nr tree; PP: 0.99, BS: 64), but to the Canina 2
clade and S. lucida in trnQ-rps16 (Fig. 1, cp tree; PP: 0.98, BS: 67).
Within the clades, considerable amounts of hard incongruence
can be found as well (see Table 3 for detailed information on support values): regarding the Canina group, S. frutescens L. is sister to
S. crithmifolia Boiss. in the cp tree, but sister to S. canina in the nr
tree; S. deserti Delile and S. syriaca from Tunisia occupy incongruent positions as well. The Scorodonia and Auriculata clades display
incongruence ‘‘vice versa’’ regarding six of their species: Scrophularia valdesii Ortega Oliv. & Devesa, S. herminii Hoffmanns. & Link,
S. oxyrhyncha Coincy, S. reuteri Daveau and S. sublyrata Brot. (referred to as ‘‘S/A taxa’’) are part of the Scorodonia clade in ITS,
but belong to the Auriculata clade in trnQ-rps16. The same is true
for the two accessions of S. viciosoi Ortega Oliv. & Devesa (‘‘A/S taxon’’), but in the opposite way, as they are part of the Auriculata
clade in ITS and the Scorodonia clade in trnQ-rps16. Single taxa
with hard incongruent placements also include S. umbrosa Dumort.
(part of the IPM clade in ITS, sister to S. scopolii and S. alpestris J.Gay
ex Benth. in trnQ-rps16), S. alpestris (part of the Nodosa clade in ITS,
sister to S. scopolii in trnQ-rps16), and S. scopolii and S. scopolii var.
grandidentata (Ten.) Boiss. (sister to the IPM clade in ITS, sister to S.
alpestris in trnQ-rps16).
As expected, the ILD test revealed severe incongruence within
the complete dataset (p = 0.001); the test with all incongruent
clades and single accessions mentioned above removed (51 accessions altogether) resulted in p = 0.445. However, when those accessions of the IPM clade which are congruent within the clade were
re-included (28 accessions), the result remained insignificant
Table 3
Bayesian posterior probabilities (PP), ML bootstrap support values (BS) and ILD test
results for four groups and four single species displaying hard incongruence among
the chloroplast (cp) and nuclear (nr) markers. Accessions/clades were added to a
pruned, congruent matrix (p = 0.144; see Sections 2.4. and 3.3.); accessions of S.
scopolii were only tested together. Asterisks indicate significance at the p = 0.05 level,
accessions with insignificant results are shown in bold. No separate support values
are given for accessions unresolved or weakly resolved within their respective clade/
group. Explanation of ‘‘S/A taxa’’ and ‘‘A/S taxon’’ see Section 3.3.; hyp., hypericifolia;
ramos., ssp. ramosissima, other abbreviations according to Table 1.
Group/accession
cp PP/BS
nr PP/BS
P value
S/A taxa
S. sublyrata
S. reuteri
S. oxyrhyncha
S. valdesii
S. herminii (za)
S. herminii (na)
1.00/99
–
–
–
–
0.66/80
0.66/80
1.00/87
–
–
–
–
–
–
0.001⁄
0.003⁄
0.001⁄
0.001⁄
0.004⁄
0.001⁄
0.002⁄
A/S taxon
S. viciosoi (tor)
S. viciosoi (ant)
0.98/62
1.00/86
–
1.00/96
–
–
0.027⁄
0.028⁄
0.027⁄
Canina 1
S. libanotica
S. deserti
S. xanthoglossa
S. syriaca ‘‘hyp.’’
S. syriaca (isr)
S. syriaca (tun)
1.00/97
–
0.99/66
0.97/70
0.97/70
0.99/66
1.00/91
0.96/83
0.64/75
0.95/90
0.95/90
0.64/75
1.00/100
1.00/100
0.008⁄
0.040⁄
0.073
0.025⁄
0.019⁄
0.070
0.008⁄
Canina 2
S. canina ramos.
S. canina
S. frutescens
S. crithmifolia
S. scopolii (x2)
S. alpestris
S. umbrosa
S. lucida
0.98/67
–
1.00/94
0.96/97
0.96/97
0.96/67
0.96/67
1.00/100
0.98/67
0.96/83
0.64/75
0.95/61
0.95/61
0.64/75
0.99/64
0.99/79
1.00/81
1.00/94
0.018⁄
0.037⁄
0.033⁄
0.009⁄
0.025⁄
0.135
0.051
0.063
0.134
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A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
(p = 0.144), suggesting that the incongruence observed regarding
the IPM clade did concern its sister groups (S. scopolii and the Canina group with S. lucida, respectively) rather than the clade itself.
Therefore, further tests were conducted using this ‘‘congruent
dataset’’ (p = 0.144) with only the remaining 23 hard incongruence
accessions removed. Results for all clades and accessions tentatively re-included into the dataset are shown in Table 3. Seven
accessions did not render the dataset incongruent when re-added;
of these, all possible pairs were tested to find suitable combinations (results not shown). The final ‘‘reduced dataset’’ was chosen
to comprise all taxa from the congruent dataset plus both accessions of S. scopolii, thereby including as much data as possible
while keeping congruence of the dataset as large as possible
(p = 0.135). The remaining 21 excluded accessions (=conflicting
taxa; see Table 3) represent app. 1/3 of the ingroup. The results
of the SLP test are shown in Table 4. Unlike ITS, the trnQ-rps16
dataset rejected most of the foreign topologies, in particular
regarding all hard incongruence taxa but also several partly unresolved/weakly supported positions, which suggests that the latter
contain explicit information and are not the mere outcome of
insufficient data.
Within the FSN constructed to visualize conflicting signals between and within markers (Fig. 2A), clades are represented in a
tree-like way where single marker trees are fully congruent (e.g.,
Arguta and Nodosa clades). However, the structure of most groups
is highly networked, and their relationships among each other are
entangled. The Scorodonia and Auriculata clades within the IPM
clade are connected by bundles of parallel edges (which indicate
different signals within the data) representing the incongruent
positions of the S/A and A/S taxa switching between clades in
trnQ-rps16 and ITS. The Macaronesia clade is situated in between
both clades, while the IPM clade itself is closely connected to S.
umbrosa (Fig. 2A, ‘‘5’’). Scrophularia scopolii (‘‘3’’) represents a connection between the IPM clade and the remainder of the sampling.
When the taxa within the FSN are diminished to those of the reduced dataset as defined above (Fig. 2B), the number of parallel
edges decreases substantially, leaving only few reticulations. Thus,
a combined analysis with conflicting taxa duplicated can be assumed to produce largely reliable results not hampered by major
incongruence issues.
3.4. Plastid haplotype inference
The haplotype network analysis revealed 18 distinct haplotypes, connected by 20 missing intermediate (unsampled) haplotypes, and with no more than seven inferred mutational changes
A
„Nodosa“
0.001
Outgroup
6
5
„Arguta“
3
„Macaronesia“
2
„Canina 1“
1
4
„Scorodonia“
„Canina 2“
„Auriculata“
„Nodosa“
B
0.001
Outgroup
„Arguta“
3
2
„Macaronesia“
1
„Scorodonia“
„Auriculata“
Fig. 2. Filtered super networks (split networks), based on each 1501 trees from the
posterior distribution from two single marker Bayesian analyses (chloroplast trnQrps16, nuclear ITS) which yielded the consensus trees shown in Fig. 1. Scale bars
represent treesize weighted mean edge weights. Major clades according to Fig. 1 are
indicated in grey. Composition of the network based on (A) all 72 accessions
included in the study (with seven outgroup taxa reduced to a single edge in the
graphics), and (B) 21 conflicting accessions (explanation see Section 2.4.), marked
by black dots on the edge tips in (A), removed using the ‘‘exclude selected taxa’’
option. 1, S. pyrenaica; 2, S. macrorrhyncha; 3, S. scopolii and S. scopolii var.
grandidentata; 4, S. lucida; 5, S. umbrosa; 6, S. alpestris.
to connect sampled haplotypes. Six haplotypes are shared among
up to eight accessions, and among one to five species, respectively.
Table 4
Results of SLP tests for hard incongruence taxa and partly unresolved groups/species (see Section 2.4.). Tests on chloroplast (cp, left side) and nuclear (nr, right side) dataset using
11 constrained topologies as provided in Supplementary Fig. S1. Length of most parsimonious tree(s), number of trees saved during heuristic search and minimum/maximum SLP
test P values are given for each marker and constraint. Asterisks indicate significance at the p = 0.05 level, ° marks cases where only one value was significant. Constraints
regarding the phylogenetic position of: 1, five ‘‘S/A taxa’’, explanation see Section 3.3.; 2, one ‘‘A/S taxon’’; 3, the Canina/Canina 1/Canina 2 clades and S. lucida; 4, S. scopolii; 5, S.
alpestris; 6, S. umbrosa; 7, the Auriculata and Macaronesia clades; 8, S. ningpoensis, S. yoshimurae, S. urticifolia and S. amplexicaulis; 9, S. grandiflora; 10, S. trifoliata; 11, S.
tanacetifolia. No., number.
Constraint
Length (cp)
No. trees
P value (nr)
Length (nr)
No. trees
P value (cp)
None
1
2
3
4
5
6
7
8
9
10
11
499
509
509
500
519
511
518
507
509
507
508
501
5000
5000
5000
5000
5000
5000
5000
5000
5000
5000
5000
4800
–
0.0016/0.0184⁄
0.0016/0.0184⁄
0.3173/0.7389
<0.0001/0.0002⁄
0.0005/0.0047⁄
<0.0001/0.0003⁄
0.0047/0.0455°
0.0075/0.0124⁄
0.0047/0.0455°
0.0027/0.0290⁄
0.1573/0.5271
544
557
557
559
549
548
551
546
550
547
547
545
3800
3400
3800
2800
4400
3800
3900
3500
5000
3600
3600
4100
–
0.0003/0.0046⁄
0.0003/0.0073⁄
0.0001/0.0011⁄
0.0956/0.2818
0.1573/0.3785
0.0348/0.1488
0.1573/0.5637
0.1533/0.3692
0.0833/0.4054
0.0833/0.4054
0.3173/0.7630
A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
In one case, haplotypes are shared among present-day allopatric
species. Of the eight species represented by more than one accession, five feature two different haplotypes. The three groups in
the network correspond to the Scorodonia, Auriculata and Macaronesia clades in the chloroplast tree (Fig. 1). Groups are separated by
at least four mutational changes, while haplotypes within groups
differ by two mutations at most. The ‘‘biggest outgroup probability’’ according to TCS, i.e., the most likely ancestral haplotype
was found in eight accessions/four species of the Macaronesia
clade.
3.5. Biogeographic reconstruction
The two runs from the Bayesian analysis of the duplicated dataset had converged after 10,000,000 generations (standard deviation of split frequencies 0.006). The best-scoring ML tree (results
not shown) largely corroborated the Bayesian majority-rule consensus. The latter mostly reproduced the relationships of the single
marker analyses, however, with reduced resolution and lower support values. Results from the RASP analyses as plotted onto the
Bayesian consensus are shown in Fig. 4. Exact marginal probabilities for each ancestral range and Bayesian posterior probabilities
for tree nodes are available from Supplementary Table S2.
The different RASP runs mostly yielded congruent results as
well; one node (node 17) with almost identical marginal probabilities for two ancestral distributions, shifted in the different runs
between including or excluding Western North Africa from the
most frequent distribution. Inferred ancestral ranges had low marginal probabilities in some, especially more basal nodes; these results should therefore be treated with caution.
4. Discussion
4.1. Reticulate evolution within Scrophularia
The Nodosa, Arguta and IPM clades exclusively consist of species from section Anastomosantes subsection Scorodoniae sensu
Stiefelhagen (= section Scrophularia subsection Scrophularia).
Species of section Tomiophyllum subsection Lucidae sensu
Stiefelhagen (or section Canina sensu G.Don) are restricted to the
Canina clade. This is in accordance with Navarro-Pérez et al.
(2013) who found a clade of semi-shrubby, sparsely foliate plants
of section Canina embedded within section Scrophularia comprising mostly herbaceous representatives with numerous and often
large leaves featuring clearly anastomosing nerves.
The species of the IPM clade (Fig. 1) as presented here can be
characterized as subshrubs or perennial (biennial) herbs, with
undivided to 3-pinnatisect, lanceolate to suborbicular leaves. A
scarious margin, more or less distinct, is always present on the calyx lobes. The corolla is usually indistinctly bicolored, with the posterior part showing predominantly purple or brownish tones,
while the anterior is greenish, brown, yellowish or reddish in color.
The staminode is suborbicular, obovate, reniform, or transversely
elliptical in shape and of considerable variability. The globose,
ovoid, or subconical capsule is often apiculate, or the base of the
style is persisting as mucro on mature capsules. However, none
of these morphological characters can be regarded as synapomorphic for the clade. Similarly, the monophyly of the IPM clade (with
or without S. umbrosa depending on the marker) is supported by
only two nucleotide synapomorphies in the ITS sequence alignment and one in trnQ-rps16. In constrast, the Auriculata and Scorodonia clades mostly receive high supports in phylogenetic
analyses, but also lack clear morphological synapomorphies. This
is best explained by the comparatively young age of the IPM clade
(inferred at around 3.7my by Navarro-Pérez et al., 2013) and is also
305
reflected in the weakly resolved relationships among and within
the IPM clade, as well as low levels of sequence divergence with
several cases of identical sequences (see Supplementary Table S1).
As a consequence, especially regarding the high frequency of
hybridization and polyploidy within the group, reticulate events
among the closely related species studied here are to be expected,
and the large amount of incongruence found between trees from nuclear and plastid markers corroborates this assumption (see Fig. 2).
Similar examples within the Lamiales are known from e.g. Plantaginaceae (Albach and Chase, 2004; Blanco-Pastor et al., 2012) or Lamiaceae (Bräuchler et al., 2010), among others. Reticulation can be due to
e.g. hybridization (Arnold, 1997; Rieseberg et al., 1996), introgression (Mason-Gamer, 2004; Rieseberg and Wendel, 1993) or incomplete lineage sorting (persistence of ancient polymorphism/deep
coalescence; Degnan and Rosenberg, 2009; Maddison, 1997), all of
which have frequently been considered as explanation for cases of
well-supported topological incongruence among phylogenetic trees
(hybridization: e.g. Albaladejo et al., 2005; Doyle et al., 2003; Nieto
Feliner et al., 2002; see further references in Vriesendorp and Bakker,
2005; lineage sorting: e.g. Jakob and Blattner, 2006; Vilatersana et al.,
2010). However, artificial factors can cause topological incongruence
as well (Wendel and Doyle, 1998); such factors were excluded as far
as possible in the present study. Sampling error was avoided by redetermination of nearly all accessions; species coverage is close to
100% for the main study regions, avoiding insufficient taxon sampling (Stockley et al., 2005). Methodical mistakes caused by model
misspecification were ruled out by performing all analyses with
two different methods. Furthermore, only robustly supported cases
of incongruence were taken into account. Undetected ITS pseudogenes can also introduce topological incongruence into datasets
(Álvarez and Wendel, 2003); however, no signs of sampled non-functional nrDNA copies were found in the sequences. Some sister groups
with long branches in the basal (and sparsely sampled) part of the
trees (see Supplementary Fig. S2) could possibly be the result of long
branch attraction (Felsenstein, 1978), so no implications are made
regarding these taxa.
In cases where incongruence reflects some kind of reticulate
evolutionary history, forcing conflicting signals into one phylogenetic tree might blur real relationships (Bull et al., 1993; Lecointre
and Deleporte, 2005); on the other hand, pruning hard incongruent
taxa from a combined analysis (Huelsenbeck et al., 1996; Johnson
and Soltis, 1998) will disregard much of the available data, and
valuable information about possible parent species will be lost in
cases where hybridization is frequent (Rieseberg and Brunsfield,
1992; see examples in Albaladejo et al., 2005; Fehrer et al., 2007;
Okuyama et al., 2005; Soltis and Kuzoff, 1995). The duplication approach (Pirie et al., 2009, 2008) as used in this study provides a
suitable solution to this problem.
In the case of Scrophularia as sampled here, the ITS phylogeny
generally seems to better reflect the relationships known from
morphological studies (e.g. Bentham, 1846; Ortega Olivencia,
2009; Stiefelhagen, 1910). Single accessions of the same species
are more often retrieved as monophyletic (S. scopolii, S. arguta, S.
nodosa). It has already been recognized that in many cases, the
ITS topology is congruent with phylogenetic hypotheses established from morphological or biogeographical data (Baldwin
et al., 1995; Fehrer et al., 2007; Kellogg et al., 1996). However, species – independent geographical structuring as described by Wolf
et al. (1997) is hardly if at all present in the trnQ-rps16 tree. Therefore, we chose to refer to the ITS topology (or the duplicated topology, Fig. 4) when inferring species relationships, and to draw
additional information from the chloroplast tree where useful.
Although detailed differentiation between evolutionary mechanisms lies beyond the scope of this study, parts of the reticulation
observed here are very likely to be the result of hybridization. As an
example, six species within the IPM clade display hard
306
A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
incongruence, changing positions between the Auriculata and the
Scorodonia clades in ITS/trnQ-rps16 (S/A and A/S taxa). Results
from Navarro-Pérez et al. (2013) match this observation in at least
two cases; three of the other species remain unresolved in the plastid tree, which is probably due to the choice of a different marker
(trnL-trnF) providing weaker resolution. In contrast, the incongruence regarding the sixth of the species, S. herminii, as found here
clearly contradicts results by Navarro-Pérez et al. (2013); unless
more specimens are analyzed, we refrain from making any conclusions regarding this species here. The evolutionary split between
the Auriculata and Scorodonia clades has been shown to be rather
young (mid-Pliocene; Navarro-Pérez et al., 2013); consequently,
lineage sorting is a possible explanation for the observed pattern
(Maddison, 1997). On the other hand, if incongruent placements
in the ITS tree resulted from sorting of ancestral polymorphisms
in the IPM clade ancestor, one would expect, from the stochastic
nature of the process, that accessions appeared at any position in
the IPM clade (Buckley et al., 2006). Here, the species change positions between two clades exclusively. Furthermore, both clades
are highly supported and well separated (Figs. 1 and 3, also see
Navarro-Pérez et al., 2013) which makes lineage sorting a less
likely cause for the incongruence (Morgan et al., 2009). Introgression by geographically close populations of the other clade cannot
be excluded due to the lack of populational sampling; however, the
‘‘Sambucifolia’’ haplotype (Fig. 3) encloses species from very different geographic regions, which precludes introgression as the only
cause for haplotype sharing and suggests common ancestry of
the respective species (Gutiérrez Larena et al., 2002). On the other
hand, viable hybrids between S. scorodonia and S. auriculata have
already been created by Grau (1976) and Dalgaard (1979), and
the natural hybrid S. moniziana Menezes was shown to be derived from S. scorodonia and S. racemosa (belonging to the Auriculata clade; Fig. 1). Therefore, it seems more likely that S. sublyrata,
S. reuteri, S. oxyrhyncha, S. valdesii and S. viciosoi have originated
through homoploid hybrid speciation (Mallet, 2007), with ancestors or members of the Scorodonia clade being the female parent
in S. viciosoi, and ancestors/members of the Auriculata clade acting
smith(tag)
smith(1)
smith(cha)
calliantha
lang(ag) hirta(r)
lang(erj) hirta(1)
hirta(2)
lang(sil)
glab(ten)
as female donor in the remaining cases, contributing the maternally inherited plastid.
Morphologically, S. sublyrata shares characters with S. sambucifolia (Richardson, 1972), and S. reuteri is similar to S. sambucifolia
(Daveau, 1892) and to some extent also S. sublyrata (Grau, 1976).
This corresponds to the identical haplotype found in the three species (Fig. 3). Morphological similarities are also present between S.
oxyrhyncha and S. sublyrata (Coincy, 1898; Stiefelhagen, 1910). Furthermore, S. oxyrhyncha is connected to S. reuteri by a distinctive
long - subconical capsule (Grau, 1976); both are local endemics of
Western Spain. Scrophularia valdesii is a threatened narrow endemic known from only 14 populations occurring in the Duero Basin
in Spain and Portugal (Bernardos et al., 2006). It shares the haplotype found in S. sambucifolia, S. sublyrata and S. reuteri (Fig. 3) and
is closely related to the latter morphologically (Ortega Olivencia
and Devesa Alcaraz, 1991). Scrophularia viciosoi is the only hybrid
with its paternal source found in (ancestors of) the Auriculata clade.
Ortega Olivencia and Devesa Alcaraz (1991) relate the species to S.
grandiflora, a local endemic of the Coimbra region in Portugal; indeed, the ITS sequence of S. viciosoi from Antequera is identical to
that of S. grandiflora (Supplementary Table S1). Morphological
similarities include the densely pubescent – glandular, pinnatisect
leaves possessing many small intercalars, and the subsessile
peduncles (Ortega Olivencia and Devesa Alcaraz, 1991).
4.2. Chromosome number evolution, origins of polyploidy and
ancestral hybridization within Iberian Scrophularia
Hybridization and polyploidization are considered as driving
forces in the diversification history of the genus Scrophularia, e.g.
in the high level polyploids occurring in North America (Carlbom,
1969; Scheunert and Heubl, 2011; Shaw, 1962); they also play an
important role in plant evolution and speciation in general (Hegarty and Hiscock, 2005; Leitch and Leitch, 2008; Otto and Whitton, 2000; Schubert, 2007). Besides the evidence for homoploid
hybrid speciation as discussed above, our phylogenetic reconstructions support allopolyploid hybridization in several cases.
„Macaronesia“
glab(pal)
scor(mad)
scor(ten)
laxi(bar)
vicio(ant)
„Scorodonia“
laxi(alg)
macro
trifoliata
grandifl
pyren
vicio(tor)
tanacet
„Auriculata“
oxyrhyn
hermi(na)
hermi(za)
hispida
sambucifolia
sublyrata
reuteri
valdesii
racemosa
auri“balb“
auriculata
eriocalyx
lyrata
Fig. 3. Statistical parsimony network obtained from analysis of the trnQ-rps16 intergenic spacer, limited to taxa from the IPM clade. Lines represent single mutational steps,
small circles represent inferred haplotypes. The size of boxes is relative to the number of accessions possessing the respective haplotype; the oval represents the most likely
ancestral haplotype as inferred by TCS. Major plastid lineages indicated by grey boxes correspond to clades in Fig. 1. Solid dots (Iberian Peninsula) and triangles (Macaronesia)
indicate the distribution of the respective (sub)species. For complete taxon names and other abbreviations see Table 1.
A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
Scrophularia alpestris, distributed in montaneous regions of Southern France and Northern Spain, with a chromosome number of
2n = 68 (Grau, 1976), is sister to S. scopolii (2n = 26; Grau, 1976)
in the trnQ-rps16 phylogeny and, according to ML estimations, sister to S. bourgaeana (2n = 42; Ortega Olivencia and Devesa Alcaraz,
1990) in the ITS topology. Regarding the long branches of S. alpestris and S. bourgaeana in ITS, their sister relationship could theoretically be a result of long branch attraction (Supplementary Fig. S2).
However, S. alpestris was already proposed to be an allopolyploid
(with S. scopolii and S. bourgaeana as progenitors) by Grau (1976)
and Ortega Olivencia and Devesa Alcaraz (1990). This hypothesis
is corroborated by morphology and by molecular phylogenetic
reconstructions as presented here.
A similar case seems to be apparent in S. auriculata which typically possesses 2n = 84 (Grau, 1976) chromosomes. Grau (1979)
suggested the species to result from allopolyploid hybridization
between (ancestors of) S. lyrata Willd. (2n = 58; Grau, 1976) and
S. umbrosa (2n = 26, 52; Vaarama and Hiirsalmi, 1967). This is supported by intermediate morphological traits connecting S. auriculata to its putative parents; e.g., bracts and bracteoles in S. lyrata are
scariously margined across their whole length as opposed to the
non-margined S. umbrosa, while the bracts of S. auriculata have
no or narrow margins generally confined to the tip of the leaf. Furthermore, the staminode is reniform to bilobed in S. umbrosa, obovate to suborbicular in S. lyrata, and subreniform in S. auriculata.
A close relationship of S. auriculata to S. lyrata is evident from the
ITS phylogeny in one of the two accessions only (Fig. 1); connections to S. umbrosa are present in neither plastid nor nuclear trees.
Possibly, this unexpected result is due to fixation of the maternal
ITS copy in the hybrid species through concerted evolution (Álvarez
and Wendel, 2003), an event that would leave no trace of a hybrid
origin as long as no additional markers are included (see e.g. Blöch
et al., 2009; Joly et al., 2006; Sang et al., 1997).
The second accession, S. auriculata ‘‘balbisii’’ (originally determined as S. balbisii Hornem, a name synonymized with S. auriculata
L. ssp. auriculata by Ortega Olivencia, 2009), does not cluster with
the first specimen, but instead is sister to the Algerian - Moroccan
endemic S. hispida (Fig. 1). This species is morphologically similar
to S. lyrata and has the same chromosome number (2n = 58; Grau,
1976). With respect to the considerable variability found within S.
auriculata (visible in e.g. S. auriculata ssp. valentina with lyrate-pinnatisect leaves, as well as several synonyms listed in Ortega Olivencia, 2009), and the great potential for hybridization, a definite
conclusion about the phylogenetic position of S. auriculata does
not seem advisable based on two inconsistently placed specimens.
However, an involvement of S. hispida in the origin of S. auriculata
should be considered, especially with regard to the third taxon in
the respective ITS clade, the Madeiran endemic S. racemosa. This
species has been related to S. auriculata and also possesses
2n = 84 chromosomes (Dalgaard, 1979); its position is clearly separated from the remaining Macaronesian perennial endemics
which are part of the Macaronesia clade.
Disregarding hybrid species with higher chromosome numbers
(S. auriculata, S. racemosa) as described above, the IPM clade is
characterized by the derived chromosome number 2n = 58 (with
aneuploidy in some taxa, e.g. S. sublyrata, S. glabrata Aiton) as already pointed out by Grau (1976). The number seems to be exclusive for the species of the IPM clade; this points toward a single
evolutionary event, which generated a presumably allopolyploid
ancestor with that particular chromosome number. Some suggestions have been made regarding its origin, but no concrete evidence was provided: Ortega Olivencia and Devesa Alcaraz (1990)
hypothesized an allopolyploid taxon with 2n = 60 derived from
progenitors with 2n = 36 (as present in S. nodosa and S. peregrina;
Grau, 1976; Vaarama and Hiirsalmi, 1967) and 2n = 24 (as present
in S. crithmifolia and occasionally found in S. canina; Ortega
307
Olivencia and Devesa Alcaraz, 1990), with subsequent chromosome number reduction to 2n = 58. Based on the sister relationships as present in the nuclear and plastid phylogeny, our
molecular data support an allopolyploid origin, by ancient hybridization involving an ancestor from the Canina group (possibly with
2n = 30 chromosomes as occasionally found in S. canina; Ortega
Olivencia and Devesa Alcaraz, 1990) as maternal parent. Interestingly, almost all species of the Canina group themselves yield
highly significant ILD test results (Table 3), and their cp and nr sequences occupy different positions in the duplicated tree (Fig. 4;
Canina clade vs. Canina 1 and 2 clades). This could be explained
by assuming reticulation present in their origin as well, possibly
involving groups not sampled for this study. Regarding the paternal source, a contribution by a taxon with 2n = 36 chromosomes
as proposed by Ortega Olivencia and Devesa Alcaraz (1990) is
not supported; instead, an S. umbrosa – like species (2n = 26) is
suggested by the duplicated tree (Fig. 4), as S. umbrosa is placed
in a polytomy with the Canina clade and S. lucida with all being sister to the IPM clade. Scrophularia umbrosa was already mentioned
as potentially involved in the origin of the group; it does not occur
in the Iberian Peninsula today (westernmost populations reach
Norway, the British Isles, and France), but could easily have done
so in the past according to Grau (1976). A natural hybrid between
S. auriculata and S. umbrosa (sub S. alata Gilib.) was already noted
by Stiefelhagen (1910); this confirms the close association of the
species. Surprisingly, when consulting the ITS tree, S. scopolii is sister to the IPM clade and S. umbrosa is nested within the latter.
However, the position of S. umbrosa is characterized by a long
branch in the phylogram (Supplementary Fig. S2); the substitutions shared with the rest of the IPM clade species could thus be
homoplasious. The low alpha value of the gamma shape parameter
in the ITS dataset (Table 2) supports this view. If we finally consider the fact that the chromosome number of S. umbrosa with
2n = 26 would be unique within the IPM clade, we can conclude
that this position as a member of, and not sister to, the IPM clade
is likely artificial.
If we assume an S. canina – like (2n = 30) and an S. umbrosa –
like species (2n = 26) as progenitors for the IPM clade ancestor
(Fig. 4), and that subsequent chromosome doubling was necessary
to enable fertility of the new hybrid, the resulting allopolyploid
should have had 2n = 56 chromosomes. Ascending aneuploidy
could then have resulted in the present-day 2n = 58 for the group;
the latter process was also proposed to account for the deviant
chromosome number of S. viciosoi, counted with 2n = 58 as well
as 2n = 64 (Grau, 1976, sub S. sublyrata; Ortega Olivencia and Devesa Alcaraz, 1990).
4.3. Biogeographic implications
The primary diversity center of the genus Scrophularia is assumed in the Irano - Turanian region (Grau, 1981; Lall and Mill,
1978). Most of the species studied here are part of a secondary center of diversity located in the Iberian Peninsula (Ortega Olivencia,
2009; Ortega Olivencia and Devesa Alcaraz, 1990). According to
ancestral area reconstructions as performed by RASP, the most recent common ancestor (MRCA) of the IPM clade was distributed in
the Western Mediterranean, however with low Bayesian support
for the underlying node (Fig. 4, node 31; for node supports and exact frequencies of occurrence see Supplementary Table S2).
Regarding the progenitors of the IPM clade as discussed above,
RASP inferred that the ancestor of the Canina group was distributed in a region ranging from Eastern Africa, Israel, Lebanon and
Syria to Southwestern Asia, the Caucasus and Turkey (node 18,
27). However, the marginal probabilities for the inferred range
are low (42.19, 43.23); a reconstruction with possible areas at each
node restricted to two, narrows the most frequent ancestral range
308
A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
A
B
Fig. 4. (A) Biogeographical optimization as performed by RASP (maxareas = 5), using the majority-rule consensus of 9002 trees from a duplicated Bayesian analysis of the ITS
region and the plastid trnQ-rps16 intergenic spacer. Conflicting taxa duplicated for the analysis are highlighted in bold and suffixed with ‘‘nr’’ and ‘‘cp’’, for the nuclear and
chloroplast sequences, respectively (see Section 2.5.). Branches with PP < 0.85 in the consensus tree are shown by dashed lines, branches with PP P 0.95 in bold. Outgroups
were reduced to one branch in the diagram, clade names correspond to those in Fig. 1. Pie charts illustrate inferred distributions of MRCAs from one of five RASP runs (only
ranges supported by all five runs are shown). Color – coded fractions represent the frequency of occurrence/marginal probability (P25) of the respective ancestral
distribution over the Bayesian sample of trees. Asterisks mark nodes where no ancestral distribution reached a marginal probability of P25 in all five runs. Areas additionally
supported by an optimization with maxareas set to two are underlined. Contemporary distribution and known chromosome numbers are denoted next to each taxon, ‘‘?’’
indicate cases where counts yielded inconsistent results; *: known autopolyploidy (2n = 26, 52; Vaarama and Hiirsalmi, 1967) within S. umbrosa. °: apart from 2n = 26,
occasionally counted numbers in S. canina also include 2n = 24 and 2n = 30 (Ortega Olivencia and Devesa Alcaraz, 1990; Vaarama and Leikas, 1970). 1: Dalgaard, 1979; 2: Ge
and Li, 1989; 3: Grau, 1976; 4: Ortega Olivencia and Devesa Alcaraz, 1990; 5: Vaarama and Hiirsalmi, 1967; 6: Murin and Sheikh, 1971; 7: Mohamed, 1997; 8: Ortega Olivencia
and Devesa Alcaraz, 1991. ramos., ramosissima; hyper., hypericifolia; gr., grandidentata; lang., langeana; other abbreviations according to Table 1. (B) Table and map showing
15 areas defined for ancestral area reconstructions, and color codes for inferred ancestral ranges.
A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
to Israel, Lebanon, Syria and Southwestern Asia with a higher frequency of occurrence (85.96, 58.58). According to the reconstructions, east–west migrations would then have expanded the
distribution range of the group to Western North Africa (nodes
14, 15, 17, and 24-26). The ancestral range inferred for the Canina
2 clade (chloroplast; node 30, marginal probability: 83.59) suggests that the ancestor of this part of the Canina group (here represented by S. canina, S. frutescens and S. crithmifolia) at some
point reached the Iberian Peninsula via the Strait of Gibraltar and
diversified in situ. This is supported by the ancestral areas inferred
for nodes 14, 28 and 29 with mostly sufficient marginal probabilities and Bayesian node supports. Similar biogeographical patterns
involving expansion from east to west have been recorded in several Mediterranean groups, e.g. in elements of the Spanish steppe
flora (Polunin and Smithies, 1973), in Asteraceae (Font et al.,
2009), Araceae (Mansion et al., 2008), Rutaceae (Salvo et al.,
2011), and in insects (Sanmartín, 2003).
For S. umbrosa, the second assumed progenitor of the IPM clade,
biogeographic reconstructions were ambiguous and marginal
probabilities insufficient; hypotheses about the biogeography of
this widespread species must remain speculative at this point. In
every case, the contact of an S. umbrosa ancestor with a taxon from
the Canina group in the Iberian Peninsula should have resulted in
the hybridization event generating the allopolyploid ancestor of
the IPM clade.
In the course of the diversification of the IPM clade, an early dispersal of S. macrorrhyncha (Humbert, Litard. & Maire) Ibn Tattou
into Northern Africa is suggested by its position within the clade.
This subshrub species is adapted to semi-arid conditions and is endemic to Morocco today. From the three main lineages derived
from the MRCA, one dispersed to Macaronesia (Macaronesia clade,
see Section 4.4. and Fig. 4, node 41); the Scorodonia clade underwent local radiation and mostly remained restricted to the Iberian
Peninsula (Fig. 4, nodes 42–44). In contrast to that, RASP reconstructions show that Northern Africa might have played a larger
role in the diversification of the Auriculata lineage (nodes 35, 36,
and 37). Both Scorodonia and Auriculata clades also contain more
widespread elements which have dispersed into Macaronesia, the
Eastern Mediterranean, and Europe (S. scorodonia, S. auriculata).
Diversification of Scrophularia in the Western Mediterranean
and especially the Iberian Peninsula as mentioned above is likely
to be, to a great extent, the result of repeated hybridization as discussed in Sections 4.1 and 4.2. In addition, there is evidence that
glacial refugia also played a role in promoting and preserving species diversity. Five taxa of the IPM clade remain unresolved in the
chloroplast tree (Fig. 1; S. pyrenaica Benth., S. macrorrhyncha, S.
grandiflora, S. trifoliata, S. tanacetifolia); their positions were shown
to contain distinct phylogenetic signal in two of three tested cases
(Table 4) and correspond to haplotypes which are isolated from the
remainder of the IPM clade by up to seven steps (Fig. 3). In the
duplicated tree (Fig. 4), these taxa are unresolved as well (S. pyrenaica, S. macrorrhyncha), are sister to the remainder of the Auriculata
clade (S. grandiflora, S. trifoliata) or part of the Scorodonia clade (S.
tanacetifolia). All except the latter species are restricted to only
small areas, and exclusively or predominantly inhabit regions classified as refugia within the Mediterranean bioclimatic region
(Médail and Diadema, 2009): the central Pre-Pyrenees and Pyrenees (S. pyrenaica; Ortega Olivencia, 2009), Sardinia and Corsica
(S. trifoliata; Gamisans and Marzocchi, 1996), Beira Litoral of Western Portugal (S. grandiflora; Ortega Olivencia, 2009), and the High,
Middle and Anti Atlas mountains of Morocco (S. macrorrhyncha; Ibn
Tattou, 2007). Given their distinctive genetic features and their
occurrence in refugia, these four species are likely to represent
more ancient lineages within the IPM clade which persisted in
the favorable conditions of the climatically stable refugial areas.
Long isolation in restricted regions likely accumulated the geno-
309
typic changes and accounts for the long branches in the phylograms
(Supplementary Fig. S2). Scrophularia tanacetifolia does not have a
restricted distribution, but is more widespread in the eastern and
southeastern parts of the Iberian Peninsula. Unlike in S. grandiflora
and S. trifoliata, the SLP test was insignificant for this species (Table 4), suggesting that its position in the chloroplast tree might also
be due to a lack of informative characters. However, it is sister to a
clade containing S. laxiflora and S. scorodonia in the combined analysis by Navarro-Pérez et al. (2013), which corroborates its isolated
position. It is also subtended by a long branch in the trnQ-rps16 phylogram (Supplementary Fig. S2), and, like S. macrorrhyncha, is characterized by 2–3 pinnatisect leaves resembling those of the Canina
group (confusions with S. crithmifolia were reported by Ortega Olivencia, 2009), a rather plesiomorphic character within the IPM clade.
Possibly, this species dispersed to its present distribution area in
Southeastern Spain from refugia located in the area.
Scrophularia species are distributed throughout nearly all regions of the Iberian Peninsula today, occurring from sea level up
to 2500 m. Hybrid species from the Scorodonia and Auriculata
clades (S. sublyrata, S. reuteri, S. oxyrhyncha, S. valdesii and S. viciosoi) are confined to granite or siliceous substrates; their distribution corresponds to a biogeographical pattern as shown by
Moreno Saiz et al. (2013), which divides the Peninsula into two distinct distributional areas characterized by different soil conditions.
Whether substrate characteristics influenced hybridization in this
area remains unclear; but furthermore, the present-day distributions of these species likely reflect the influence of the varied topography in the region, also in the context of the climatic conditions
during their formation. Divergence time estimations by NavarroPérez et al. (2013) suggest their very recent divergence in the Pleistocene; this is corroborated by identical haplotypes in three of five
cases (Fig. 3) and very low levels of nuclear sequence divergence
among each other and to closely related non-hybrid species
(Supplementary Table S1) as shown here. Possible parental taxa
and hybrid offspring are allopatrically distributed within the Peninsula in several cases; thus, it is conceivable that range shifts
in the parental lineages, promoted by climate fluctuations during
the Pleistocene (Hewitt, 2000), enabled hybridization in contact
zones. The narrow distributions of three of the hybrids, (including
one threatened species; Bernardos et al., 2006) indicate that geographic features might also have had special influence, by isolating
new species e.g. on the sierras of the Cordillera Central (S. reuteri)
and the Sierra Morena (S. oxyrhyncha). Isolation is regarded essential for survival of homoploid hybrids (Rieseberg and Willis, 2007)
as it prevents backcrossing with the already established lineages.
Likewise, the role of the sierras of Central Spain as refugia for plant
and animal species has been highlighted (Crochet et al., 2004;
Médail and Diadema, 2009). Isolation and range shifting in glacial
refugia have promoted speciation in several genera, amongst
others Erodium (Geraniaceae; Fiz-Palacios et al., 2010) and Armeria
(Plumbaginaceae; Gutiérrez Larena et al., 2002).
4.4. The origin of the Macaronesian taxa
Although our sampling of the Macaronesian taxa does not allow
detailed inferences on colonization pathways among the islands,
some general patterns are clearly supported by the present data
(Fig. 5). According to phylogenetic reconstructions, at least four
distinct lineages of Scrophularia have colonized the Macaronesian
archipelago; these roughly correspond to the three groups defined
by Dalgaard (1979). Multiple independent introductions into
Macaronesia have also been shown in e.g. Asteriscus (Asteraceae;
Goertzen et al., 2002), Ilex (Aquifoliaceae; Cuénoud et al., 2000),
Lavatera (Malvaceae; Fuertes-Aguilar et al., 2002), or Plantago
(Plantaginaceae; Rønsted et al., 2002), and have been reviewed in
Carine et al. (2004). Regarding Scrophularia, Madeira was colonized
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A. Scheunert, G. Heubl / Molecular Phylogenetics and Evolution 70 (2014) 296–313
at least four times (by members of the Scorodonia, Auriculata,
Macaronesia and Arguta clades, see Figs. 4 and 5). At least two dispersals to the Canary Islands can be assumed (by members of the
Arguta clade and the ancestor of the Macaronesia clade). In particular, the perennial endemics of the Canary Islands (S. smithii Hornem., S. glabrata, S. calliantha) are shown to be the result of a
dispersal event from the Western Mediterranean mainland to the
Atlantic islands (Fig. 4, node 32, marginal probability: 91.50). The
dated phylogeny by Navarro-Pérez et al. (2013) places the split between mainland taxa of the Scorodonia clade and Macaronesian
perennial endemics in the late Pliocene. Indeed, many species
and lineages of the Macaronesian islands have been shown to be
recently derived from continental ancestors, rather than being
relictual elements of the flora (Barber et al., 2002; Carine et al.,
2004, see also studies reviewed therein; Francisco-Ortega et al.,
1997; Helfgott et al., 2000); this also seems to be the case for Scrophularia. The ancestors of the Madeiran perennial endemic S. hirta
were inferred to have originated on the Canary Islands (Fig. 4, node
41, marginal probability: 71.76). Colonization routes from the Canary Islands to Madeira were also found in e.g. Sonchus (Asteraceae;
Lee et al., 2005), Bystropogon (Lamiaceae; Trusty et al., 2005) or
Micromeria (Lamiaceae; Meimberg et al., 2006). A second colonization event, from the Western Mediterranean or possibly also Western North Africa (Fig. 5), was inferred for the other perennial
endemic on Madeira, S. racemosa (Fig. 4, node 36, marginal probabilities: 58.20 and 35.06, respectively). Two of the species occurring in Macaronesia are more widespread (S. scorodonia, S.
auriculata); therefore, their biogeography should be examined
using more specimens from different parts of their distribution
range, and no conclusions are made here. The annual taxa occurring in Macaronesia today are part of a clade whose closest relatives remain unclear; accordingly, no informative ancestral
distributions could be inferred.
4.5. Conclusions and perspectives
This study provides an initial framework in understanding the
complex evolutionary history of Scrophularia lineages from the
Western Mediterranean, Northern Africa and Macaronesia.
Interspecific hybridization and polyploidization have significantly
influenced the diversification of the genus in this area and explain
the major incongruences found between nuclear and chloroplast
datasets. Hybrid speciation is favored by the pollination biology
of the genus and the absence of reproductive barriers among closely related taxa. The comparatively young age of the lineages
might explain the lack of resolution among Macaronesian and Iberian groups as well as single species (on the other hand, haplotypes
are not necessarily identical within species). This also indicates
that apart from hybridization, other reticulate processes like introgression and lineage sorting may have occurred among and within
species. The sampling and methods employed in the present study
are not intended for assessing these topics or for disclosing interisland colonization patterns across the Atlantic archipelagoes in
detail. A different approach involving an extensive geographic
and intraspecific population-level sampling and appropriate markers (e.g. SSR, ISSR, AFLP etc.), together with additional chromosome
counts, would help to further unravel the evolutionary history of
the genus.
Acknowledgments
The authors wish to thank the herbaria and curators of A, GH,
HU/HZU, M, MA, MJG, MSB, and W for leaf material and for permitting the examination of their specimens, and the Botanical Gardens
in Madeira, Erlangen and Madrid as well as Christian Bräuchler for
providing seed material from Scrophularia racemosa, S. lowei, S.
grandiflora and S. herminii. Cheng-xin Fu is acknowledged for making available leaf material from S. urticifolia and Ramón Morales for
leaf material from S. viciosoi and S. bourgaeana; Jürke Grau for providing seed material from S. auriculata ‘‘balbisii’’, Matthias Erben for
seed material from S. arguta from Lanzarote, S. smithii ssp. smithii
from Chamorga/Tenerife, S. scorodonia from Tenerife, and S. hirta
from Pico de Ruivo/Madeira. We thank Tanja Ernst for laboratory
assistance, Dirk Albach and Daniel H. Huson for helpful correspondence, and two anonymous reviewers for valuable comments
improving the manuscript.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2013.09.
023.
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Fig. 5. Colonization of the Canary Islands and Madeira by different lineages of
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4.4. Article IV
Against all odds: reconstructing the evolutionary history of Scrophularia
(Scrophulariaceae) despite high levels of incongruence and reticulate evolution.
by Agnes Scheunert & Günther Heubl
Organisms Diversity and Evolution, Online First (2017)
IV
The final publication is available at Springer via
http://dx.doi.org/10.1007/s13127-016-0316-0
95
96
Org Divers Evol
DOI 10.1007/s13127-016-0316-0
ORIGINAL ARTICLE
Against all odds: reconstructing the evolutionary history
of Scrophularia (Scrophulariaceae) despite high levels
of incongruence and reticulate evolution
Agnes Scheunert 1
&
Günther Heubl 1
Received: 4 August 2016 / Accepted: 30 November 2016
# Gesellschaft für Biologische Systematik 2017
Abstract The figwort genus Scrophularia (Scrophulariaceae),
widespread across the temperate zone of the Northern
Hemisphere, comprises about 250 species and is a taxonomically challenging lineage displaying large morphological and
chromosomal diversity. Scrophularia has never been examined in a large-scale phylogenetic and biogeographic context
and represents a useful model for studying evolutionary history in the context of reticulation. A comprehensively sampled
phylogeny of Scrophularia was constructed, based on nuclear
ribosomal (ITS) and plastid DNA sequences (trnQ-rps16
intergenic spacer, trnL-trnF region) of 147 species, using
Bayesian inference and maximum likelihood approaches.
Selected individuals were cloned. A combination of coding
plastid indels and ITS intra-individual site polymorphisms,
and applying Neighbor-Net and consensus network methods
for adequate examination of within-dataset uncertainty as well
as among-dataset incongruence, was used to disentangle phylogenetic relationships. Furthermore, divergence time estimation and ancestral area reconstruction were performed to infer
the biogeographic history of the genus. The analyses reveal
significant plastid-nuclear marker incongruence and considerable amounts of intra-individual nucleotide polymorphism in
the ITS dataset. This is due to a combination of processes
including reticulation and incomplete lineage sorting,
Electronic supplementary material The online version of this article
(doi:10.1007/s13127-016-0316-0) contains supplementary material,
which is available to authorized users.
* Agnes Scheunert
agnes.scheunert@gmx.net
1
Systematic Botany and Mycology, Department Biology I, and
GeoBio-Center LMU, Ludwig-Maximilians-University Munich,
Menzinger Strasse 67, 80638 Munich, Germany
possibly complicated by inter-array heterogeneity and
pseudogenization in ITS in the presence of incomplete concerted evolution. Divergence time estimates indicate that
Scrophularia originated during the Miocene in Southwestern
Asia, its primary center of diversity. From there, the genus
spread to Eastern Asia, the New World, Europe, Northern
Africa, and other regions. Hybridization and polyploidy
played a key role in the diversification history of
Scrophularia, which was shaped by allopatric speciation in
mountainous habitats during different climatic periods.
Keywords Scrophularia . Incongruence . Reticulate
evolution . Intra-individual polymorphism . 2ISP . Allopatric
speciation
Introduction
In recent years, an increasing number of phylogenetic studies
in plants, based on molecular sequence information from numerous independent loci, have revealed discordance among
chloroplast and nuclear gene trees as well as gene trees in
general. Although methodological issues in data collection
or analysis might be responsible for some of these observations, incongruence may also be due to conflicting genealogical histories (e.g., Rokas et al. 2003; van der Niet and Linder
2008). These can be caused by gene duplications or losses, or
by incomplete lineage sorting (ILS; Maddison 1997; Degnan
and Rosenberg 2009). Furthermore, processes involving reticulation, i.e., gene flow among species, have been identified,
e.g., horizontal gene transfer, introgression, and homo- or
polyploid hybridization. These phenomena are common in
plants (Rieseberg and Wendel 1993; Rieseberg et al. 1996;
Wendel and Doyle 1998; Mason-Gamer 2004; Richardson
and Palmer 2007); reticulation is now even regarded as a
Scheunert A., Heubl G.
major driving force in the evolution and diversification of
plant lineages (Ellstrand and Schierenbeck 2000; Seehausen
2004; Soltis and Soltis 2009), as is polyploidization (Leitch
and Bennett 1997; Abbott et al. 2013).
Researchers have aimed to trace reticulations within the
evolutionary history of plants by examining incongruences
among gene trees, often a combination of uniparentally (usually plastid) and biparentally (nuclear) inherited molecular
markers (e.g., Rieseberg 1991; Baumel et al. 2002;
Okuyama et al. 2005; Marhold and Lihová 2006; Fehrer
et al. 2007; Scheunert and Heubl 2011, 2014). However, it
has to be noted that the information content of phylogenetic
reconstructions can be severely impeded in the presence of
hybridization. Additionally, ILS/deep coalescences lead to incongruent patterns identical to those from hybridization and
introgression. The use of nuclear ribosomal internal transcribed spacer (ITS) sequence information, although most
popular due to its considerable advantages (Baldwin et al.
1995), can possibly mislead phylogenetic inference
(reviewed by Álvarez and Wendel 2003; Nieto Feliner and
Rosselló 2007). The arrangement of the 35S rDNA genes in
tandem arrays at (sometimes several) separate ribosomal loci
results in thousands of repeats per genome. Consequently, any
ITS sequence will contain the summarized signal from many,
often non-orthologous, not necessarily identical units. This
leads to intra-individual site polymorphism, which can have
a variety of origins (see BDiscussion^ section). Although these
variabilities in rDNA units are often homogenized by the process of concerted evolution (Brown et al. 1972; Arnheim et al.
1980; Nei and Rooney 2005), several cases are known where
it is slowed down, incomplete or non-operational (Grimm
et al. 2007; Rosselló et al. 2007; Grimm and Denk 2008;
Xiao et al. 2010; Hodač et al. 2014). All of the phenomena
described above can have substantial impact on phylogenetic
reconstruction (Álvarez and Wendel 2003; Linder and
Rieseberg 2004; Beiko et al. 2008; Degnan and Rosenberg
2009), challenging traditional approaches which aim to depict
evolutionary history and its underlying events using dichotomously branching phylogenetic trees. In such cases, the incorporation of network construction methods (e.g., Huson and
Bryant 2006) into the analyses is a useful alternative.
T h e g e n u s S c ro p h u l a r i a L . , 1 7 5 3 ( L a m i a l e s :
Scrophulariaceae) represents a useful model for studying the
influence of reticulation on evolutionary history and how it
affects the inference of the latter. The genus consists of about
250 species, with estimates ranging from about 200 species in
Mabberley (1997) and Fischer (2004), to more than 300 in
Willis (1973). Plants are mostly herbaceous perennials, less
often suffrutescent perennials or subshrubs and, more rarely,
biennial or annual herbs. They are characterized by quadrangular, sometimes winged stems and considerably variable,
undivided to 3-pinnatisect, generally opposite leaves. The inflorescence typically is a thyrse or a raceme with
chasmogamous flowers. The five lobes of the calyx frequently
possess a scarious margin, the often brownish, purplish or
greenish corolla is sympetalous, bilabiate, and generally tubular or ventricose in shape. Apart from the four fertile stamens,
the fifth (adaxial) stamen, if not completely absent, is generally sterile and forms a scale-like staminode of various shapes.
The fruit is a septicidal, globose to subconical capsule containing numerous small seeds.
The most recent taxonomic treatment by Stiefelhagen
(1910) divided the genus into two sections, Scrophularia sect.
Anastomosantes Stiefelh. (= S. sect. Scrophularia) and
Scrophularia sect. Tomiophyllum Benth. (= S. sect. Canina
G.Don). Scrophularia extends throughout the temperate zone
of the Northern Hemisphere (Asia, Europe and Northern
America), with very few species expanding into tropical regions (e.g., the Greater Antilles). Based on floristic studies,
species are concentrated in the Irano-Turanian floristic region
sensu Takhtajan (1986), with particularly high species diversity found in Iran and Turkey (42 and 59 species according to
Lall and Mill 1978; Grau 1981; Davis et al. 1988), but a large
number also accounted for in the Flora of the USSR
(Gorschkova 1997). Secondary centers of species richness
are located in the Himalayan region and China (more than
36 species; Hong et al. 1998), as well as the Iberian
Peninsula and adjacent areas including Macaronesia (28
species; Dalgaard 1979; Ortega Olivencia 2009).
Representatives of the genus mainly inhabit highland plateaus
and mountainous regions (all centers of species diversity comprise mountainous regions) but also coastal and lowland areas.
Many species prefer shady and/or moist habitats, while others
are xerophytic (especially within S. sect. Tomiophyllum) and
can tolerate drier conditions; true desert plants are however
rare. Most importantly, the genus is characterized by frequent
natural hybridization, expressed in a variety of polyploid species and also linked to great morphological plasticity
(Stiefelhagen 1910; Grau 1981). Natural hybrids in
Scrophularia have been reported or even described as species
by Menezes (1903, 1908), Stiefelhagen (1910), Pennell
(1943) or Grau (1981). Artificial crossings were successful
according to Goddijn and Goethart (1913), Shaw (1962),
Carlbom (1964), Grau (1976), and Dalgaard (1979), and several cases of homoploid and allopolyploid speciation have
been reported by Scheunert and Heubl (2011, 2014), who also
found evidence for substantial tree incongruence.
Until now, phylogenetic relationships were mainly addressed on a restricted geographical scale, regarding e.g., species of the New World (Scheunert and Heubl 2011), Iran
(Attar et al. 2011), and the Mediterranean and Macaronesia
(Scheunert and Heubl 2014). Based on a time-calibrated phylogeny, Navarro Pérez et al. (2013) recently found support for
monophyly of the genus and its divergence in the Miocene.
However, a robust phylogenetic framework comprising all
important lineages has been missing to date.
Evolutionary history of Scrophularia
Here, we use sequences from the nuclear ribosomal ITS
region and two plastid DNA regions (the trnQ-rps16
intergenic spacer and the trnL-trnF region) to infer phylogenetic relationships. We aim to test the extent to which it is
possible to reconstruct evolutionary history in the face of reticulation and incomplete lineage sorting without resorting to
cloning or whole genome analyses. The main objectives of
our study are (1) to establish a comprehensive evolutionary
framework for Scrophularia based on a broad taxon sampling;
(2) to assess the amount of intra-individual polymorphisms in
ITS sequence data and to explore their possible causes; (3) to
identify inconsistencies between nuclear and plastid DNA
phylogenies and to examine their relation to reticulate evolution; (4) to reconstruct the biogeographic history of
Scrophularia and to reveal which processes account for its
current distribution patterns and species diversity.
Materials and methods
A broad range of methods has been applied in the present
work; these are generally outlined below. Additionally, as
the intent of this study is also to provide a workflow for researchers dealing with similarly complex groups, the information provided here is complemented by detailed descriptions
including settings and procedures, available from Online
Resource 1.
Taxon sampling
The taxon sampling is the most comprehensive presented so
far in a molecular study on the genus and comprises 147 of the
approximately 250 extant Scrophularia species. Sampled taxa
include representatives from throughout the distribution area
and cover all proposed sections and subsections. Known hybrid taxa were only exceptionally included to avoid unnecessary introduction of further conflicts into the dataset. Five
widespread or morphologically diverse species (S. vernalis
L., 1753; S. scopolii Hoppe ex Pers., 1806; S. heterophylla
Willd., 1800; S. canina L., 1753; S. variegata M.Bieb., 1798)
were sampled with additional subspecies and/or varieties. To
investigate intraspecific variability, five species were included
with up to four representatives (S. auriculata L., 1753;
S. lyrata Willd., 1805; S. arguta Sol., 1789; S. nodosa L.,
1753; S. olympica Boiss., 1844). Altogether, the
Scrophularia ingroup consisted of 162 accessions. Based on
previously established relationships, 18 taxa, from the
Scrophulariaceae (represented by five species) and other families within Lamiales (Calceolariaceae, Gesneriaceae,
Plantaginaceae, Stilbaceae, Bignoniaceae, Verbenaceae), were
selected as outgroups (Kornhall et al. 2001; Albach et al.
2005; Oxelman et al. 2005; Nie et al. 2006; Datson et al.
2008; Schäferhoff et al. 2010). In addition, the sampling
included one species from Oreosolen Hook.f., 1884
(Scrophulariaceae), a genus which comprises one to four species endemic to the Himalayas and the Tibetan Plateau and
was found to be most closely related to Scrophularia (Albach
et al. 2005; Oxelman et al. 2005). Complete information on
voucher specimens is provided in Online Resource 2 alongside accession numbers for all analyzed sequences.
DNA extraction, PCR, sequencing, and cloning
Leaf material for DNA extraction was obtained from herbarium specimens (169 accessions from collections in A, B, E,
GH, HAL, HU/HZU, HSNU, KUN, KSC, LE, M, MA, MSB,
W, WU, and WUK) and in nine cases from plants cultivated
by the authors in the greenhouse of the Botanical Garden
Munich (vouchers deposited in MSB). DNA extraction,
PCR, purification and sequencing reactions were performed
according to methods described in Scheunert and Heubl
(2011, 2014). Two well-established loci from these studies
were used, the non-coding chloroplast (Bcp^) trnQ-rps16
intergenic spacer and the nuclear (Bnr^) ribosomal ITS region
(internal transcribed spacer 1, 5.8S rRNA gene, internal transcribed spacer 2). Additionally, the plastid trnL-trnF region
(consisting of the trnL intron, the trnL 3′ exon, and the trnLtrnF intergenic spacer; Taberlet et al. 1991) was used (see also
Navarro Pérez et al. 2013). All primer sequences alongside
references are provided in Online Resource 3. DNA sequences generated by Scheunert and Heubl (2011, 2014),
Navarro Pérez et al. (2013) and others, as well as sequences
from selected outgroup taxa were downloaded from NCBI’s
GenBank (http://www.ncbi.nlm.nih.gov, accessed 10 January
2014; see Online Resource 2). For further investigation of the
considerable amount of intra-individual nr DNA variability
and to support identification of putative hybrid species, six
selected individuals (from S. auriculata; S. incisa Weinm.,
1810; S. lyrata; S. musashiensis Bonati, 1911; S. ruprechtii
Boiss., 1879; and S. villosa Pennell, 1923) were additionally
cloned (for detailed information and PCR protocols see
Online Resource 1). All clones were included into a separate
phylogenetic analysis together with uncloned sequences (see
Phylogenetic inference).
Data matrix composition and coding of chloroplast indels
and ITS intra-individual site polymorphisms
Raw DNA sequence reads were edited and, where necessary,
assembled into contigs with the CLC Main Workbench v. 6
(CLC Bio, Aarhus, Denmark). Ambiguously specified
basepairs (due to poor sequence read quality or site polymorphism) were recorded using IUPAC ambiguity codes. Contigs
were aligned using MAFFT v.7.110 (Katoh and Standley
2013; online version available at http://mafft.cbrc.
jp/alignment/server/, accessed 13 October 2013); used
Scheunert A., Heubl G.
settings are reported in Online Resource 1. Manual
refinements were done in BioEdit v. 7.1.11 (Hall 1999).
Mononucleotide repeats and ambiguously aligned regions
were excluded from further analysis. ITS sequences were
checked for potential pseudogeny according to J-S Liu and
Schardl (1994), Jobes and Thien (1997), and Bailey et al.
(2003). Chloroplast indels, which have been shown to contain
phylogenetic information in Scrophularia (Scheunert and
Heubl 2011, 2014), were coded as binary states for the
ingroup only, using the simple indel coding algorithm
(Simmons and Ochoterena 2000) as implemented in
SeqState v. 1.4.1 (Müller 2005).
In order to make optimal use of the information
contained in ITS sequence data, all sequences were examined for the presence of polymorphic sites (PS). Then, two
methods were applied (with minor adaptations), which are
intended to incorporate information from PS into phylogenetic analyses. Using the ad hoc 2ISP-informative maximum likelihood (ML) approach from Potts et al. (2014), all
IUPAC codes including polymorphic sites (there termed
2ISPs, intra-individual site polymorphisms) are treated as
unique characters, by recoding the complete alignment as a
standard matrix, which is then analyzed using the multistate analysis option for categorical data in RAxML (see
below). This method is similar to some approaches described in Grimm et al. (2007); we complemented the
Potts et al. (2014) method by the application of Bayesian
inference (BI) to our coded dataset as well, based on
Grimm et al. (2007). Detailed information on the coding
procedure and the original methods can be found in Online
Resource 1.
A different approach, pursued by Fuertes Aguilar and
Nieto Feliner (2003), concentrates on BAdditive
Polymorphic Sites^ while ignoring the remainder of intraindividual polymorphisms. According to their definition, a
site is referred to as an BAPS^ when both bases involved in
the polymorphism can each be found separately at the same
site in at least one other accession. To investigate the usefulness of APS in phylogenetic reconstruction, these were
also extracted from the dataset. A subset of 17 alignment
positions with high numbers of APS, here termed Bhighly
polymorphic alignment positions^ (BHPPs^), was then
recoded according to the procedure described above and
added to the original DNA alignment. This data matrix
was likewise analyzed using ML and BI. More detailed
explanations are available in Online Resource 1; used
codes for all HPPs are listed in Online Resource 4.
Phylogenetic inference
Five datasets were used, one containing the combined
trnQ-rps16 and trnL-trnF region data and coded indels
from both markers (Bcp dataset^), one based on uncoded
nr DNA sequence data (Buncoded dataset^), one with nr
sequence data alongside coded HPPs (BAPS-coded
dataset^), one with the complete nr sequence alignment
recoded following the 2ISP-informative approach (B2ISPcoded dataset^), and one corresponding to the 2ISP-coded
dataset, but also comprising cloned sequences (Bnr+clones
dataset^). Datasets were analyzed separately using ML and
BI. For comparison purposes, additional analyses of the
uncoded dataset were conducted, once excluding the 17
highly polymorphic positions themselves and once excluding 17 accessions with high amounts of APS in their sequences (see BResults^ section). Appropriate nucleotide
substitution models were selected using MrModelTest v.
2.3 (Nylander 2004), which suggested GTR+Γ with four
rate categories as best fit to the data according to the
Akaike information criterion, adding a proportion of invariant characters for the ITS dataset only (GTR+I+Γ).
Bayesian analyses were performed with MrBayes v. 3.2.2
(Ronquist et al. 2012) on a local PC. Maximum likelihood
analyses were carried out with RAxML v. 7.4.2
(Stamatakis 2006) on a local PC using raxmlGUI v. 1.3
(Silvestro and Michalak 2012), and RAxML v. 8.0.24
(Stamatakis 2014) on the CIPRES Science Gateway (MA
Miller et al. 2010; available at https://www.phylo.
org/portal2/login!input.action, accessed 02 December
2015). Settings for all runs and the different datasets are
described in Online Resource 1.
Identifying phylogenetic conflict and uncertainty
within and among datasets
The amount of ambivalent signal contained within the ITS
raw data was illustrated using SplitsTree v. 4.12.3. (Huson
and Bryant 2006). To this end, a Neighbor-Net splits graph
(NN; Bryant and Moulton 2004) was created, based on a
pairwise distance matrix obtained from the ITS sequence
alignment (non-excluded characters, see Table 1). In order
to incorporate information from polymorphic sites, polymorphism p-distances (see Potts et al. 2014) were calculated for the ingroup. Online Resource 1 provides information
on required software and optimal settings. Additional networks were computed excluding columns containing gaps
and/or accessions with high numbers of B?^ and BN^ in
their sequences, to assess the impact of missing data on
the results.
For the visualization of incongruence among datasets,
consensus networks (CN; Holland and Moulton 2003;
Holland et al. 2004) were constructed in SplitsTree, from
each 1001 trees of both BI runs of the cp dataset and the
2ISP-coded dataset. Importantly, edge lengths in these CNs
are proportional to the split frequency within the sampled
topologies (BCOUNT^ option). A trees threshold of 0.33
was used for displaying splits, and splits were transformed
Evolutionary history of Scrophularia
polymorphic alignment positions.^ Alpha, tree score, and length in ITS
given for the uncoded and 2ISP-coded dataset. Values marked with
asterisks are based on the nr+clones dataset; values with circles refer to
results from the concatenated cp dataset. Alpha the alpha value of the
gamma shape parameter, avg average, bp basepairs, no number
Table 1 Alignment characteristics and Maximum Likelihood-based
tree statistics for the plastid trnQ-rps16 and trnL-trnF markers and
nuclear ITS. Average G+C contents and parsimony-(un)informative
characters calculated excluding outgroups, other characteristics include
outgroup values. Percentage of parsimony-informative characters
referable to non-excluded characters; the latter inclusive of Bhighly
trnQ-rps16
trnL-trnF region
ITS
ITS (clones)
No. of taxa (including outgroups)
Sequence length (avg.)
180
427–1584 bp(961 bp)
94
100–818 bp(725 bp)
181
247–667 bp(580 bp)
Aligned length
Non-excluded characters
2063 bp
2046 bp
946 bp
930 bp
763 bp
738 bp
62
436–602 bp
(571 bp)
763 bp
738 bp
Parsimony-uninformative characters
139 bp
51 bp
69 bp
98 bp*
Parsimony-informative characters
Average G+C content
134 bp (6.55%)
27.00%
32 bp (3.44%)
33.3%
125 bp (16.94%)
60.12%
150 bp (20.33%)*
60.87%
ML tree score
ML tree length
−15,802.897°
1.835°
–
–
−9001.089/−15,043.732
6.273/26.544
–
–
Alpha
0.979°
–
0.718/1.089
–
as outlined in Online Resource 1. Congruence among the
sequence datasets was also tested with the Incongruence
Length Difference (ILD) test (Farris et al. 1995) implemented as the Partition Homogeneity Test in PAUP v.
4.0b10 (Swofford 2003). Accessions or clades exhibiting
hard incongruence (HI) were identified by visual inspection
of the cp and (2ISP-coded) nr phylogenetic trees for wellsupported conflicting placements (Mason-Gamer and
Kellogg 1996), using a threshold of ≥0.90 Bayesian posterior probability (PP) in both topologies. Further information can be found in Online Resource 1.
Molecular clock analyses
Divergence times were estimated for the cp dataset using
BEAST v. 1.8 (Drummond and Rambaut 2007). For information on input matrices and how information from coded binary
indels was incorporated see Online Resource 1.
Divergence dating using fossils was impossible for this
dataset of Scrophularia (possible reasons for the failure of
fossil-based dating and additional information on the used
fossils are given in Online Resource 1). Thus, rather than
completely relying on secondary calibration constraints
for relaxed clock analyses, an approach which is generally
error-prone (see e.g., Hipsley and Müller 2014), we decided to resort to a first-step strict clock analysis in order
to obtain information about a reasonable age for the
ingroup which could be used as a starting point for further
analyses. For the strict clock, a fixed substitution rate of
8.1E−4 per site per million years was used (Lavin et al.
2005). Then, divergence times of the ingroup and the
closest outgroup genus only (reduced dataset) were inferred under a relaxed clock with log-normally distributed
rates, using the estimated ingroup age from the strict
clock run as secondary calibration point. Analyses were
performed with BEAST v. 1.8 on the CIPRES Science
Gateway; exact procedures, prior values, and settings are
provided in Online Resource 1. The relaxed clock analysis
was repeated without data (prior-only) on a local PC, to
review effective prior distributions and assess the decisiveness of the data.
Biogeographic analyses
Ancestral area optimization relied on the Bayesian Binary
Markov Chain Monte Carlo (BBM) algorithm as implemented in RASP v. 2.0b (Yu et al. 2010, 2014). Biogeographic
areas are mapped on the world map in Fig. 1b; detailed definitions can be found in Online Resource 5. Distributions of
species (including those of known synonyms) were then
assigned to the respective areas. Further information on the
classification of areas, the determination of species distributions, and the RASP analyses is available from Online
Resource 1.
BMaxareas,^ the maximum number of ancestral areas inferred at each node, was constrained following Ronquist
(1997). We assumed equal dispersal ability for ancestors and
their present-day descendants (Sanmartín 2003) and therefore
set maxareas to two (83% of the Scrophularia ingroup species
occur in one or two areas only). The number of maximum
areas was kept at five during one additional run for comparison purposes. Inferred ancestral distributions were mapped on
the majority-rule consensus tree from Bayesian analysis in
Fig. 1a, using a threshold of 25% marginal probability (frequency of occurrence of the respective range over the
Bayesian sample of trees).
Scheunert A., Heubl G.
2
V. nigrum ABCDEFGHIJKLMNO
V. arcturus ABCDEFGHIJKLMNO
moellendorffii N
72
5
chrysantha GJ
vernalis ssp. vernalis BCFG
J
7
kotschyana J
IJ
J J
vernalis ssp. clausii IJ
8
peregrina BCDEHJ
tenuipes D
14
D
buergeriana N
13
yoshimurae N
12
ningpoensis N
N
11
koraiensis N
ruprechtii J
N
17
chasmophila N
16
hypsophila N
N
macrocarpa N
15
henryi N
18 N
lijangensis N
Hengduan
N
10
spicata N
21
LN OREOSOLEN WATTII LN
20
przewalskii LN
N
Central
N
N
19
52 3 souliei
modesta N
N 22
kansuensis N
stylosa N
altaica MN
4 multiflora O
25
4 desertorum O
O
4 californica O
4a. California
atrata O
51 4
4 villosa O
27
4 laevis O
O
26
4 parviflora O
4d. New Mexico
4 montana O
O
28
24
4 micrantha O
4b. Eastern North America
4 domingensis O
O
4 marilandica O
N
4 grayana N
4 musashiensis N
4 duplicatoserrata N
31 4 nodosa cult. BCFGJMN
* 4 nodosa Germany BCFGJMN
30
USA BCFGJMN
33 44 nodosa
23
aestivalis C
CJ
4
bosniaca
C
C
32
4 kurdica ssp. glabra I
29
J
4 chlorantha IJ
34
IJ
J
IJ 44 pegaea
lateriflora J
4 divaricata GJ
4 cryptophila J
35
5 orientalis IJ
5 nervosa IJ
IJ
calycina KL
38
IJ/I
pauciflora LN
37
LN heucheriiflora MN
39
yunnanensis N
LN/N
amplexicaulis IJ
N
36 KL
elatior KLN
himalensis KL
edgeworthii K
nikitinii KM
urticifolia LN
2 delavayi N
a
6
3.6
7.7
9. VERNALIS
10. PEREGRINA
2. NINGPOENSIS
3.3
3. CHINA
6.1
3
[14.6]
4. NW / JAPAN
IJ
ANASTOMOSANTES
6.4
1
23.1
5. NODOSA
4.6
9.9
3.1
9
18. ORIENTALIS
6. CALYCINA
1.6
4
TOMIOPHYLLUM CLADE
42
41
I
3.7
I
40
trnQ-rps16
r Q- p 1 + trnL-trnF
r Ltn
85
(ABDEI)
I
61
AI
87
3.3
86
I
25
20
15
10
5
Fig. 1 a Dated phylogeny and ancestral area reconstruction for 147
Scrophularia species, on a majority-rule consensus tree obtained from
Bayesian analysis of combined plastid trnQ-rps16 intergenic spacer and
trnL-trnF region alongside coded indels. Branches indicate levels of support, based on posterior probabilities (PP) and plotted bootstrap support
values (BS) from Maximum Likelihood optimization; bold PP ≥ 95 or
BS ≥ 85, semi-bold PP ≥ 90 or BS ≥ 75, thin PP < 90/BS < 75. Seven
additional nodes only supported by ML (BS ≥ 50) were added manually
but not incorporated into further analyses. Gray bars on the right denote
Clades 1–18 and main species groups as discussed in the text. An arrow
indicates the position of the Himalayan-Tibetan endemic genus
Oreosolen. Single accessions displaying hard incongruence among
(2ISP-coded) nuclear and plastid trees are marked in bold; Clades 7 and
5 (excluding S. chlorantha; plus S. cryptophila) as a whole are also hardly
incongruent. The occurrence of large indels as defined in Table 2 is
indicated next to each accession with the respective length type number;
*
2
2
2
56 2
2
2
arguta Morocco ABDEI
arguta El Hierro ABDEI
arguta Lanzarote ABDEI
arguta Tunisia ABDEI
megalantha I
scopolii var. grandidentata BCFGHIJK
scopolii BCFGHIJK
taygetea C
crenophila I
umbrosa BCFGHIJKMN
1. ARGUTA
8. SCOPOLII
ANAST.
I
0
no number means length type = 1. Node heights represent mean ages and
were inferred under a Bayesian relaxed clock with log-normally distributed rates, using one calibration point at node 3. Important clade crown
ages are given in million years. Ancestral area optimization is based on
9002 trees from the Bayesian analysis, distribution ranges of single taxa
are provided after the respective names, area codes and colors are as
defined in (b) and Online Resource 5. Pie charts at nodes indicate inferred distributions of MRCAs from run four of four RASP runs
(maxareas = 2); asterisks mark nodes where no ancestral distribution
reached a marginal probability of 25%. At nodes 9, 37, 61, and 63,
separate runs differed with respect to the most probable ancestral area.
Alternative distributions shown in brackets derive from an additional
analysis with maxareas = 5. Exact values for each node including highest
posterior density intervals for inferred ages are listed in Online Resource
7. NW New World, V Verbascum, cult cultivated. b World map showing
areas defined for ancestral area reconstruction analyses
Evolutionary history of Scrophularia
BD 47
auriculata England ABCDF
racemosa A
sambucifolia BD
hispida D
BD
Crete BCD
49 lyrata
hirta A
48
smithii ssp. smithii A
glabrata
A
D
A
auriculata Azores ABCDF
50
lyrata Spain BCD
scorodonia ABDF
45 B BD
laxiflora BD
macrorrhyncha D
B
pyrenaica B
52
ssp. canina BCDEFG
BC canina
crithmifolia B
53
heterophylla ssp. heterophylla CH
heterophylla ssp. laciniata C
spinulescens C
C
51
canina ssp. hoppii „juratensis“ BCF
lucida BCHJK
pinardii HJ
54
imerethica J
44
floribunda J
J
55
sareptana G
cretacea G
J
G
donetzica G
variegata var. bulgarica CIJK
sosnowskyi J
myriophylla CJ
tadshicorum KM
crassicaulis I
I
subaphylla I
58
libanotica EHIJ
59
glauca I
IJ
pruinosa IJM
62
sinaica I
61
EHM
64 xanthoglossa
EH
syriaca DEHIJ
(EHIJ) EH/HI
deserti EHIK
63
IJKM
(D)EHIJ)
65 striata
syriaca „hypericifolia“ DEHIJ
HI/EI/EH
EI leucoclada IKM
60
xylorrhiza HIJ
66
peyronii CHIJ
thesioides J
IJ IJ
decipiens EHJ
59
zvartiana IJ
elegantissima K
67
farinosa I
IJ
I
frigida ssp. haussknechtii I
catariifolia IJ
71
variegata ssp. variegata CIJK
57
70
variegata ssp. cinerascens IJK
J
zuvandica IJ
IJ
IJ
IJ
IJ
69 IJ IJ atropatana
olgae J
73 puberula IJ
68 72 J
takhtajanii J
2 subaequiloba J
J/IJ
76 69 hierochuntina H
tagetifolia H
75
scariosa HJ
H
H 6 rubricaulis
nabataeorum H
74
olympica Armenia GIJ
IM
78
56
scoparia IKM
M
kabadianensis M
IJ 77
I
M
79 integrifolia
kiriloviana MN
I 80 IJ
rostrata IJ
gorganica I
I
2 bheriensis L
2 polyantha K
2 obtusa K
IJ
82
2 multicaulis M
2 incisa MN
K
2 vvedenskyi M
I 43
81
2 decomposita KL
nudata K
koelzii KL
K
griffithii KM
83
ilwensis IJ
olympica Georgia GIJ
84
IJ
dentata KN
depauperata J
J
scabiosifolia KM
gracilis I
minima J
lepidota J
46
A
G
M
F
B C J
H K
D
L
E
I
4.5
N
b
7. IPM
3.3
11. CANINA
2.3
12. LIBANOTICA
2.3
16. STRIATA
TOMIOPHYLLUM
O
13. VARIEGATA
3.2
2.0
17. SCARIOSA
14. SCOPARIA
4.0
3.2
7.8
trnQ-rps16
r Q- p 16 + trnL-trnF
r L t nF
25
20
15
10
5
15. POLYANTHA
0
Fig. 1 (continued)
Results
Alignment characteristics
Altogether, 281 sequences were generated for this study, 125
for trnQ-rps16, 123 for ITS, and 33 for the trnL-trnF region.
A total of 62 cloned ITS sequences was obtained from six
accessions (available from GenBank under accession numbers
KY067709–KY067770) and included into phylogenetic reconstructions, raising the number of ITS sequences to 243 in
the nr+clones dataset. Data coverage for trnQ-rps16 and ITS
is complete for all accessions except Aragoa, where sequencing of trnQ-rps16 failed and no sequence was available in
GenBank; the genus was coded as missing for the respective
marker but contributed a trnL-trnF sequence to the plastid
alignment. For the trnL-trnF region, which was used as
supplementary marker, only selected accessions were sequenced; the remainder was likewise coded as missing for
trnL-trnF. Detailed information on sequence and alignment
statistics including average G+C contents and proportions of
parsimony-informative characters is given in Table 1. Twentynine ingroup indels were coded for the trnL-trnF region and
84 for trnQ-rps16. The latter is characterized by the occurrence of larger indels ranging from 312 to 839 characters in
length; Table 2 shows the positions of all observed indels
>300 characters (the respective accessions are marked in
Fig. 1a).
PCR products of ITS showed clear single bands in most
cases. However, polymorphic sites were present in almost
three quarters of the ingroup accessions (121 of 163).
Additive polymorphic sites (APS) as defined above were recorded in 95 accessions (58%), from which 17 had five or
Scheunert A., Heubl G.
more APS in their sequence (BAPS-rich accessions,^ see
Fig. 2a). Fourteen of these APS-rich accessions are members
of the BTomiophyllum^ clade (see below). Generally, an unequivocal differentiation between artificial double peaks, PS
and APS, was not always possible, which means that the reported numbers rather represent best possible estimates.
Phylogenetic relationships
Fig. 2 Majority-rule consensus trees (cladograms) for 147 Scrophularia
species, obtained from Bayesian analysis of the nuclear internal
transcribed spacer region (ITS), with a intra-individual site
polymorphisms (IUPAC codes) treated as informative using 2ISP
coding or b using uncoded, unmodified sequences. Downsized
phylograms are shown beside each tree, with six taxa having
exceptionally long branches in the 2ISP-coded dataset marked in bold.
Outgroup taxa are reduced to the closest genus. Posterior probabilities
(PP) are given above branches, plotted bootstrap support values (BS)
from Maximum Likelihood (ML) optimization below. Double dashes
state that the node was not present in the fully resolved best-scoring
ML tree. Asterisks denote cases with deviating ML topologies
illustrated beside the tree; five/twelve additional nodes only supported
by ML (BS ≥ 50) were added manually. Branches indicate levels of
support as defined in Fig. 1. Gray bars denote Clades 1–18 and main
species groups. Arrows indicate the position of the Himalayan-Tibetan
endemic genus Oreosolen. Single accessions displaying hard
incongruence among (2ISP-coded) nuclear and plastid trees are marked
in bold; Clades 7 and 5 (excluding S. calycina and S. pauciflora Benth.,
1835) as a whole are also hard incongruent. Accessions obtaining
different positions in both phylogenies are connected across trees.
Amounts of intra-individual polymorphism are indicated to the left of
each accession using the following symbols: no symbol no polymorphic
sites (PS), cross PS but no APS (additive polymorphic sites, see
BMaterials and methods^ section), star APS present in the sequence,
square ≥ 5 APS present, BAPS-rich accession.^ Distribution ranges of
single taxa are provided after the respective names, area codes are as
defined in Fig. 1b and Online Resource 5. NA North America, V
Verbascum, cult cultivated, grand grandidentata, hausskn haussknechtii,
varieg variegata, cin cinerascens, het heterophylla
Bayesian analyses of the cp and nr datasets had reached convergence at the end of the runs (standard deviation of split
frequencies below 0.01). The majority-rule consensus topologies, with all outgroups except Verbascum L., 1753 pruned,
are shown in Figs. 1a and 2. Statistics on ML analyses are
given in Table 1. The best ML trees only very rarely
contradicted the Bayesian consensus trees with respect to
nodes with bootstrap support (BBS^) ≥ 50; supports were generally lower when using ML. Relative to the Bayesian tree
from the uncoded ITS dataset (Fig. 2b, 64 nodes with
PP ≥ 0.5), removal of the 17 HPPs yielded a tree with 18 nodes
collapsed and weakened support values in 22 cases; removal
of the APS-rich accessions from the uncoded dataset had less
impact but led to generally lower supports in basal nodes
(results not shown). Conversely, including the coded HPP
matrix into calculations (APS-coded dataset) resulted in a tree
with 19 new nodes (PP ≥ 0.5; not shown); using the 2ISPinformative approach and coding all double peaks present in
the sequences (2ISP-coded dataset, Fig. 2a) yielded a tree with
35 new nodes relative to the uncoded phylogeny. By contrast,
resolution did not change much in ML trees and was even
slightly reduced.
Both 2ISP-coded nuclear and plastid tree (Figs. 2a and 1a)
support a monophyletic clade of all accessions from
Scrophularia with moderate to maximum support (cp PP
1.00, BS 100/nr PP 1.00, BS 80). However, the accession
from Oreosolen wattii Hook.f. is deeply nested within the
ingroup at similar positions depending on the dataset, rendering Scrophularia paraphyletic with respect to this genus. For
convenience and to avoid confusion with other clade names,
Stiefelhagen’s (1910) section names are adopted here to name
the two main phylogenetic entities: the highly supported
Tomiophyllum clade (cp PP 1.00, BS 99/nr PP 1.00, BS 93)
largely corresponds to, but is not exactly identical with,
Scrophularia sect. Tomiophyllum. The remainder of the
Table 2 Nine characteristic indels, corresponding to eight sequence
length types, in the trnQ-rps16 intergenic spacer alignment created
from sequences of 162 Scrophularia accessions. Length type B1^ no
larger indels present, full alignment length 3325 basepairs. BIndel
position^ is referable to aligned length. Clade numbers as defined in the
main text. Assessment of phylogenetic value as diagnostic character is
given for each indel type. No acc number of accessions possessing the
respective indel, bp basepairs
Length type
Indel position
Indel length
No acc
Species/clade
Diagnostic?
1
2a
2b
2c
–
485–796
518–845
527–845
–
312 bp
328 bp
319 bp
118
7
1
1
–
Clade 15
Delavayi
Subaequiloba
No
Yes
No
No
2d
528–845
527–844
688–1118
551–1147
251–1014
207–1045
318 bp
318 bp
431 bp
597 bp
764 bp
839 bp
5
1
1
25
2
1
Clade 8
Arguta El Hierro
Souliei
Clades 4+5
Clade 18
Rubricaulis
Yes?
No
No
Yes
Yes
No
3
4
5
6
Evolutionary history of Scrophularia
2. NINGPOENSIS
0.58 6
--
5
54
64
0.93 9
1 8 63
92
0.83 7
--
0.90
1
12
11 67
85
3. CHINA
1
10
<50
1
84
1
13
61
1
4a. CALIFORNIA
4b. EASTERN NA
4c. JAPAN
4d. NEW MEXICO
5. NODOSA
1
80
0.70
<50 14
0.80
-- 15
1
1
17 94
0.87
92
<50 16
0.75
65
1
0.87
20 100
<50
0.98
1
25 65
88
24
0.92
0.86 0.95
27 <50
<50
<50
18
19
21
26
28
0.52
-- 23
3
0.91
22
<50
0.96
<50
6. CALYCINA
0.85 29
<50
30
0.83
0.57 36 93 37
60
0.78
35
0.97
*
0.98
38
34
*1
86
0.97
39
33
98
-1 40
92 51
1
32
0.72 41
84
51
1
42
1
100
31
50
0.99
44
1 43 63
84
auriculata Az
37 racemosa
36
sambucifolia
35*
lyrata Sp
94
39 lyrata Cr
38*
34
56
hispida
auriculata En
7. IPM
8. SCOPOLII
9. VERNALIS
10.PEREGRINA
0.63/ --
0.78
50
0.88 49 -1
63
48
98
0.70 47
0.76
51
<50
66
46
0.99
60 55
0.99
54
<50
11. CANINA
12. LIBANOTICA
1
59
0.95
58 <50
0.96
57 -0.70 56 ---
1
4
66
1
60
--
0.57 61
-0.54
-- 62
1 80
0.89 64 76 65
-0.90
0.65
66
63
---
13. VARIEGATA
0.96 68
1
70
-0.95
69 871
-71
--
0.94
-- 67
0.70
--
0.99
73
0.69
72 62
<50
45
0.68
0.78
-- 74
-- 76 1 77
0.56 75
71
<50
14. SCOPARIA
bheriensis
79* polyantha
71
obtusa
59 80*
kabadianensis
78
nudata
0.68
-- 53
78
1
15. POLYANTHA
0.80 79
*
95
0.73 80
*
0.91 57
0.53
-- 82
-- 81
1
-0.87
-0.84
-0.78
<50
1
93 52
16. STRIATA
84
85
86
1 90
0.99
89 <50
0.98
59
91
-88
1
0.61
0.78 92 96 93
--0.80
-- 94
0.65 87
--
17. SCARIOSA
ITS
2ISP-coded
83
18. ORIENTALIS
0.94
95
<50
0.52
-- 96
0.87 97
<50
0.78
98
82
0.64 99
53
V. nigrum
V. nigrum A-O
V. arcturus
V. arcturus A-O
arguta Morocco
arguta Morocco ABDEI
arguta El Hierro ABDEI
arguta El Hierro
arguta Lanzarote
arguta Lanzarote ABDEI
arguta Tunisia
arguta Tunisia ABDEI
koraiensis
koraiensis N
ningpoensis
ningpoensis N
buergeriana
buergeriana N
yoshimurae
yoshimurae N
yunnanensis
OREOSOLEN WATTII LN
amplexicaulis
yunnanensis N
amplexicaulis IJ
chasmophila
delavayi N
hypsophila
chasmophila N
delavayi
hypsophila N
macrocarpa
macrocarpa N
henryi
henryi N
lijangensis
lijangensis N
spicata
spicata N
modesta
modesta N
kansuensis
kansuensis N
przewalskii
przewalskii LN
souliei
souliei N
stylosa
stylosa N
OREOSOLEN WATTII
multiflora O
multiflora
desertorum O
desertorum
californica O
californica
atrata O
atrata
villosa
villosa O
micrantha O
micrantha
domingensis O
domingensis
marilandica O
marilandica
grayana N
grayana
musashiensis N
musashiensis
duplicatoserrata N
duplicatoserrata
laevis O
laevis
montana
montana O
parviflora O
parviflora
nodosa cult. BCFGJMN
nodosa cult.
nodosa Germany BCFGJMN
nodosa Germany
nodosa USA BCFGJMN
nodosa USA
divaricata GJ
divaricata
bosniaca C
bosniaca
aestivalis C
aestivalis
pegaea IJ
megalantha
cryptophila J
pegaea
kurdica ssp. glabra I
cryptophila
lateriflora J
kurdica ssp. glabra
calycina KL
lateriflora
pauciflora LN
calycina
pauciflora
racemosa A
auriculata Azores ABCDF
racemosa
sambucifolia BD
auriculata Azores
auriculata England ABCDF
auriculata England
hispida D
hispida
lyrata Spain BCD
sambucifolia
lyrata Crete BCD
lyrata Spain
hirta A
lyrata Crete
smithii ssp. smithii A
hirta
glabrata A
smithii ssp. smithii
macrorrhyncha D
glabrata
umbrosa BCFGHIJKMN
macrorrhyncha
scorodonia ABDF
umbrosa
laxiflora BD
scorodonia
pyrenaica B
laxiflora
crenophila I
pyrenaica
scopolii var. grand. BCFGHIJK
crenophila
scopolii BCFGHIJK
scopolii var. grand.
scopolii
taygetea C
moellendorffii N
taygetea
chrysantha GJ
moellendorffii
chrysantha
vernalis ssp. vernalis BCFG
vernalis ssp. vernalis
kotschyana J
kotschyana
vernalis ssp. clausii IJ
vernalis ssp. clausii
peregrina BCDEHJ
peregrina
tenuipes D
tenuipes
chlorantha IJ
chlorantha
megalantha I
canina ssp. canina
canina ssp. canina BCDEFG
spinulescens
spinulescens C
thesioides
thesioides J
canina ssp. hoppii „juratensis“ BCF canina ssp. hoppii j.
decipiens
decipiens EHJ
frigida ssp. hausskn.
frigida ssp. hausskn. I
subaphylla I
subaphylla
libanotica EHIJ
libanotica
glauca
glauca I
catariifolia IJ
catariifolia
griffithii KM
griffithii
olgae J
olgae
zvartiana IJ
zvartiana
elegantissima K
elegantissima
leucoclada IKM
leucoclada
syriaca DEHIJ
syriaca
ruprechtii J
ruprechtii
imerethica J
imerethica
olympica Georgia GIJ
olympica Georgia
olympica Armenia GIJ
olympica Armenia
scabiosifolia KM
scabiosifolia
heterophylla ssp. laciniata C heterophylla ssp. laciniata
gorganica
gorganica I
variegata ssp. varieg.
variegata ssp. varieg. CIJK
variegata ssp. cin.
variegata ssp. cin. IJK
cretacea
cretacea G
donetzica
donetzica G
atropatana
atropatana IJ
zuvandica
zuvandica IJ
puberula
puberula IJ
farinosa
farinosa I
scoparia
scoparia IKM
dentata
dentata KN
integrifolia
integrifolia M
kiriloviana
kiriloviana MN
multicaulis
multicaulis M
incisa
incisa MN
bheriensis
kabadianensis M
polyantha
bheriensis L
obtusa
polyantha K
kabadianensis
obtusa K
nudata
nudata K
crassicaulis
crassicaulis I
xylorrhiza
xylorrhiza HIJ
heterophylla ssp. het.
heterophylla ssp. het. CH
variegata var. bulgarica
variegata var. bulgarica CIJK
syriaca “hypericifolia“
syriaca „hypericifolia“ DEHIJ
peyronii
peyronii CHIJ
lepidota
lepidota J
rostrata
rostrata IJ
sareptana
sareptana G
pinardii
pinardii HJ
gracilis
gracilis I
crithmifolia
crithmifolia B
vvedenskyi
vvedenskyi M
pruinosa
pruinosa IJM
depauperata
depauperata J
decomposita
decomposita KL
sosnowskyi
sosnowskyi J
takhtajanii
takhtajanii J
minima
minima J
sinaica
sinaica I
xanthoglossa
xanthoglossa EHM
deserti
deserti EHIK
striata
striata IJKM
floribunda
floribunda J
subaequiloba
lucida BCHJK
tagetifolia
myriophylla CJ
hierochuntina
subaequiloba J
koelzii
tagetifolia H
rubricaulis
hierochuntina H
scariosa
koelzii KL
nabataeorum
rubricaulis H
lucida
scariosa HJ
myriophylla
nabataeorum H
ilwensis IJ
ilwensis
elatior
elatior KLN
edgeworthii K
edgeworthii
orientalis
orientalis IJ
nervosa
nervosa IJ
nikitinii
nikitinii KM
heucheriiflora MN
heucheriiflora
tadshicorum
tadshicorum KM
altaica
altaica MN
urticifolia
urticifolia LN
himalensis
himalensis KL
1
99
1
58 5
92
2
b
9 0.51
56 8 1
88
12a
0.96
0.98
94 11
76
0.81
10b 52
54
10
13a
0.95
75
0.92
<50
C
0.71
<50
B
60
56
14 0.64
<50
18
19
21
1
97 17
1
0.85
<50
0.98
76
0.77
A <50
1
0.58
95 16
<50
0.94
68
1
0.65
100 20
<50
80 1
25 87
0.62
28 <50
0.66
27a --
24a
0.63
--
3
1
72
0.94
<50 0.95
64
0.51
96
36a 54 0.65
35 59
51
1
34
95
0.99
0.85
39
33
89
<50
40 0.54
0.55
41b <50
79 85
ANASTOMOSANTES
94
2
29
30
53
42
44
72
32
1
99
1
86
31
0.99
80 43 1
87
0.99
54
49 0.55
70
1
48
96
0.97
51
79
55 0.98
0.95
54
54
<50
4
0.79
60
61b 0.63
54
0.77
61a <50
70b 0.79
84 63
TOMIOPHYLLUM
99
1
1. ARGUTA
0.69
53b -0.52
77a
<50
79a 0.72
88 72 78 1
0.89 95
80a
63
0.62
83b 52
0.58
83a <50
53a
0.76
86 <50
89 0.85
59
0.85
<50
52
1
93
95 0.89
54
1
93
96
1
57
1
85
99 0.64
52
97
98
ITS
uncoded
ANAST.
1
a
Scheunert A., Heubl G.
1
1
Outgroup
Majority-rule consensus tree (cladogram) for 224 accessions of
147 Scrophularia species, including 62 cloned sequences from six
accessions, and obtained from Bayesian analysis of 2ISP-coded nuclear
ITS. Detailed relationships are shown for clades which include clones and
where deviating from Fig. 2a; dashed lines represent one or more
accessions removed for clarity. Posterior probabilities ≥ 0.90 are given
above branches. Clones and corresponding direct sequences (DS) are
highlighted in gray; brackets on the right denote the clade they belong
to. Distinct positions of clones and DS for each species are indicated by
arrows. NA North America
Fig. 3
1. ARGUTA
2. NINGPOENSIS
OREOSOLEN WATTII
0.99
3. CHINA
1
1
0.94
1
0.94
0.99
1
1
0.99
4a. CALIFORNIA
1
multiflora
desertorum
californica
atrata
villosa DS
villosa clone 5
villosa clone 11
villosa clone 13
villosa clone 17
villosa clone 19
villosa clone 22
villosa clone 24
villosa clone 25
villosa clone 27
villosa clone 20
villosa clone 26
villosa clone 18
villosa clone 21
S. villosa
4b. EASTERN NA
grayana
musashiensis DS
musashiensis clone 6
musashiensis clone 18
musashiensis clone 1
musashiensis clone 8
musashiensis clone 10
musashiensis clone 5
musashiensis clone 12
musashiensis clone 22
musashiensis clone 11
musashiensis clone 21
musashiensis clone 23
duplicatoserrata
4d. NEW MEXICO
4c. JAPAN
0.95
S. musashiensis
5. NODOSA
0.95
0.97
1
1
1
1
1
1
1
8. SCOPOLII
9. VERNALIS
10. PEREGRINA
1
0.99
0.96
11. CANINA
12. LIBANOTICA
0.93
1 0.95
1
0.99
0.97
0.94
13. VARIEGATA
1
1
0.98
1
0.98
1
1
ruprechtii DS
imerethica
olympica Georgia
ruprechtii clone 1
ruprechtii clone 2
ruprechtii clone 8
ruprechtii clone 15
ruprechtii clone 3
ruprechtii clone 4
ruprechtii clone 9
ruprechtii clone 17
ruprechtii clone 19
ruprechtii clone 20
olympica Armenia
scabiosifolia
gracilis
heterophylla ssp. laciniata
1
0.90
0.99
0.95
scoparia
dentata
integrifolia
kiriloviana
incisa DS
incisa clone 6
incisa clone 15
incisa clone 9
incisa clone 8
incisa clone 10
incisa clone 3
incisa clone 7
incisa clone 12
multicaulis
15. POLYANTHA
S. ruprechtii
TOMIOPHYLLUM
1
14. SCOPARIA
0.95
6. CALYCINA
racemosa
auriculata Azores
auriculata England clone 1
auriculata England clone 3
auriculata England clone 4
auriculata England clone 9 S. auriculata
auriculata England clone 10
auriculata England clone 14
auriculata England DS
hispida
sambucifolia
lyrata Spain DS
lyrata Spain clone 1
S. lyrata
lyrata Spain clone 2
lyrata Spain clone 3
lyrata Spain clone 5
lyrata Spain clone 7
lyrata Spain clone 8
lyrata Spain clone 13
lyrata Spain clone 14
lyrata Spain clone 15
lyrata Spain clone 22
lyrata Crete
hirta
smithii ssp. smithii
glabrata
auriculata England clone 2
auriculata England clone 6
auriculata England clone 8
auriculata England clone 11
macrorrhyncha
umbrosa
scorodonia
laxiflora
pyrenaica
7. IPM
0.95
S. incisa
xylorrhiza
heterophylla ssp. heterophylla
variegata var. bulgarica
pinardii
16. STRIATA
17. SCARIOSA
18. ORIENTALIS
species mainly belong to Scrophularia sect. Anastomosantes;
these species do not form a monophyletic clade, but are
paraphyletic with respect to the Tomiophyllum clade.
Nevertheless, they are united by a number of shared characteristics and are therefore referred to as the BAnastomosantes
group.^
Several clades can be recognized in both trees (see boxes in
Fig. 1a and 2a): for example, BArguta^ (Clade 1) comprises all
four accessions of this annual species and receives high to
maximum support in the analyses. A clade of mainly
Chinese endemics (BChina,^ Clade 3) receives maximum support by BI in ITS only. In the cp tree, its composition is slightly different and includes the BNingpoensis^ clade (Clade 2).
The most widespread representative of the genus, the type
species S. nodosa, forms a monophyletic clade with seven to
nine other taxa (BNodosa,^ Clade 5) and is moderately to
highly supported by BI. In the cp tree, it includes the
Southwestern Asian/Turkish - Caucasian S. chlorantha
Kotschy & Boiss., 1879, in ITS the Turkish endemic
S. cryptophila Boiss. & Heldr., 1853, and the mainly
Southern Asian Calycina clade (BCalycina,^ Clade 6). Clade
7, the BIPM^ clade (BIberian Peninsula–Macaronesia^ as introduced by Scheunert and Heubl 2014), is supported in all
analyses and differs between cp and nr only with regard to
S. umbrosa Dumort., 1827. Within the Tomiophyllum clade,
fewer clades are present in both trees, and consistency regarding their members is much less pronounced. Taxa from the
New World group differently in the analyses: the plastid tree
supports monophyly of all New World species and three
BJapanese taxa^ (S. grayana Maxim. ex Kom., 1907;
S. duplicatoserrata Makino, 1906; S. musashiensis) in a
BNew World (BNW^)/Japan^ clade (Fig. 1a, Clade 4).
Within the clade, three subclades from the New World are
supported, one with mainly lowland taxa centered in
California (BCalifornia,^ 4a), one with subalpine taxa distributed in New Mexico and Arizona (BNew Mexico,^ 4d), and
one comprising species from the Greater Antilles, and in the nr
tree the mainly lowland Eastern North American
S. marilandica L., 1753 (BEastern North America,^ 4b). In
the nr trees, the NW/Japan clade collapses into its subclades,
with S. grayana being associated with Clade 4b and two of the
Japanese taxa forming Clade 4c (BJapan^). Clades 4a–d remain unconnected using the 2ISP-coded dataset, but based on
Evolutionary history of Scrophularia
Fig. 4 Neighbor-Net splits graph
for 163 accessions of
Scrophularia, based on
polymorphism p-distances (i.e.,
treating intra-individual site
polymorphisms as informative)
inferred from the ITS sequence
data set. Scale bar corresponds to
split weights based on ordinary
least squares estimates. Clades 1–
18 are marked, square brackets
below denote main species
groups. Asterisks highlight six
taxa having exceptionally long
branches in the 2ISP-coded
dataset, e.g., S. auriculata from
England, the accession with the
highest number of polymorphic
sites in this study, within Clade 7.
The Auriculata subclade of Clade
7 is indicated by an arrow, an
assemblage of taxa (node 87 in
Fig. 2a) which also includes the
Striata (16) and Scariosa (17)
clades is marked by the square
bracket above. sar sareptana, rost
rostrata, luc/myrio lucida/
myriophylla, umbr umbrosa
10.0
9.
1.
10.
18.
*
2.
megalantha
*
luc./myrio.
4b.
17.
16.
11.
olgae
sar.
13.
4d.
4a.
Oreosolen
rost.
*
12.
6.
ilwensis
3.
14.
umbr.
*
*
7.
*
5.
8.
ANASTOMOSANTES
the uncoded dataset, three of them form a weakly to very
weakly supported grade leading towards the China clade
(Fig. 2). By contrast, in the cp tree the NW/Japan clade is
sister to the Nodosa clade and S. cryptophila with moderate
to maximum support. Altogether, the members of 11 of the 18
main clades vary due to low resolution and/or incongruence.
Relationships among clades and the backbones of the trees are
only poorly resolved.
Bayesian analysis of the nr+clones dataset resulted in the
majority-rule consensus presented in Fig. 3. The topology is
largely similar to the 2ISP-coded ITS tree, with generally
equal or slightly lowered support values in clades without
clones. The clade containing S. ruprechtii (Fig. 2a, node 63)
collapsed into its subclades when clones were included.
ITS raw data network and incongruence
among chloroplast and nuclear markers
The Neighbor-Net splits graph computed from polymorphism
p-distances (Fig. 4) is extremely non-treelike; high complexity
and substantial ambiguity are present throughout the whole
ingroup. Its dumbbell-shaped structure traces the two main
phylogenetic groups also seen in tree reconstructions.
Accessions recovered in well-supported clades in Fig. 2a
mostly group together in the NN. Although distance-based
methods are often sensitive to missing data, their exclusion
did not improve the resulting network distinctly; rather, negative effects were observed as the amount of discarded data
15.
TOMIOPHYLLUM
increased. Thus, the NN in Fig. 4 is based on the full ingroup
dataset.
Although several clades are found in both plastid and nuclear trees, their composition is often considerably incongruent. Considering only cases of well-supported incongruence
(among the plastid and 2ISP-coded nr dataset) however, only
28 single accessions display hard incongruence as defined
above (HI accessions; highlighted in bold in Figs. 1a and 2).
These conflicts are based on switches within the same clade in
almost 60%; swaps among Clades 1–18 are observed in
S. umbrosa, S. kabadianensis L.Fedtsch., 1913, and
S. gorganica Rech.f., 1955. In S. ruprechtii, incongruence
involves a switch from a clade within Tomiophyllum in ITS
towards the Ningpoensis clade within a large clade of
Anastomosantes taxa in cp. In addition to the single HI accessions, the IPM clade (Clade 7) as a whole is incongruent as
well: it is embedded in the Tomiophyllum clade (cp) while
being sister to the BScopolii^ clade (Clade 8) of
Anastomosantes in ITS (see also its position in Fig. 5a).
Similarly, S. cryptophila and the Nodosa clade as shown in
the cp tree (excluding S. chlorantha) are also hardly incongruent. The detected HI accessions thus sum up to a total of 47.
The ILD test did not detect significant incongruence between the two chloroplast markers at the p = 0.05 confidence
level (p = 0.180), so combined analysis of the plastid data was
justified. However, as expected from visual inspection of the
trees, the test revealed severe incongruence among nuclear
and plastid datasets (p = 0.002). Deleting the 47 HI accessions
Scheunert A., Heubl G.
Fig. 5 Consensus networks
(CNs) based on each 1001 trees
from both runs of both single
marker Bayesian analyses of the
2ISP-coded nuclear ITS and
plastid datasets (which yielded the
consensus trees shown in Figs. 2a
and 1a). Scale bar units reflect the
frequency of splits within the
entered trees (COUNT option),
trees proportion threshold for
displaying splits = 0.33. a CN
containing all accessions. Square
brackets denote main species
groups, Clades 1–18 are marked.
Dashed lines highlight exemplary
taxa supported within the
indicated clade by one (the
plastid) dataset only. Asterisks
mark three species with
intermediate positions between
Clades 14 and 15. Hard
incongruent accessions are
highlighted by dots, APS-rich
accessions by crosses (terms as
defined in BMaterials and
methods^ and BResults^ sections).
b CN with 28 hard incongruent
accessions, the IPM clade, the
Nodosa clade without S. calycina/
S. pauciflora/S. chlorantha, and
17 APS-rich accessions removed
using the Bexclude selected taxa^
option. gorg gorganica
7. IPM
a
10000.0
TOMIOPHYLLUM
2. Ningpoensis
8. Scopolii
11. Canina
ruprechtii
16. Striata
1. Arguta
umbrosa
3. China
12 Libanotica
(16)
Oreosolen
17. Scariosa
18. Orientalis
gorg.
6. Calycina
(14/15)
4c. Japan
**
*
kabadianensis
OG
13. Variegata
15. Polyantha
4a. California
14. Scoparia
chlorantha
10. Peregrina
b
4b. Eastern NA
4d. New Mexico
5. Nodosa
9. Vernalis
1000.0
ANASTOMOSANTES
from the matrix did not change the result (p = 0.002); incongruence between datasets apparently is not limited to wellsupported conflicts. This is evident also from the consensus
network constructed with Splitstree (Fig. 5a). Although the
clades defined above can, at least partly, be traced in the CN,
the relationships among them are inextricably entangled, often
with similar numbers of trees supporting each of the conflicting splits. Bundles of parallel edges indicate conflicting signals or uncertainty also within several clades, and some are
even split in the CN (examples see dashed lines in Fig. 5a).
When HI accessions (labeled by black dots) were removed
from the network, the number of parallel edges decreased
considerably; the effect was much less pronounced when
APS-rich accessions (indicated by black crosses) were excluded (not shown). But even upon exclusion of all marked accessions, a large number of reticulations remained in the consensus network (Fig. 5b, 99 non-trivial splits left compared to 167
in Fig. 5a).
chlorantha
Divergence time estimation
The chronogram from the strict clock analysis is available in
Online Resource 6. Compared with the results from the
BEAST run with the prior only, posterior distributions differed from prior distributions in most cases, indicating that
the priors did not illegitimately influence the result. The
effective prior distributions of the prior-only run matched
the expected ones without any conflicts among priors.
Figure 1a shows the respective chronogram, inferred by relaxed clock analysis of the reduced dataset, on the majorityrule consensus topology derived from the MrBayes runs.
Node ages for all nodes alongside 95% highest posterior
density (HPD) intervals are provided in Online Resource
7. The mean substitution rate was calculated at 7.8592E−4.
The standard deviation of the uncorrelated log-normal clock
and the coefficient of variation were 0.47 and 0.49 (95%
HPD 0.26–0.71), indicating medium rate heterogeneity
Evolutionary history of Scrophularia
within the dataset. The origins of the genus and its basal
lineages can be traced back to the Miocene (23.1–11.2 million years ago, Bmya^, according to Fig. 1a); however, 11 of
the 18 clades did not start to diversify before the beginning
of the Pliocene (4.6–3.1 mya), and four clades even date to
the Pleistocene (2.3–1.6 mya). Three of those belong to the
Tomiophyllum clade, while old clades with crown ages in
the late Miocene (7.7–6.1 mya) include the BPeregrina^
(Clade 10), China and NW/Japan clades of the
Anastomosantes group.
Ancestral area optimization
Results of the four independent RASP runs were largely similar; all runs, including that with maxareas set to five, reached
convergence (standard deviations of split frequencies 0.0013–
0.0018). In Fig. 1a, pie charts at nodes represent the inferred
distributions; exact marginal probabilities for each range are
available from Online Resource 7. Southwestern Asia,
Turkey, and the Caucasus were inferred as the most likely
ancestral areas of Scrophularia (I and J, Fig. 1a, node 3) with
low marginal probability (32.70). The same ancestral distribution was inferred for the Tomiophyllum clade (node 43) with
high probability. Some clades within the Tomiophyllum clade
have other most probable ranges of origin, including the
Levant which is here defined as present-day Lebanon, Syria,
Israel, and Jordania (BScariosa^ clade 17, node 74), or
Afghanistan to the Western Himalayas (BPolyantha^ clade
15, node 82) with high frequencies of occurrence (89.22/
68.72, respectively). The ancestor(s) of the New World taxa
were reconstructed as distributed in Eastern Asia (node 24)
with high marginal probability. The uncertainty in ancestral
areas at nodes 61 and 63 argues for a more widespread occurrence of the most recent common ancestor, as also inferred by
the five maxareas analysis (results in brackets in Fig. 1a).
Discussion
Implementation of ITS intra-individual polymorphism
and chloroplast indel information
The genus Scrophularia provides a striking example of incongruence and ambiguity among, but also within, gene trees and
the corresponding DNA sequences. Although tree reconstruction per se seems appropriate to adequately illustrate the treelike parts of the genus’ phylogenetic history, it is hampered by
the widespread abundance of intra-individual site polymorphisms in the ITS sequences, which introduces considerable
conflict into the dataset. The problem of reduced tree resolution when analyzing polymorphic sequences has been known
for some time and has been noticed by several authors (e.g.,
Eidesen et al. 2007; Grimm et al. 2007), especially in cases
where hybrids (or more generally, taxa bearing APS) were
included into tree reconstructions (McDade 1995; Campbell
et al. 1997; Whittall et al. 2000; see also the weakly resolved
ITS phylogeny from dataset B in Fuertes Aguilar and Nieto
Feliner 2003). Several suggestions have been made on how to
deal with intra-individual polymorphisms. Among the mostused is pruning the respective taxa (e.g., Whittall et al. 2000;
Fuertes Aguilar and Nieto Feliner 2003); another possibility is
exclusion of polymorphic alignment positions (e.g., Scherson
et al. 2008). However, in Scrophularia, attempts to infer a
stable backbone using a representative subset of completely
monomorphic sequences did not improve the result (not
shown). Neither did the removal of APS-rich accessions and
exemplary deletion of the 17 HPPs considerably reduced the
resolution of the resulting phylogeny (see BResults^ section).
Further strategies include the replacement of polymorphisms
by missing data or the most common nucleotide, their resolution in favor of the stronger signal (Fehrer et al. 2009), statistical haplotype phasing methods (e.g., Stephens et al. 2001,
employed in Lorenz-Lemke et al. 2005), or cloning (see Nieto
Feliner and Rosselló 2007; however, cloning all accessions
may not be feasible in species-rich genera).
Rather than discarding or substituting sequence site variabilities, including them as phylogenetically informative characters seems to be a better solution. This can be achieved by
the approach employed here, using 2ISP coding and the ad
hoc implementation in ML from Potts et al. (2014) with additional adaptation of the method for BI. The procedure is
straightforward to apply, does not require the use of step
matrices, and allows the choice of appropriate DNA
substitution models. As in Grimm et al. (2007) and Potts
et al. (2014), recoding resulted in considerably increased resolution and enhanced support values in the phylogenetic tree:
for instance, the BLibanotica^ clade (Clade 12), corroborated
by morphological similarities of its taxa (Boissier 1879; Grau
1981) is only recovered in the Bayesian topology using 2ISP
coding (see Fig. 2a, node 58). Furthermore, the results obtained only very rarely contradict the topology generated without
additional coding, but mostly strengthen existing clades. This
corroborates the applicability of the method to the
Scrophularia dataset.
The latter seems to be characterized by polymorphisms
derived from several processes, including hybridization
resulting in APS according to Fuertes Aguilar and Nieto
Feliner (2003) but also others like e.g., inherited ancestral
polymorphism (which might lead to ILS). Furthermore,
homoeologous rDNA arrays might be subjected to differential
silencing after interspecific hybridization. This can produce
pseudogenes (Bailey et al. 2003; Volkov et al. 2007) recognizable by certain characteristics (Mayol and Rosselló 2001;
Grimm and Denk 2008). Although G+C contents were slightly lowered in nine uncloned accessions, only one of them
(S. nervosa Benth., 1846) showed a long edge in the NN
Scheunert A., Heubl G.
indicating increased distance (Fig. 4, Clade 18, see asterisk),
and no ingroup accession had substitutions or length changes
in the conserved parts of ITS1 or the 5.8 rDNA. However, it is
still possible that incipient pseudogeny accounts for some of
the observed sequence ambiguities. Finally, the rDNA marker
used here can be affected by recombination (see Álvarez and
Wendel 2003). No clear evidence of recombination was found
in the sequences; however, respective patterns would be difficult to detect in a complex dataset like this (see below), which
is additionally characterized by low sequence divergence.
Consequently, an influence of recombination processes cannot
be ruled out.
Due to the large number of polymorphisms, the processes
involved in their formation cannot be distinguished in the
Scrophularia dataset: informative 2ISPs are scattered across
the alignment, with very few coherent mutation patterns identifiable among them. This also excludes the explicit detection
of hybrids by comparing APS patterns as done by Fuertes
Aguilar and Nieto Feliner (2003). Instead, APS were tentatively used for character coding, to assess their influence on
the result. However, as reliable detection of all APS could not
be achieved, only selected putative APS within 17 HPPs were
coded. Analysis of the APS-coded dataset (results not shown)
yielded an intermediate topology with respect to those from
the 2ISP-coded and uncoded dataset. Supports of individual
nodes matched those of one or the other dataset (e.g., nodes
10, 20, 68, 95, see Online Resource 7), were intermediate
between them (nodes 19, 28, 40) or were worse or better
(nodes 4, 33, 61b, 80a). This suggests that the approach is
biased, and the results are dependent on the chosen subset of
alignment columns and their APS. We thus conclude that
restricting the coding procedure to APS only is not possible
in Scrophularia. By contrast, the 2ISP-informative approach
is particularly suitable for such datasets, as it does not discard
polymorphisms based on their origin. The improvement in
phylogenetic results seen here suggests that the information
contained in intra-individual polymorphisms can be used even
in cases where their sources are not exactly known, as also
emphasized by Potts et al. (2014).
An inherent disadvantage of the approach is that if artificial
ambiguities due to bad read quality are present, these are also
coded and could possibly blur relationships. In the present
study, discrimination of artificial double peaks from Breal^
polymorphism cannot be assumed to be completely reliable
(although low-quality sequences are not too frequent), which
was why all ambiguous bases were subjected to coding. In
such cases, results regarding accessions with potential data
quality issues, which have long branches in the coded tree
compared to the uncoded tree, should be regarded with caution. From the six long-branch accessions marked in Fig. 2
(thumbnail trees; the respective accessions are marked by
asterisks in the NN in Fig. 4), four can be assumed to be
influenced by artificial ambiguities, while long branches seen
in S. lateriflora Trautv., 1866, and S. auriculata from England
rather also reflect real polymorphism. However, although cautionary interpretation regarding taxa with many artificial characters seems justified, the fact that both trees are largely congruent and that nodes strongly supported in the uncoded tree
are only rarely weakened using 2ISP coding, indicates that the
method is robust to a certain amount of Bnoise.^ Thereby, the
tips of the phylogeny seem to benefit most from coding, while
more basal relationships remain unchanged or may even collapse (Fig. 2b, nodes A and B, node 53b).
It is important to note that although 2ISP coding improves
the resolution of the phylogenetic tree, it will not solve the
problem of inherent conflict present within the data, among
others incompatible signals after hybridization (Potts et al.
2014). Here, examination of the NN is useful as it represents
all information and all conflict contained in the sequences
(Bryant and Moulton 2004; Morrison 2010). Its highly interwoven structure as presented in Fig. 4 suggests that irrespective of the method applied, any bifurcating tree will suffer
from an insufficiently resolved backbone as well as an amount
of weakly supported nodes regarding certain relationships.
Clades showing high amounts of ambiguity in the NN
(Fig. 4) are likely to be sensitive to the type of analysis conducted (BI vs. ML), and will often remain insufficiently resolved (see Fig. 2a with inserts: Clade 15, Clade 7 with the
BAuriculata^ subclade, Clade 3, clade node 87, highlighted by
a square bracket in Fig. 4, S. ilwensis K. Koch, 1844). This
also means that no conclusions should be made based on weak
Bayesian support values in these cases (compare e.g., the
weakly supported sister clade of S. rostrata Boiss. & Buhse,
1860, and S. sareptana Kleop. ex Ivanina, 1972, to their positions in the NN, or the weak association of S. olgae Grossh.,
1932, with the Libanotica clade 12).
It is remarkable that generally, results from ML are
much less resolved (see Fig. 2); many nodes supported
by BI are not found in the best-scoring ML tree or are
supported by BS < 50. Furthermore, 2ISP coding does
not improve the situation, it seems to have only very little
effect. One possible reason for this is that ML reconstruction in RAxML per se is not completely naive concerning
polymorphic sites as outlined in Potts et al. (2014), and that
coding thus does not make much difference. The different
way site ambiguities are handled are also visible from the
different positions of the highly polymorphic S. auriculata
from England. Another explanation might be that in a
dataset with not too many, but clearly structured informative polymorphisms, bootstrapping more often produces
deviating topologies while BI quickly achieves one
Boptimal solution.^
Apart from the coding of ITS polymorphisms, additional
information can also be drawn from plastid trnQ-rps16 indels.
This marker is particularly suitable for Scrophularia, regarding both its high information content in terms of parsimony-
Evolutionary history of Scrophularia
informative characters (Table 1) as well as the occurrence of
diagnostic indels as already described in Scheunert and Heubl
(2011). Both NW/Japan (Clade 4) and Nodosa (Clade 5)
clades are exclusively characterized by one particular indel
of 597 bp length (Table 2). The same accounts for Clade 18,
the BOrientalis^ clade (764 bp), while a shorter indel (312 bp)
occurs in Clade 15 (Polyantha). These length differences constitute a reliable diagnostic tool for the respective clades; quick
discrimination can be achieved by a simple PCR reaction (see
Fig. 2 in Scheunert and Heubl 2011). Other indels might be of
limited phylogenetic value: the Scopolii clade is characterized
by a 318-bp indel, however, in the respective region of the
alignment (length types 2b–d, ranging roughly from position
518 to 845), indels also seem to arise spontaneously in single
unrelated taxa or even accessions (Table 2, Fig. 1a).
Utility of gene tree discordance, cloned sequences,
and Neighbor-Net splits graphs for tracing reticulate
events in Scrophularia
In addition to intra-individual variability, many taxa obtained
conflicting positions among nr and cp phylogenies. Moreover,
the number of incongruent accessions is probably
underestimated. As hard incongruence is by definition dependent on high supports of the associated nodes, it must be
assumed that lowered resolution, caused by large amounts of
PS in certain ITS sequences, prevented taxa from being recognized as hard incongruent. This is corroborated by the fact
that the removal of HI accessions did not render the ILD test
insignificant. Accordingly, the consensus network of both
markers showed a considerable amount of reticulation even
after excluding all HI accessions and APS-rich accessions
(Fig. 5b). In consequence, weakly supported cases of incongruence should not be ignored completely (see for example
the position of S. chlorantha between Clades 5 and 9/10).
Conflicts among datasets can be dealt with in various ways:
ignoring the incongruence altogether (concatenation approach; Gadagkar et al. 2005; L-Y Chen et al. 2014; but see
Rokas et al. 2003; Kubatko and Degnan 2007; Weisrock et al.
2012), pruning conflicting taxa prior to combined analysis
(Huelsenbeck et al. 1996), or duplicating them (Pirie et al.
2008, 2009). While incongruence in the present dataset is far
too widespread for pruning or using the taxon duplication
approach, it is also quite obvious from the CN from ITS and
plastid trees that combining both datasets in a concatenation
approach would yield a rather uninformative tree. Comparison
of individual plastid and nuclear gene trees including phylogenetic relationships of cloned sequences, and additional examination of the corresponding networks seems more suitable
in this case.
Similar to the ambiguity present within the ITS dataset, the
complex relationships found among the phylogenetic trees
suggest that a combination of different processes has
influenced the evolutionary history of Scrophularia. With
the exception of S. arguta, conspecific accessions are never
monophyletic, and sequences from three of the six cloned
accessions group with other taxa. Unequivocal identification
of evolutionary events like ILS and reticulation is not possible
based on the current dataset, but some inferences can nevertheless be made. For example, while earlier studies have determined the placements of S. auriculata and S. lyrata within
the Auriculata subclade (as in Fig. 2a, node 34), two of the
accessions unexpectedly group with S. scorodonia L., 1753
and S. laxiflora Lange, 1878, in the cp tree. Although lineage
sorting effects cannot be ruled out in this relatively young
group, the observed pattern is more likely to reflect geographic proximity: on the Azores, where the accession from
S. auriculata was sampled, S. scorodonia occurs as an introduced species, and the accessions from S. lyrata from Spain
and S. laxiflora were collected app. 150 km apart, while
S. lyrata (Crete) was collected as far as 2500 km away.
Together with the fact that hybridization of S. scorodonia
and S. auriculata is possible (Grau 1976; Dalgaard 1979), this
suggests that the observed pattern might be due to introgressive hybridization. Interestingly, no APS were found in the
ITS sequences of both accessions, which might be explained
by repeated backcrossing towards S. auriculata/S. lyrata
(chloroplast capture). Geographic patterns in plastid phylogenies, as opposed to those in ITS which are often morphologycorroborated, have been found in several other plant genera,
including Phlomis (Lamiaceae; Albaladejo et al. 2005),
Mitella (Saxifragaceae; Okuyama et al. 2005), or
Antirrhinum (Plantaginaceae; Vargas et al. 2009). However,
analysis of plastid data may also correctly infer relationships
which are blurred in ITS. This is the case in e.g., the Scariosa
clade, which is expected to include S. hierochuntina Boiss.,
1853 based on morphological evidence (Boissier 1879, Flora
Orientalis; Eig 1944; Grau 1980). Correct interpretation of
phylogenetic relationships in Scrophularia therefore requires
careful comparisons of all results.
Recent or ancient hybridization can result in deviating ITS
copies (see above) as well as incongruence among markers
(Fuertes Aguilar and Nieto Feliner 2003; Vriesendorp and
Bakker 2005; Peng and Wang, 2008; Vargas et al. 2009),
and obviously had an important impact on the speciation process in Scrophularia (as demonstrated in Scheunert and Heubl
2014). In such cases, cloned sequences are particularly useful
as they can provide information on putative parent lineages
(Fig. 3). In the species studied here, clones in general
contained all variation corresponding to the extracted APS
from the Bdirect sequences^ (i.e., obtained from direct sequencing), and often even more. This means that cloning
should be favored over direct sequencing as far as possible,
especially when high polyploids are present and polymorphic
sites are more easily missed (Joly et al. 2006). Scrophularia
auriculata (with 2n = 84 chromosomes) was already proposed
Scheunert A., Heubl G.
to be an allopolyploid resulting from hybridization between
S. lyrata or S. hispida Desf., 1798 (both 2n = 58) and
S. umbrosa (2n = 26) or their ancestors (Grau 1979;
Scheunert and Heubl 2014). The English voucher specimen
of S. auriculata, with its chromosome number determined to
match the typical number for the species, has as much as 13
PS in the direct sequence. Cloned sequences are separated into
two distinct clades: six clones obtain a position similar to that
of the accession in the cp tree; they are part of a clade also
containing both direct sequences of S. auriculata, S. racemosa
Lowe, 1831, and S. hispida. The fact that the direct sequence
of S. auriculata England is sister to S. hispida using the 2ISPcoded dataset, and that none of the clones is found within the
monophyletic clade of clones from S. lyrata, argues for
S. hispida as potential parent. The remaining ITS clones are
situated in a monophyletic clade at the basal polytomy of the
IPM clade, but without depicting a sister relationship to
S. umbrosa. This might suggest that the hybridization event
is more ancient, with more time for accumulation of
autapomorphisms in S. auriculata. In such cases, parental lineages would be more difficult to trace due to a greater accumulation of autapomorphies (Wolfe and Elisens 1994;
Baumel et al. 2002; Vargas et al. 2009). The distinct status
of S. auriculata England is also illustrated in the NN, where
it is isolated in the Auriculata subclade and positioned closer
towards the Scopolii clade (Clade 8), another potential parental lineage related to S. umbrosa (Fig. 4, species marked by an
asterisk within the subclade). It remains unclear why traces of
the hybrid ancestry of S. auriculata can be found in only one
of the sampled accessions. However, the variable,
intermingled phylogenetic relationships and weakly defined
species boundaries among the closely related taxa of the
IPM clade have not been fully recovered yet.
The Caucasian endemic S. ruprechtii is part of a predominantly Turkish-Caucasian clade in ITS. It was cloned to elucidate a possible hybrid origin concerning the Ningpoensis
clade, with which it is closely associated in the cp tree,
resulting in a clearly intermediate placement in the CN
(Fig. 5a). However, although two diverging ITS ribotypes
were found, neither of them bear signs of relationships with
lineages from outside Tomiophyllum. Close relationships with
S. olympica and S. imerethica Kem.-Nath., 1955, are corroborated by six of the clones, a clade of four clones remains
unresolved.
The six species of the BScoparia^ clade (Clade 14) in ITS
occur in partly overlapping distribution areas ranging from
Central Asia across Afghanistan and the Western Himalayas
to China and Siberia. They represent a rather homogeneous
assemblage of subshrubs featuring few-flowered cymes, exserted stamens, linear staminodes, and leaves which are divided to various extents. Scrophularia multicaulis Turcz., 1840, a
perennial species with included stamens, does not fit into this
pattern, and also is the only species without any PS in the ITS
sequences, while two (in S. kiriloviana Schischk., 1955) to
eight (in S. incisa) PS are found in all other members. The
clones from S. incisa reveal two main ribotypes, one of which
is associated with S. multicaulis. Three of the clones remain
unresolved within the clade. It seems reasonable to assume
that S. incisa was the result of a hybridization event that involved S. multicaulis and a second member from the Scoparia
clade, which would explain the morphological similarities.
Shared PS in the ITS sequences connect S. incisa to
S. scoparia Pennell, 1943, but also to S. dentata Royle ex
Benth., 1835. Further evidence can be drawn from the occurrence of a sequence length polymorphism within ITS2, which
is characteristic for the Scoparia clade and was excluded from
calculations. The morphologically very variable S. scoparia
(as all other sampled Scrophularia accessions) has a clear
sequence containing a GTG motif, while S. multicaulis shows
a clear sequence with a GTGTG motif at the respective position. Scrophularia incisa (as well as all other members of the
clade) features a length polymorphism, with some ITS copies
having GTG (here present in clones 6 and 15) and others
having GTGTG. This might support the hypothesis that
S. scoparia, which is likewise unresolved in Fig. 3, acted as
second parent for S. incisa. Apart from the complex relationships within the clade, the Scoparia clade is also entangled
with the Polyantha clade, whose species according to the
ITS tree are distributed from Central Asia and Afghanistan
southeastwards as far as the Eastern Himalayas. Three taxa
(among those S. incisa as well as S. multicaulis) switch positions between the two clades in plastid and nuclear reconstructions, resulting in their intermediate position in the CN
(Fig. 5a, marked by asterisks).
The origin of Scrophularia
Although divergence dating should rely on results from different cell compartments whenever possible, we refrained
from performing molecular clock analyses on the ITS dataset.
In Scrophularia, the respective sequences are highly polymorphic and considerably influenced by reticulation. This means
that they cannot provide an entirely tree-like signal, which
makes them unsuitable for molecular clock or ancestral area
inferences. Results are therefore based on one, the plastid,
marker only. However, it still should be kept in mind that
estimation of divergence times might be impaired by the presence of a reticulate history. For example, for a hybrid lineage,
one distinct time of divergence may not represent its true history which might have involved several periods of gene flow
(Payseur and Rieseberg 2016) or several independent hybridization events. Furthermore, gene flow between two species
might lead to underestimation of their divergence time, as
demonstrated by Leaché et al. (2014) for species trees.
According to the molecular dating analysis, Scrophularia
diverged from Verbascum around the Oligocene/Miocene
Evolutionary history of Scrophularia
boundary (approximately 23 mya), with diversification of major lineages starting in the Miocene, within approximately the
last 15 my. These results are comparable to those of a recently
published time-calibrated phylogeny based on ndhF sequence
data of Lamiales, where the authors used a combination of
several fossil and secondary calibrations (Navarro Pérez
et al. 2013). Ages as inferred here are in clear contrast with
considerably older divergence times obtained in a small-scale
study on New World species based on secondary calibration
of the root only (Scheunert and Heubl 2011). This is probably
due to too small taxon sample size and the choice of method
and information source for obtaining secondary calibration
points. Ancestral area reconstructions revealed that
S c rop h u l a r i a o r i g i n a t e d i n a re g i o n c o m p r i s i n g
Southwestern Asia and Turkey (Fig. 1a, node 3), which corresponds to its present-day primary center of diversity. This
contradicts Stiefelhagen (1910), who promoted the Himalaya
as ancestral region for the genus.
In the Miocene, Southwestern Asia was under strong influence of processes initiated by the collision of the Arabian with
the Eurasian plate, which had started in the late Eocene and led
to the formation of the main mountain ranges within the region (Djamali et al. 2012a). During that period, Asian biota
also were affected by substantial climatic and environmental
changes, including a shift towards increased seasonality (An
et al. 2001) and the aridification of interior Asia (Guo et al.
2002, 2004; Fortelius et al. 2006; Miao et al. 2012). A combination of these processes is likely to have triggered the divergence and diversification of Scrophularia. At the time inferred for the origin of the genus, the final closure of the
Tethyan seaway and the retreat of the Paratethys ocean
(Ramstein et al. 1997; Mouthereau et al. 2012) created additional suitable land area for plant colonization in Southwestern
Asia, e.g., in the Zagros region. While the Greater Caucasus
was still largely submerged at that time, the Alborz and
Anatolian highlands already existed for a longer time
(Popov et al. 2004). Also, according to time estimates
presented here, diversification of Scrophularia into its
main lineages began during a period where new suitable
habitats for species adapted to montane environments
came into existence in its ancestral region; by the emergence of the Greater Caucasus and its transformation into
a mountain chain (approximately 14–13 mya and 9 mya;
Meulenkamp and Sissingh 2003; Popov et al. 2004;
Olteanu and Jipa 2006), the uplift of the Iranian Plateau
(from 15–12 mya; Mouthereau et al. 2012; Djamali et al.
2012a and references therein), and the formation of the
Western Alborz (approximately 12–10 mya) and Zagros
(approximately 15–10 mya) mountains (Dercourt et al.
1986; Popov et al. 2004; Guest et al. 2007; Mouthereau
et al. 2012). Additionally, the establishment of continental
climate conditions (Berberian and King 1981) with increasing aridity (Ballato et al. 2010) during this period
could have triggered the colonization of more moderate,
humid habitats of higher elevations (Roe 2005).
Establishment of the genus in mountainous regions might
have been accelerated by its potential for frequent and successful interspecific hybridization and (allo-)polyploidization.
Both evolutionary processes enable adaptation to new or hitherto unsuitable environments by the rapid formation of new
phenotypic combinations, promoting adaptive radiation and
colonization, and thus have been associated with speciation
events and diversification (Stebbins 1959; Seehausen 2004;
Mallet 2007; Estep et al. 2014). While Verbascum resembles
its sister Scrophularia in many respects including habitat
requirements and a Southwestern Asian center of diversity,
the former genus apparently lacks the aforementioned
peculiarities. Although species easily hybridize wherever
they grow together, the resulting offspring is always sterile
according to Murbeck (1933) and Huber-Morath (1978).
Reported chromosome numbers within Verbascum mostly
range between 2n = 30–36, with the highest count being
2n = c. 64 (Goldblatt and Johnson 1979–). In contrast,
Scrophularia features numbers from 2n = 18 (in S. altaica
Murray, 1781) up to 2n = 96 (in the NW/Japan clade), with
several clades being characterized by (high) polyploidy (Shaw
1962; Goldblatt and Johnson 1979–; Scheunert and Heubl
2014). It seems worth mentioning that the highest polyploids
within Scrophularia are concentrated in regions where only
few or no Verbascum species occur (i.e., China and the New
World). Carlbom (1969) concluded that polyploidy in
Scrophularia has triggered migration into the rough environments of higher altitudes as well as regions of higher latitudes.
The predisposition for polyploidy and hybridization found in
Scrophularia i s shared by other lineages within
Scrophulariaceae (e.g., Diascia or Nemesia; Steiner 1996;
Datson et al. 2006) and related families (e.g., Clay et al.
2012 and references therein; Rojas Andrés et al. 2015).
Apart from that, mountainous regions themselves promote
plant species diversification: diversification rates have been
shown to be elevated in regions subjected to active orogeny
(for a short overview see Hoorn et al. 2013 and references
therein), and mountain ranges provide complex, heterogeneous habitats on small geographical scales, which facilitate
adaptive speciation and allopatric divergence (Lobo 2001;
Djamali et al. 2012a and b and references therein; Wen et al.
2014), and can also stabilize new hybrids by isolating them
from their parents. These conditions might be assumed to also
have produced the high levels of endemicity in Scrophularia
as mentioned by Vaarama and Hiirsalmi (1967). Even in the
absence of detailed distribution mapping, it seems likely that a
combination of geographic isolation/habitat fragmentation
and successful hybridization has been the key factor in the
diversification of Scrophularia. This has also been suggested
for other genera occurring in the Tibetan Plateau or mountainous regions of the Mediterranean (e.g., Senecioneae,
Scheunert A., Heubl G.
Fig. 6 Biogeographic history of
Scrophularia. The ancestral
region of the genus is marked in
gray; red arrows indicate main
dispersal routes as inferred from
plastid DNA data. Note that
arrows in some cases may
illustrate more than one dispersal.
Boxes next to arrows denote the
approximate time of the migration
event in million years ago, drawn
from crown ages of the respective
clades
6-2
1.5/2.5
6/6.5
4.5
2.5
3.5
2
Asteraceae, J-Q Liu et al. 2006; Linaria, Plantaginaceae,
Blanco-Pastor et al. 2012; Meconopsis, Papaveraceae, Yang
et al. 2012; Rhodiola, Crassulaceae, Zhang et al. 2014).
A remarkable result in the ITS tree is the basal position of
the annual S. arguta as sister to the remainder of the generally
persistent Scrophularia (Fig. 2a, node 3). However, upon a
closer look, this position might likely be artificial.
Potentially higher evolutionary rates in annuals can result in
support of a sister relationship of the former to their perennial
relatives (e.g., Laroche and Bousquet 1999; Andreasen and
Baldwin 2001; Tank and Olmstead 2008; Müller and Albach
2010; J-X Yue et al. 2010). But, although rarely, other annual
species are found in Scrophularia which obtain inconspicuous
placements, e.g., in Clade 6 or 10 (S. calycina Benth., 1835,
S. peregrina L., 1753). More importantly however, and unlike
most of the other Scrophularia species, self-pollination is
common in S. arguta, and the species is unique within the
genus in possessing a mixed mating system of chasmogamous
flowers occasionally complemented by small cleistogamous
flowers near the ground. High substitution rates have been
correlated with a selfing breeding system (Glémin et al.
2006), although this is subject of debate (Wright et al. 2002;
Müller and Albach 2010). Moreover, mating shifts seem to
create strong interspecies isolation barriers which effectively
prevent hybridization (Wright et al. 2013). The Arguta lineage
might thus owe its isolated position to the accumulation of
mutations due to breeding system effects. It is likely that the
particular reproductive traits of S. arguta have enabled colonization of habitats otherwise unsuitable for Scrophularia. In
the hot and dry environments of e.g., the Sudan, Eritrea,
Somalia and Oman, S. arguta consequently is the only representative of the genus. In the plastid tree, the species is shown
as an earlier diverging lineage and is sister to the
1.5
4.5
4.5
Tomiophyllum clade. Its ancestral area could not be satisfactorily determined; analyses with a maximum of five areas
yielded an ancestral range corresponding to its present-day
distribution (areas ABDEI; Fig. 1a, node 85). A recent study
on S. arguta, based on representatives from several populations although not covering the whole distribution range,
found evidence for a westward expansion from the east of
its distribution range (Valtueña et al. 2016), which reaches
its limit on the Arabian Peninsula.
Reconstruction of major evolutionary events
Based on individual clade ages and ancestral areas (Fig. 1a),
several expansions of the genus in various directions can be
hypothesized (Fig. 6). Eastward migration events to China
and the Tibetan Plateau region are inferred at approximately
6 mya (nodes 10 and 24) and later. Given the preference for
mountainous habitats in Scrophularia, dispersal along higher
mountain chains as corridors for colonization seems reasonable. Evidence for growth of the Kunlun Shan in the northern part of the Tibetan Plateau has been reported since
Eocene times (see Yuan et al. 2013), while uplifts of the
Tian Shan located to the northwest presumably occurred
from the late Oligocene through the Miocene until approximately 7 mya or later (Abdrakhmatov et al. 1996; Sobel
et al. 2006; citations in Miao et al. 2012). The Hindu
Kush, which spreads farther west into Afghanistan,
underwent uplift around the Oligocene-Miocene boundary
(Hildebrand et al. 2000), with tectonic processes in the region occurring much earlier (Dercourt et al. 1986;
Hildebrand et al. 2001). The Kopet Dag finally, roughly
situated between the Hindu Kush in the east and the
Alborz of Iran in the west, only re-emerged as a mountain
Evolutionary history of Scrophularia
chain at approximately 10 mya or later, after submergence
following higher-altitude phases from the late Oligocene until the early Miocene (Dercourt et al. 1986; Popov et al.
2004). Altogether, this means that by the time inferred for
the first eastward migrations of figworts, a more or less continuous mountain belt should have existed, which connected
the Southwestern Asian Alborz to the Himalayas and the
Tibetan Plateau, and presumably provided a suitable pathway
for dispersal of Scrophularia. Transitions of this kind from
Western/Central Asia to Eastern Asia (or vice versa) have
been reported in several other genera, including Incarvillea
(Bignoniaceae; S-T Chen et al. 2005), Rhodiola (Zhang et al.
2014), or Solmslaubachia (Brassicaceae; J-P Yue et al.
2009); the latter genus inhabits alpine scree-slope habitats
similar to several Scrophularia species.
China has been colonized at various times and by different
lineages; the most important secondary diversity center of
Scrophularia harbors, among others, species from the
BVernalis^ (Clade 9) and Calycina clades, S. umbrosa, and
species from the Scoparia/Polyantha clade of Tomiophyllum.
These mainly alpine taxa have extended their distribution
areas from Central Asia, Siberia, or Southern Asia into
China. In contrast, the China and Ningpoensis clades almost
exclusively consist of Chinese species. When disregarding
altitude, Chinese Scrophularia have rather similar habitat requirements (especially within the China clade s.str. as shown
in Fig. 2); they occur in humid conditions in mountainous
forests or grasslands, often in crevices and among rocks.
The Ningpoensis clade (Clade 2), with its crown age estimated at approximately 3 my, constitutes an eastern group of
mainly non-alpine taxa including the pharmaceutically important S. ningpoensis Hemsl., 1899. Its distribution extends from
the eastern parts of China to Korea, Japan, and Taiwan; repeated contacts between those landmasses from the late
Miocene onwards offered opportunities for dispersal
(references in Qiu et al. 2011). In contrast, the taxa of the
China clade s.str. (Clade 3 in the ITS tree) are mostly alpine
species with mostly narrow distributions. Most species from
the BHengduan^ subclade resolved in the plastid tree are restricted to the Hengduan mountains (located in parts of
Sichuan and Yunnan provinces with adjacent Tibet), which
are considered one of the world’s biodiversity hotspots
(Mittermeier et al. 2004). The BCentral^ subclade comprises
high-alpine and subalpine species distributed in central parts
of China.
Crown ages of both subclades were estimated at about
4.5 my during the Pliocene. Many authors have attributed
diversification events in the region to the uplift of the
Tibetan Plateau (see review by Qiu et al. 2011 and references
in Y-S Sun et al. 2012), often without checking for exact
spatial and temporal concordance among inferred divergence
times and geological events. This however seems to be
indispensible given the complex geological history of the
Tibetan Plateau and the ongoing debates on appropriate
models for its formation (Yuan et al. 2013; C-S Wang et al.
2014; J-J Li et al. 2015). Diversification of the Hengduan
subclade from the early Pliocene on coincides with a period
of uplift hypothesized for the Hengduan Shan by B-N Sun
et al. (2011) and Ming (2007). However, it may not be necessary to invoke uplift as a cause for diversification when its
result seems to be more important: the extreme topography
with alternating high peaks and deep ridges and the variety
of vegetation types and climatic conditions which characterize
this biodiversity hotspot (see Boufford et al. in Mittermeier
et al. 2004) effectively stimulates speciation and leads to high
species diversity and endemism. Generally, high species numbers have been linked with the Bextreme physiographical heterogeneity of temperate eastern Asia^ by Qian and Ricklefs
(2000). Finally, the present-day distribution patterns of the
Hengduan and Central subclade species might also be the
result of recolonization after the Last Glacial Maximum (approximately 24,000–18,000 years ago) from suitable refugial
areas in the region; such have been recognized in the
Hengduan Shan (e.g., for Metagentiana, Gentianaceae, S-Y
Chen et al. 2008; Angelica, Apiaceae, Feng et al. 2009;
Lepisorus, Polypodiaceae, L Wang et al. 2011) and, for the
Central subclade, in the Qinling mountains or, more generally,
the BNortheast Qinghai-Tibetan Plateau edge^ (Qiu et al.
2011).
According to our reconstructions, North America was colonized up to three times independently from Eastern Asia
(Figs. 1a, 2). The Japanese and most of the New World taxa
are connected to the Nodosa clade (plastid tree) as well as taxa
from the China clade, with whom they share a clade in the
uncoded ITS tree (Fig. 2b, node A) and the coded ITS+clones
tree (Fig. 3). Divergence from the Asian ancestors has happened approximately 6 mya at the earliest (Fig. 1a, node 24).
This corresponds to a general Eastern Asian-North American
disjunct pattern found in many plant genera, e.g., Picea
(Pinaceae; Lockwood et al. 2013), Gleditsia (Fabaceae;
Schnabel et al. 2003), Triosteum (Caprifoliaceae; Gould and
Donoghue 2000), and also Scrophulariaceae (Hong 1983),
among many others (see H-L Li 1972; Boufford and
Spongberg 1983; Hong 1993; Xiang et al. 1998; Wen et al.
2010, 2014). Similar divergence times of North American
from Eastern Asian lineages have been reported in Kellogia
(Rubiaceae, 5.42 ± 2.32 mya; Nie et al. 2005) and Rhodiola
(5.3 mya, 95% HPD 2.3–9.1 mya; Zhang et al. 2014). Both
studies suggest long-distance dispersal for colonization of
North America. Zhang et al. (2014) additionally hypothesize
migration across the Bering Land Bridge (BLB; Tiffney and
Manchester 2001); this was also proposed for Angelica
genuflexa max. 4.3 mya, by Liao et al. (2012). During which
periods and how long the BLB was available for plant migrations remains a matter of debate. Estimates for the opening of
the Bering Strait range from 3.3 to 9 mya (Brigham-Grette
Scheunert A., Heubl G.
2001; Denk et al. 2011); however, even after the final
flooding, intermittent short-time closures of the Strait have
been assumed, among others at 4.9, 4.0, 3.3, and 2.5 mya
(KG Miller et al. 2005). Cold-adapted Scrophularia taxa
could have spread to the New World during times when landmasses were connected; long-distance dispersal however is
likely to have played a role as well, especially during later
periods (see Fig. 1a, nodes 25, 26, 28). Scrophularia seeds
do not possess special adaptations favoring any mode of dispersal; however, they are easily dispersed by wind due to their
small size and weight, and thus may not have been dependent
on suitable land bridges.
In accordance with results obtained by Scheunert and
Heubl (2011), the New World taxa of Scrophularia are divided into three geography-based clades, whose distribution
ranges do not overlap much and which receive stronger supports in the plastid tree. Most of the North American species
are closely related; they have been successfully hybridized
(Shaw 1962) or even intergrade naturally in contact zones
(e.g., S. parviflora Wooton & Standl., 1913 and
S. californica Cham. & Schltdl., 1827; Kearney and Peebles
1951). Shaw (1962) emphasized that rather than reproductive
isolation, geographic barriers seem to play an important role in
maintaining the species (compare Carlbom 1969), an assumption that exactly fits the general diversification mechanisms
discussed above and is corroborated by the characteristics of
the three clades found here. A similar situation was reported
for Jamesbrittenia, another Scrophulariaceae genus prone to
successful interspecific hybridization, where geography helps
to maintain species identity (Verboom et al. 2016). Species
diversity is greater in the west (10 species) than in the east
of the North American mainland (two species), possibly due
to greater geographic heterogeneity in the former (Qi and
Yang 1999) but also the California floristic province biodiversity hotspot located there (Mittermeier et al. 2004).
Apart from eastward migrations by Scrophularia, resulting
in the China and New World clades, westward movements
from the ancestral region led to the colonization of the
Mediterranean, Northern Africa, and Europe. Scheunert and
Heubl (2014) recently found that the IPM clade (Fig. 2a, node
32/Fig. 1a, node 45), which comprises the majority of Iberian
and Macaronesian species, is of hybrid origin, involving progenitors both of the Scopolii clade or S. umbrosa, and the
BCanina^ clade (Clade 11) or allies (Fig. 2a, node 31 or 32/
Fig. 1a, node 44). According to reconstructions using the
plastid dataset, these ancestors were distributed in
Southwestern Asia and in the Turkey-Caucasus region, respectively (Fig. 1a, node 40 or 86/44). The approximate time
of the hybridization event, limited by divergence from the
parental lineage and diversification of the hybrid lineage, is
assumed at around 5 mya; the ancestor of the IPM clade was
most likely distributed in the Western Mediterranean and diversified from about 4.5 mya (Fig. 1a, node 45; Scheunert and
Heubl 2014). The role of the Irano-Turanian floristic region as
a key source for colonization of the Mediterranean has been
emphasized (Comes 2004; Djamali et al. 2012b), especially
for temperate elements (Quézel 1985; Thompson 2005;
Mansion et al. 2008).
The Tomiophyllum clade
The relative ages of the two main lineages within Scrophularia
have been discussed by various authors. Scrophularia sect.
Tomiophyllum might be regarded as Bprimitive^ due to putatively ancestral traits like the often xerophytic, subshrubby habit of
its members and the general lack of polyploid chromosome
numbers (Carlbom 1969). The section is centered in the
Caucasus, Iran, Iraq, and Turkey, with approximately half of
the sampled taxa distributed there, and is completely absent from
Macaronesia and the New World. On the other hand, the mainly
herbaceous, richly foliated, often meso- or hygrophytic members
of S. sect. Anastomosantes (Stiefelhagen 1910) are characterized
by a large number of polyploid species, a wide ecological amplitude and a geographic distribution which exceeds that of S.
sect. Tomiophyllum by far. Regarding molecular results, the
Tomiophyllum lineage is highly supported as a distinct clade in
both analyses and is nested within clades of Anastomosantes
taxa (Figs. 1a and 2a). This reveals S. sect. Tomiophyllum to be
derived from within S. sect. Anastomosantes.
The factors leading to the evolutionary success of the
Tomiophyllum lineage remain uncertain. One might speculate
that changes in aridity in its ancestral region during the second
half of the Miocene and later (Ballato et al. 2010) had an
influence on its divergence and diversification (which started
approximately 8 mya). While ecological preferences of members of Scrophularia sects. Anastomosantes and
Tomiophyllum overlap, their habitats reveal a certain shift
from moist sites on riverbanks and in forests (in the former)
towards rock crevices and gravelly substrates with low humidity in the latter, illustrating a greater tolerance of dry conditions. This is also reflected in differences in their respective
distributions, with Tomiophyllum species predominantly
inhabiting the dry parts of e.g., Iran and Turkey, while not
necessarily being absent from other areas. Several of the xerophytic species also take advantage of the lower temperatures
at higher altitudes; the few desert representatives tend to flower during early spring, with their rhizomes persisting in crevices during hot periods.
It is noteworthy that the phylogenetic (and also morphological) boundaries between the Anastomosantes and
Tomiophyllum groups are also not altogether strict. For example, the position of S. megalantha Rech.f., 1955 in the NN,
with no unequivocal connection to any of the Anastomosantes
clades and closer to the center of the graph (Fig. 4), indicates a
certain affinity to the Tomiophyllum clade. This is also supported by the trees in Figs. 1a and 2a, however, with
Evolutionary history of Scrophularia
consistently weak supports. On the other hand, similarities to
S. sect. Anastomosantes can be found in some members of the
Tomiophyllum clade, e.g., in the mainly Turkish and
Caucasian S. ilwensis, which was considered part of S. sect.
Anastomosantes by Stiefelhagen (1910) and occurs in more
humid habitats like forests or near water. While plastid reconstructions of the Tomiophyllum clade support a main split into
two, once more geography-based, main clades, this species is
sister to the remainder of Tomiophyllum in the ITS trees
(Fig. 2). Its distinct status is reflected also in its intermediate
position in the NN (Fig. 4). Apart from S. ilwensis, 14 further
taxa (including the BStriata^ (Clade 16) and Scariosa clades)
obtain basal positions within the Tomiophyllum clade in the
uncoded ITS tree. In the 2ISP-coded tree, these species form a
clade; both placements are however weakly supported
(Fig. 2a, node 87; Fig. 2b, nodes 52 and 53a) due to inherent
ambiguity in the data as discussed above. The NN (Fig. 4,
square bracket) clearly depicts the complex relationships of
this assemblage and also a certain shift towards
Anastomosantes. Scrophularia nabataeorum Eig, 1944 from
the Scariosa clade indeed features morphological traits typical
for S. sect. Anastomosantes as well: Eig (1944) described its
ambiguous characteristics in between the two main sections
and stated himself being Bundecided as to the affinity of this
species.^ Interestingly, the Striata clade comprises some of the
few species that have colonized truly arid environments, occurring in steppe and desert habitats.
Both Striata and Scariosa clade give evidence for migration
into areas west and southwest of present-day Iran, which was
facilitated since the dry-up of the Mesopotamian Basin in the
late Miocene (Popov et al. 2004). This has led to the colonization of the Levant, the Arabian Peninsula south of Iraq, and
also Eastern North Africa by members of these clades.
Notably, while several IPM clade species also extend into
(or are endemic for) western regions of North Africa, the three
species confined to its eastern part are found within the Striata
and Libanotica clades. Other species occur throughout
Northern Africa (S. canina, S. peregrina, S. syriaca Benth.,
1846, S. arguta).
methodical workflow as presented here is suitable for any plant
group where similar problems are encountered and laborious
search for (potentially likewise problematic; Nieto Feliner and
Rosselló 2007) low-copy nuclear markers or cloning of all taxa
cannot be considered.
The molecular phylogenies revealed two large groups of
species (of which only one is monophyletic) corresponding to
previously described taxonomic entities. The emergence of
Scrophularia in the Miocene and its diversification are closely
linked to geological and climatic events in the Irano-Turanian
region and Central/Eastern Asia. Most diversification events
as well as further successful dispersals to other regions were
dated to the colder Pliocene-Pleistocene period.
The inferred spatio-temporal framework will provide a solid basis for future studies focusing on specific clades or morphological questions. It can be assumed that the considerable
morphological variability is linked to the complex evolutionary history of the genus; this is relevant also regarding previous taxonomic concepts, which need to be re-evaluated. A
survey of the relevant morphological traits, together with karyological analyses, will complement the present study from a
more taxonomic perspective, also with respect to the small
Himalayan genus Oreosolen, which has to be transferred to
Scrophularia (Scheunert and Heubl in preparation).
Acknowledgements The authors wish to thank the herbaria and curators of A, B, E, GH, HAL, KUN, M, MA, MSB, W, and WU, for providing loans, for access to the specimens for study and for help in
obtaining leaf material. Dirk Albach, Cheng-Xin Fu, Mark Mayfield,
Søren Rosendal Jensen, Andrej Sytin, and Lang-Ran Xu are acknowledged for sending plant material from specimens deposited in GOET,
HU/HZU, KSC, HSNU, LE, and WUK. Christian Bräuchler, Matthias
Erben, and the Botanical Garden Munich kindly contributed seeds or
plants required for study. We are grateful to Dirk Albach for sending
photos and specimens and for continued support and helpful suggestions;
to Guido Grimm for the generous time given for help with and discussion
of the ITS data; to Susanne Renner and Lars Nauheimer for advice in
divergence dating and BEAST; and to Ingo Michalak for help with
RAxML and substitution model details. Alastair Potts and three anonymous reviewers are acknowledged for helpful comments which improved
the manuscript. We thank Tanja Ernst for invaluable help in the lab and
Wei Jie for providing translations from Chinese language.
Compliance with ethical standards
Conclusions
The present paper represents the first comprehensive phylogenetic study of the genus Scrophularia, based on a broad taxon
sampling including representatives of all sections. This study
has confirmed the monophyly of the genus but has also provided
evidence for significant phylogenetic incongruence and ambiguity among and within sequence datasets. Exemplary cloning of
taxa showed that intra-individual site polymorphism in ITS is
widespread. Our study suggests that conflicting signals in
Scrophularia derive from a variety of sources, most importantly
reticulation, due to frequent hybridization and introgression. The
Conflict of interest The authors declare that they have no conflicts of
interest.
This article does not contain any studies with human participants or
animals performed by any of the authors.
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5. General Discussion
5.1. Phylogenetic relationships in Rhinantheae and taxonomic implications
Our analyses of tribe Rhinantheae yielded two phylogenetic trees partially
supporting differing relationships. However, pruning of five incongruent taxa (see
below) and subsequent combination of the data resulted in a better resolved phylogeny
with a highly supported backbone. The phylogenies of Rhinantheae, as presented in
Article I (and summarized in Fig. 9), corroborated previous findings in several ways.
Melampyrum was sister to all other taxa within Rhinantheae in accordance with Bennett
and Mathews (2006), and Rhinanthus, Lathraea and Rhynchocorys were grouped
together in what we referred to as the "RRL clade". Bartsia alpina was the firstbranching taxon within the core group of Rhinantheae (in agreement with Těšitel et al.,
2010). This result was consistent among four accessions covering the geographic range
of the species. It has already been known for some time that the New World species of
Bartsia s.l. do not cluster with the generic type, but with species of Parentucellia (see
below). However, our analyses for the first time showed that the East African species of
Bartsia s.l. are distinct as well: they shared a clade with the monotypic Hedbergia
abyssinica, far from B. alpina and New World Bartsia s.l.
As a consequence, some taxonomic rearrangements were necessary for the
polyphyletic Bartsia (changes are mapped in Fig. 9). In the circumscription of Molau
(1990), the genus contained 49 annual or perennial, herbaceous to rarely suffrutescent
species: Bartsia alpina in Bartsia sect. Bartsia, two afromontane species within B. sect.
Longiflorae Molau, B. trixago in B. sect. Bellardia (All.) Molau, and 45 South American
species in four sections, B. sects. Orthocarpiflorae Molau, Strictae Molau, Laxae Molau
and Diffusae Molau. According to our taxonomic concept, the generic type Bartsia alpina
(photo see Fig. 9) is left alone in the traditional genus Bartsia, which is now defined as a
monotypic genus containing one perennial, obligate hemiparasite with the following
characteristics: geophytic, possessing a persistent subterraneous rhizome and annual
aerial shoots, distributed in alpine and subarctic regions of Europe and Northeastern
North America. The two afromontane species of Bartsia s.l., native to Eastern Africa from
Ethiopia to Tanzania, are clearly connected to Hedbergia Molau by their vegetative
morphology and palynological characters. Hedbergia abyssinica, a perennial
hemiparasitic subshrub distributed in montane Western (Nigeria and Cameroon) and
Eastern Africa (from Ethiopia to N-Zambia and Malawi), was separated from Bartsia s.l.
by Molau (1988) due to its distinct flower morphology (rotate instead of bilabiate
corolla, see photo in Fig. 9). However, this characteristic trait might simply represent an
adaptation to a special pollinator. Species of B. sect. Longiflorae sensu Molau (1990)
were therefore transferred to Hedbergia, which now consists of perennial hemiparasitic
subshrubs with a rotate or bilabiate corolla and an afromontane distribution.
The third group within the polyphyletic Bartsia s.l. is unequivocally associated
with Parentucellia and Bellardia as discovered earlier (Bennett and Mathews, 2006;
Těšitel et al., 2010): the chiefly Mediterranean Parentucellia latifolia (L.) Caruel is sister
to a clade of Bartsia s.l. from the Andean montane habitats of Colombia, Bolivia, Peru,
and Chile to Northern Argentina. Species of Bellardia and Parentucellia are annual,
facultative hemiparasites with a native distribution range predominantly in the
Mediterranean (with Parentucellia also reaching farther east into Asia). Apart from that,
both genera are introduced as noxious weeds to e.g. Australia and America. The
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Fig. 9. Phylogenetic relationships in Rhinantheae. Consensus tree yielded from analysis of a combined
plastid and nuclear dataset. Only nodes with supports PP ≥ 0.80 or BS ≥ 75 are shown. Bold lines
represent nodes with supports of PP ≥ 95 or BS ≥ 85. Divergence of the core group of Rhinantheae is
marked by an arrow. Species which were subject to taxonomic changes are given with their former names
in brackets. Four accessions showing plastid-nuclear marker incongruence are connected to their
respective sister groups in the chloroplast (cp) or nuclear (nr) trees by dashed lines. Photos of
representative species, from top to bottom: Lathraea squamaria L. (photo by Andreas Fleischmann),
Bartsia alpina L. (photo by Andreas Fleischmann), Hedbergia abyssinica (Benth.) Molau (photo by Jakub
Těšitel), Bellardia trixago (L.) All., Odontites viscosus (L.) Clairv. (photo by Andreas Fleischmann). RRL,
Rhynchocorys-Rhinanthus-Lathraea clade
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monotypic Bellardia itself used to be part of Bartsia s.l. sensu Molau (1990; see above),
but later again was regarded different because of a deviating corolla, calyx and seed
morphology. Although not visible from the phylogenetic tree, consensus networks reveal
a certain relationship of Bellardia and Parentucellia viscosa (L.) Caruel (see Fig. 4 of
Article I). This is corroborated by morphological similarities, overlapping distribution
ranges, reports of putative hybrids among the two species, and also by results of UribeConvers and Tank (2016). As there seems to exist a continuous pattern of shared
characteristics leading from Bellardia to P. latifolia, we included Parentucellia into
Bellardia. To avoid paraphyly of Bellardia, Andean Bartsia s.l. were also transferred into
this genus (however we limited taxonomic combinations to the species actually
analyzed). This classification implies a long-distance dispersal of the Mediterranean
ancestor of the Neotropical lineage with subsequent adaptive radiation, resulting in a
disjunct pattern which is not uncommon (see e.g. Calviño et al., 2008; Bräuchler et al.,
2010; Kadereit and Baldwin, 2012) and has its counterpart in the establishment of an
African lineage from Mediterranean ancestors (or, more general, European ones, as put
by Uribe-Convers and Tank, 2015) in the Hedbergia clade, as inferred by Těšitel et al.
(2010). Bellardia thus now comprises annual as well as perennial hemiparasites. A
different approach was taken by Uribe-Convers and Tank (2016), who accepted
Bellardia viscosa (L.) Fisch. & C.A.Mey but transferred Bellardia latifolia (L.) Cuatrec.
back to Parentucellia. For the Andean species of Bartsia s.l., they proposed a new genus,
Neobartsia Uribe-Convers and Tank, with 47 species. Although this classification
encompasses all of the New World species (while Article I only included two of them), it
leaves important questions regarding the considerable similarities of the involved taxa
unanswered.
In relation to Bornmuellerantha, which was found to be nested in Odontites by
Těšitel et al. (2010), our results revealed that another two of the small genera
segregated from Odontites by Rothmaler (1943) and Bolliger (1996), based on divergent
corolla morphology and pollen characters, are in fact part of Odontites: Bartsiella and
Macrosyringion. Bornmuellerantha and Bartsiella were re-included into a broad
circumscription of Odontites; however, no final decision was made on Macrosyringion:
although M. glutinosum (M.Bieb.) Rothm. was nested in Odontites as sister to
Bornmuellerantha in ITS, its placement was incongruent regarding the plastid markers,
where it was weakly supported as sister of Odontites s.l. (Fig. 9). Several cases of
reticulate evolution have been reported in Rhinantheae, so hybridization (possibly also
involving a parent from outside Odontites s.l.; see Pinto Carrasco et al., accepted) could
explain the observed pattern. The fourth Odontites-like genus, Odontitella, surprisingly
was found to be sister to Nothobartsia, a genus consisting of two perennial species from
the Iberian Peninsula and neighboring regions, which was separated from Bartsia s.l. by
Bolliger and Molau (1992). (Ancient) Hybridization likely played a role in the origin of
this clade; it is incongruently placed as sister to the Hedbergia clade in the chloroplast
tree but sister to Odontites s.l. in ITS (Fig. 9). A hybrid origin of the NothobartsiaOdontitella clade from those two lineages is corroborated by morphology: Nothobartsia
shares characters with Odontites (e.g. the elongate-spicate inflorescence, the obovoid
capsules, pendulous ovules, and relatively narrow-winged seeds) as well as Bartsia s.l.
(e.g. the broad ovate stem leaves and the entire galea) according to Bolliger and Molau
(1992). Shared characters are also found in Odontitella.
Interestingly, besides these common morphological traits, which are shared by
Nothobartsia and Odontitella, but also other lineages, there are few characters which are
exclusively synapomorphic for the two genera; rather, there is actually a number of
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morphological differences (Pinto Carrasco et al., accepted). This illustrates a general
problem linked to the analysis of morphological characters in Rhinantheae: while
individual genera have accumulated autapomorphic features which make them easily
diagnosable (e.g. Euphrasia, Hedbergia, Macrosyringion, Bornmuellerantha),
synapomorphies for groups of species are often missing (a problem which is also
observed in Rhinantheae as a whole). Plesiomorphic characters resulting in the
"'Bartsia-like' general morphology" found in many lineages (Těšitel et al., 2010) impede
the correct assignment of taxa to their respective lineage, with hybridization (possibly
resulting in intermediate morphologies) further complicating the situation. In
consequence, morphological characters as traditionally used (like corolla shape, anther
indumentum or pollen types, see Rothmaler, 1943; Bolliger & Wick, 1990; Bolliger,
1996) are not entirely suitable for generic classification in Rhinantheae. Regarding
phylogenetic reconstruction based on molecular markers, our study clearly shows that a
careful assessment of topological incongruence is essential in the group, with respect to
correct inference of the relationships of taxa or lineages affected by reticulate evolution
(Nothobartsia-Odontitella, Macrosyringion), but also to node supports in combined
marker trees which might be affected if pruning of incongruent taxa is omitted.
Altogether, according to the updated classification presented in Article I, the core group
of Rhinantheae now consists of four monophyletic, distinct lineages (Hedbergia,
Bellardia, Odontites s.l., and Nothobartsia-Odontitella; Fig. 9) plus the monotypic genera
Bartsia s.str. and Tozzia, as well as Macrosyringion and the large genus Euphrasia.
5.2. The biogeographic history of Scrophularia
The biogeographic history of the genus Scrophularia as a whole had never been
studied in detail. Our comprehensively sampled phylogeny (Article IV) revealed that the
genus originated around the Oligocene / Miocene boundary at app. 23 million years ago
(mya), when it diverged from its sister genus Verbascum. Its most likely geographic
region of origin lies in Southwestern Asia s.l. (including Iran, Iraq and part of the Arabian
Peninsula but also Turkey and the Caucasus; depicted by the dashed line in Fig. 4), which
largely corresponds to its present-day primary center of diversity. These results
contradict Stiefelhagen (1910) who, in his monographic treatment of the genus,
promoted the Himalayas as ancestral region for Scrophularia. From Southwestern Asia,
several lineages of the genus spread to the east as well as the west, finally resulting in
the broad northern hemispheric distribution observed today (see Fig. 4). A similar
situation involving an Irano-Turanian (sensu Takhtajan, 1986) diversity center and
clades with both more easterly (to Yunnan) and westerly (to the Canary Islands)
distributions can be found in Ferula L. (Apiaceae; Kurzyna-Młynik et al., 2008). These
patterns are indicative of some characteristic features of the Irano-Turanian region in
general: especially the west-central part of the region, which comprises most of Iran,
Armenia, Azerbaijan, Afghanistan, and parts of Turkey, Iraq, Turkmenistan and Pakistan,
is "a major center of speciation and endemism" (review by Djamali et al., 2012b, there
referred to as the "IT2 sub-region"). This seems to be due to its high diversity of habitats
(forests, scrubs, alpine grasslands, steppes etc.), its heterogeneous topography (see
chapter 5.3.2.) and its climatic distinctness (Djamali et al., 2012b). High species richness
and endemism in the Irano-Turanian region are combined with a high tendency to
spread elsewhere, which means a pronounced representation of Irano-Turanian
elements in neighboring regions, especially to the west (Mediterranean and SaharoArabian region; Djamali et al., 2012b; see also Mansion et al., 2008 and references
128
Fig. 10. Phylogenetic relationships in Scrophularia, based on (a) analysis of combined plastid data and (b)
nuclear ITS data, including a dated phylogeny (in million years) and ancestral area reconstruction for the
plastid tree. Nuclear tree computed with sequence polymorphisms treated as informative; important
relationships supported by the uncoded tree added by dashed lines. Branches indicate levels of support
according to Figs. 1 and 2 of Article IV (bold: PP≥ 95 or BS ≥ 85, semi -bold: PP ≥ 90 or BS ≥ 75, thin: PP <
90 / BS < 75). Small arrows indicate the position of Oreosolen Hook.f., a large arrow the large basal
polytomy of the ITS tree. Pie charts represent marginal probabilities of inferred ancestral distributions;
area codes at the nodes as explained on the left side. The three sampled Japanese taxa are highlighted by
red branches. ENA, Eastern North America clade, IPM, Iberian Peninsula – Macaronesia clade
129
therein). However, plant migrations from Central Asia to the east have also been
documented in several cases (see e.g. S-T Chen et al., 2005; Yue et al., 2009).
5.2.1. Eastward migrations
In Scrophularia, early eastward migrations to Eastern Asia were inferred at app. 6
mya (Fig. 10a, and Fig. 1a of Article IV) and were followed by diversification of the
"China" clade into three lineages with several species in China and surrounding regions,
at about 4.5 and app. 3 mya. This resulted in the genus' most important secondary
center of diversity (Fig. 4). Our analyses presented in Article II and IV also revealed that
the New World was colonized (possibly repeatedly and maybe also involving backdispersals) from Eastern Asia, with the New World taxa diverging from their Asian
ancestors app. 6 mya at the earliest. Colonization might have happened through
migration via the Bering Land Bridge (BLB; Tiffney, 1985) as depicted in Fig. 4; during
the later Tertiary, this passage was increasingly limited to cool-temperate taxa (Tiffney
and Manchester, 2001), which would however not have posed a problem for
Scrophularia. Even after the closure of the BLB (estimates range from 3.3 to 9 mya;
Brigham-Grette, 2001; Denk et al., 2011), migration might have been successful, using
short-time openings (assumed e.g. at 4.9 mya, 4.0 mya, 3.3 mya, and 2.5 mya; Miller et
al., 2005). Furthermore, Scrophularia may also have reached the New World via longdistance dispersal: although the seeds do not possess special adaptations, they are easily
dispersed by wind due to their small size and weight, and might have overcome the
Bering Strait in this way. Generally, it can be stated that the Bering region was one of the
most important connections between the two continents, with several lineages
continously distributed from Eastern Asia to North America until the global climatic
cooling of the late Pliocene and the Pleistocene (Xiang et al., 2000; Wen et al., 2016).
Multiple dispersals in either direction and repeated back-colonizations often necessitate
a broad sampling to identify numbers and times of dispersal correctly.
Divergence of North American from Eastern Asian lineages is known from several
groups, e.g. Kellogia Torrey ex Benth. (Rubiaceae; Nie et al. 2005), Rhodiola L.
(Crassulaceae; Zhang et al. 2014), Angelica L. (Apiaceae; Liao et al., 2012) or Aralia L.
(Araliaceae; Wen et al., 1998). In Scrophularia, three North American lineages are
supported. In several of the reconstructions, Eurasian and New World clades share
sister relationships; the Japanese species (highlighted by red branches in Fig. 10) often
even are more closely related to (Eastern) North American species than to other Eastern
Asian taxa, a scenario discussed in Donoghue et al. (2001) and found in other lineages as
well (see e.g. Li et al., 2003). These close relationships correspond to general Eastern
Asian–North American disjunct patterns which are known for a long time and from
many plant genera and can involve Eastern as well as Western North America (Boufford
and Spongberg, 1983; Kadereit and Baldwin, 2012; Wen et al., 2016; for examples see
e.g. Gould and Donoghue, 2000; Schnabel et al., 2003). In family Scrophulariaceae, both
patterns are found (Hong, 1983).
5.2.2. Westward migrations
Westward expansion in Scrophularia led to the colonization of the Mediterranean,
Northern Africa and Europe, which is typical for Irano-Turanian elements as outlined
above. The Iberian Peninsula and Macaronesia host another secondary diversity center
and are represented by a large clade of mostly Iberian taxa, which has a crown age of
about 4.5 my (Article IV). Within this "Iberian Peninsula - Macaronesia" = "IPM" clade
(Fig. 10), three subclades can be found (Article III). One is largely confined to the Iberian
130
Peninsula ("Scorodonia") while another reaches into Western North Africa with several
species ("Auriculata"). Interestingly, while these species do not extend beyond Tunisia,
other taxa are confined to Eastern North Africa. These belong to lineages from the
Tomiophyllum clade ("Striata" and "Libanotica") which migrated into areas west and
southwest of Iran and eventually colonized Eastern North Africa (Article IV); others
today are found exclusively in the Levantine region ("Scariosa" clade). Some species
again can be found throughout Northern Africa (S. syriaca Benth. from the Striata clade
and S. canina, S. peregrina and S. arguta Sol.). Migration into more southern areas was
largely precluded by the increasing aridity in these regions during the Pliocene and
Pleistocene (Axelrod and Raven, 1978). An east-west split regarding plant lineages
distributed in Africa was also observed in Digitalis L. (Bräuchler et al., 2004) and
Ranunculus L. sect. Ranunculastrum (Paun et al., 2005).
The third lineage of the IPM clade exclusively consists of Macaronesian taxa
("Macaronesia" subclade). Members from the Auriculata, Scorodonia and "Arguta"
clades are also represented in Macaronesia, summing up to a total of four phylogenetic
lineages which have colonized the islands of Madeira, the Azores, the Cape Verde and the
Canary Islands according to reconstructions in Article III (and IV). Madeira comprises
representatives from all four lineages: two of them presumably have entered Madeira
from the Western Mediterranean or also Western North African mainland (i.e., the
Iberian Peninsula or Morocco), and one from the Canary Islands. The Canary Islands
were colonized two times independently, including one dispersal from the Western
Mediterranean (Fig. 5 of Article III). No conclusions were possible for the Azores (where
S. auriculata L. is native) and the Cape Verdes (which host S. arguta). For S. arguta in
general, no migration pathways could be determined; it is presently also found on
Madeira and the Canary Islands. Valtueña et al. (2016), using a larger sampling, inferred
three different colonization events to the Canaries, evidenced by different haplotypes on
the western and eastern islands and Gran Canaria. Colonization possibly happened from
Morocco in the latter two groups.
A second example for colonization of islands from the adjacent mainland (and
subsequent diversification) in Scrophularia is found in the Greater Antilles, which
constitute one of the very few examples where the genus advances into tropical regions.
The species distributed on the islands were revealed to be the result of a dispersal event
from the North American mainland (Florida) to the Caribbean (Cuba), by an ancestor of
the Eastern North American S. marilandica L. (Article II); all species involved are part of
the "Eastern North America" ("ENA") clade (Fig. 10). Dispersal is a frequent mechanism
of Antillean colonization with several examples in flowering plants (amongst many
others e.g. Styrax L. sect. Valvatae, Styracaceae, Fritsch, 2003; Lythraceae, Graham,
2003). Hedges (2006) stated that dispersal likely represents the key factor in Antillean
colonization by terrestrial vertebrates and often leads to adaptive radiation on the
islands. This seems to be true in Scrophularia as well: while only two species are native
in the eastern parts of the USA (and only S. marilandica in Florida), the Greater Antilles
harbor seven species, one widespread in forests of Cuba, Hispaniola, Puerto Rico and
Jamaica, and six endemic to the Hispaniolan pine forests above 800 m; this underlines
the status of these regions as biodiversity hotspot (Mittermeier et al., 2004; Francisco
Ortega et al., 2007).
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5.3. The influence of geography and topography on diversification
5.3.1. Origin and expansion of Scrophularia
The evolutionary and biogeographic history of Scrophularia has been heavily
influenced by geologic processes since its divergence at the beginning of the Miocene. It
can be hypothesized that the emergence of the genus was a result of substantial
geological reorganizations, and climatic changes, in its ancestral region. The Miocene
brought about a shift towards increased seasonality (An et al., 2001) and the
aridification of interior Asia (Guo et al., 2002, 2004; Miao et al., 2012), with substantial
environmental effects on Asian biota. Furthermore, the collision of the Arabian with the
Eurasian plate, which had been initiated in the late Eocene (Berberian and King, 1981;
Mouthereau et al., 2012), exerted strong influence on Southwestern Asia during the
Oligocene and Miocene. Around the time inferred for the origin of the genus (Article IV),
the final closure of the Tethyan seaway and the retreat of the Paratethys ocean
(Ramstein et al., 1997; Mouthereau et al., 2012) created additional suitable land area for
plant colonization, e.g. in the Zagros region. Highlands had been already existing for a
longer time in Anatolia and the Alborz (Dercourt et al., 1986; Popov et al., 2004). The
Arabia - Eurasia collision eventually led to the uplift of the Iranian plateau and the
formation of the main mountain ranges of the region during the Miocene (see Djamali et
al., 2012a). Diversification of Scrophularia into its main lineages consequently began
during a period where new suitable habitats for plants adapted to montane
environments were created; by the emergence of the Greater Caucasus and its
transformation into a mountain chain (app. 14-13 mya and 9 mya; Popov et al., 2004;
Olteanu and Jipa, 2006), the uplift of the Iranian Plateau (from 15-12 mya, Mouthereau
et al., 2012; Djamali et al., 2012a and references therein) and the formation of the
Western Alborz (app. 12-10 mya) and Zagros (app. 15-10 mya) mountains (Dercourt et
al., 1986; Popov et al., 2004; Guest et al., 2007; Mouthereau et al., 2012). The
establishment of continental climate conditions (Berberian and King, 1981) with
increasing aridity (Ballato et al., 2010) during this period might additionally have
promoted the colonization of more moderate, humid habitats at higher elevations (Roe,
2005); temperate elements additionally benefitted from the temperature decrease at
app. 14-13.5 mya, after the mid Miocene climatic optimum (Zachos et al., 2001; Tiffney
and Manchester, 2001). The pronounced emphasis on mountainous regions in the
present-day distribution of Scrophularia lends support to this scenario.
Mountain formation also possibly triggered the spread of the genus into remote
areas, by providing suitable migratory pathways. Given the habitat preferences
mentioned above, dispersal along higher mountain chains seems reasonable. By the time
inferred for the first eastward migrations of figworts at 6 mya, the rise of the Eastern
and Central Asian mountain chains, the Kunlun Shan, the Tian Shan and the Hindu Kush
from the Eocene through the Miocene (Dercourt et al., 1986; Abdrakhmatov et al., 1996;
Hildebrand et al., 2000, 2001; Sobel et al., 2006; references in Miao et al., 2012; Yuan et
al., 2013), together with the re-emergence of the more westerly Kopet Dag at app. 10
mya or later (Dercourt et al., 1986; Popov et al., 2004), had formed a more or less
continuous mountain belt, which connected the Southwestern Asian Alborz to the
Himalayas and the Tibetan Plateau. Migrations from Western or Central Asia to Eastern
Asia or vice versa are also known from e.g. Incarvillea Juss. (Bignoniaceae; S-T Chen et
al., 2005), Rhodiola (Zhang et al., 2014), or Solmslaubachia Muschl. (Brassicaceae; Yue et
al., 2009).
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5.3.2. Diversification in the Irano-Turanian region and the Mediterranean
The most important factor regarding diversification in Scrophularia is however
that tectonic processes with associated mountain building generated habitats which
themselves promote speciation. In the Iranian region, the center of Scrophularia species
diversity, pronounced topographic barriers also act as boundaries separating
biogeographical or climatic regions (e.g. the Alborz mountain system, see Djamali et al.,
2012b). Furthermore, the tectonic history of Southwestern Asia created a complex
topography of several mountain ranges, which provide heterogeneous habitats on small
geographical scales. This facilitates adaptive speciation and allopatric divergence, and
has led to the extraordinary species diversity and endemism found in the region
(Djamali et al., 2012b), for example in Astragalus (Fabaceae; Podlech and Zarre, 2013) or
Cousinia Cass. (Asteraceae), which has been found to be most diverse "in regions with
the widest range of topographical variations and the highest frequency of elevations
above 2000 m" (Djamali et al., 2012a). Geological heterogeneity in the Irano-Turanian
region was also invoked as explanation for diversification of a large clade of
Haplophyllum A.Juss. (Rutaceae; Manafzadeh et al., 2014). The connection of
topographical complexity and high species richness is of course not confined to the
Irano-Turanian region. Similar patterns are found in other parts of the world, and
remarkably often coincide with diversity centers of Scrophularia. Indeed, all
Scrophularia diversity centers comprise mountainous regions (Fig. 4), and high levels of
endemism are found throughout the genus (Vaarama and Hiirsalmi, 1967).
A good example for "vicariance" caused by fragmentation of habitats is found in
the Mediterranean, where plate movements in combination with marine regression /
transgression events explain part of the large species diversity and number of endemics,
through the repeated formation of contacts and barriers for plant lineages (e.g. Mansion
et al., 2008). On a smaller scale, topographical complexity was suggested to promote
diversification and speciation processes, often in combination with climatic changes
during the Quaternary glaciations (Thompson, 2005). Lobo (2001) examined patterns of
plant species richness in the Iberian Peninsula and found that diversity is significantly
related to maximum elevation and altitude range (= environmental heterogeneity). This
might also explain the secondary diversity center of Scrophularia found in the Iberian
Peninsula with 22 species, 12 of those endemic to the Peninsula including the Pyrenees.
While the origin of the IPM clade (by ancient hybridization, see chapter 5.4.3.) was
inferred at around 5 mya and its crown age at app. 4.5 mya (Article IV), i.e. well before
the establishment of the Mediterranean climate rhythm (around 3 mya, Suc, 1984),
diversification of the main lineages was dated to have happened from 2 mya on (see also
Navarro Pérez et al., 2013). This implies that speciation in the fragmented habitats was
additionally reinforced by expansions, contractions or shifts of distribution ranges
created by climatic fluctuations (Thompson, 2005). In Scrophularia, two Iberian
alongside two Mediterranean endemic species were found to obtain isolated positions in
a haplotype network (Fig. 3 of Article III) and were unresolved in the respective tree.
These species are restricted to only small areas, and exclusively or predominantly
inhabit regions classified as refugia within the Mediterranean bioclimatic region (Médail
and Diadema, 2009). These, putatively more ancient, lineages might have got isolated
while retreating into the favorable conditions of the climatically stable refugial areas.
Many examples for this mode of speciation are known from the Iberian Peninsula, e.g.
from Erodium L'Hér. (Geraniaceae; Fiz Palacios et al., 2010) or Reseda L. (Resedaceae;
Martín Bravo et al., 2010). Hybridization of lineages in secondary contact zones has been
another important factor, see chapter 5.4.
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Apart from topography and climate, a spatial structuring of genetic variation
might also be generated by habitat islands due to different soil types. An example is
found in the Iberian gypsophyte Gypsophila struthium L. (Caryophyllaceae; Martínez
Nieto et al., 2013). Habitat fragmentation by soil types is also a driver of speciation in the
Strait of Gibraltar region. The climatically stable Algeciras and Tanger Peninsulas are
characterized by a landscape comprising deep gorges and a mosaic of limestone
outcrops and siliceous sandstone patches producing large numbers of narrow endemics,
mostly originating from recent speciation (Rodríguez Sánchez et al., 2008; Lavergne et
al., 2013). Some narrow endemic Scrophularia species are found in the region, amongst
those S. fontqueri Ortega Oliv. & Devesa (not sampled for this thesis), native to
calcareous substrates in the Rif mountains of Northern Morocco; S. viciosoi Ortega Oliv.
& Devesa (see chapter 5.4.2.) of Málaga province, Spain which prefers similar habitats in
calcareous outcrops; and S. laxiflora Lange (Fig. 10, Scorodonia subclade), distributed on
sandstone soils on both sides of the Strait of Gibraltar, in Southern Cádiz province of
Spain and the Tanger region, Morocco; the latter also houses another narrow endemic, S.
papillaris Boiss. & Reut. (not sampled), which is closely related to S. scorodonia (Ortega
Olivencia and Devesa Alcaraz, 1996, 1998; Ibn Tattou, 2007; Ortega Olivencia, 2009). As
yet, no evidence for an association of these species with the edaphic characteristics of
the Strait of Gibraltar region has been provided. However, Lobo (2001) found general
evidence for an (albeit subordinate) influence of bedrock geology variables on Iberian
vascular plant diversity.
5.3.3. Diversification in Eastern Asia and the New World
In Eastern Asia, another secondary diversity center of the genus, where more
than half of the 36 Scrophularia species listed in the Flora of China (Hong et al., 1998)
ascend to alpine levels of 3000 m or more, the extreme relief found in mountain regions
also limits gene flow effectively. Qian and Ricklefs (2000) attributed considerably higher
species numbers in Eastern Asia compared to Eastern North America (the Eastern Asian
- Eastern North American species diversity bias) to "the extreme physiographical
heterogeneity of temperate eastern Asia". Apart from that, similar to the Mediterranean
region, climate fluctuations could have enabled repeated fragmentation and extensions
of distribution areas, further pushing diversification (Qian and Ricklefs, 2000). In a
review on plant diversification on the Tibetan Plateau, Wen et al. (2014) highlighted the
"extremely complex topography with diverse habitats", which fosters allopatric
divergence, as a main diversification mechanism on the plateau. Like for other mountain
systems (Hughes and Atchison, 2015), many authors have attributed diversification
events in the region to the uplift of the Tibetan Plateau (examples e.g. in Wen et al.,
2014), although not always legitimately so (Renner, 2016). In Scrophularia,
diversification of the Hengduan subclade (Fig. 10), with most of its members restricted
to the Hengduan mountains and adjacent areas, might possibly have been triggered by a
phase of uplift of the Hengduan Shan. However, the general great uncertainty in dating
both divergence times and geologic events makes conclusions based on the present data
somewhat questionable. Diversification of the Hengduan and also the Central subclade
in China seems to be more obvious to relate to the extreme topography and the variety
of vegetation types and climatic conditions which characterize the Mountains of
Southwest China biodiversity hotspot (Mittermeier et al., 2004; Boufford, 2014). Apart
from that, the distribution patterns seen today might also have been generated by
survival of plant lineages in suitable refugia and recolonization of China from these
refugia after the Last Glacial Maximum (app. 24,000-18,000 years ago; Liu et al., 2012).
The two predominantly alpine Hengduan and Central subclades can both be associated
134
with important refuges, which have been recognized in the Hengduan Shan (see S-Y
Chen et al., 2008; Feng et al., 2009; L Wang et al., 2011) and the Qinling mountains or,
more generally, the "Northeast Qinghai - Tibetan Plateau edge" (Qiu et al., 2011),
respectively (Article IV). Unfortunately, unequivocal identification of the underlying
diversification mechanisms is not possible based on the sampling used here. The third
Chinese (sub)clade, "Ningpoensis", predominantly comprises non-alpine species
distributed from the eastern parts of China to Korea, Japan and Taiwan and seems
clearly separated from the Hengduan and Central subclades mostly consisting of alpine
taxa with narrow distributions. An arid zone separating the two regions throughout the
Miocene (Tiffney and Manchester, 2001) could possibly have constituted a barrier for
Scrophularia, resulting in vicariant speciation; this was also proposed for Thuja L.
(Cupressaceae) by Peng and Wang (2008).
The Eastern Asian - Eastern North American species bias as investigated by Qian
and Ricklefs (2000) and Xiang et al. (2004) is found in many plant groups, e.g. in
Lespedeza Michx. (Fabaceae; Xu et al., 2012), Panax L. (Araliaceae; Wen and Zimmer,
1996) and also Scrophularia (36 species in China compared to 19 species in entire North
America plus the Caribbean). In the New World itself, species diversity in the U.S.A. plus
Canada is again lower in the east than in the west. Only one species is distributed
throughout the region (S. lanceolata Pursh, see chapter 5.4.2.). The eastern part harbors
only one additional (also widespread) species; its possible phylogenetic relationship to a
recently discovered narrow endemic Northeastern Mexican species (Mayfield and
Nesom, 2012) still needs confirmation. On the other hand, of the nine species confined to
the west, only two are distributed in more than two states, and four are limited to one to
three counties only. A clade of five taxa (Fig. 10, "California" clade) is distributed in the
California floristic province, a Mediterranean-type hotspot (Mittermeier et al., 2004). Qi
and Yang (1999) analyzed plant diversity in California and found a positive correlation
of mean elevation and speciation capacity, as well as a relation of spatial variability of
elevation to plant diversity. However, only one Scrophularia species from the California
clade ascends to alpine elevations. Diversity related to spatial heterogeneity is rather
found in the "New Mexico" clade, which includes one alpine and three subalpine species.
Notably, distribution ranges of the markedly geography-based, two western and one
eastern clades of North American Scrophularia do not overlap much. Here, geographic
isolation seems to obtain another role, in maintaining already established species: most
North American species are closely related - they can be artificially crossed (Shaw,
1962) or intergrade naturally in contact zones (Kearney and Peebles, 1951). This means
that rather than reproductive isolation, geographic barriers might be important in
sustaining the identity of the species (Shaw, 1962; see also Carlbom, 1969, who however
advocates a different species concept for Scrophularia). A similar effect has been
observed in the Scrophulariaceae genus Jamesbrittenia Kuntze, which is also capable of
successful interspecific hybridization (Verboom et al., 2016).
5.4. The influence of hybridization on diversification
5.4.1. Phylogenetic tree incongruence and intra-individual polymorphism
The genus Scrophularia is characterized by a high level of natural hybridization
and hybrid speciation as revealed in Articles II, III and IV. This process can be detected
using molecular sequence data, e.g. by examining gene trees for well-supported
incongruence. Usually, a combination of uniparentally (plastid) and biparentally
135
(nuclear) inherited molecular markers is used, and the respective position of a hybrid
lineage next to one or the other of its parents might allow conclusions on the hybrid
speciation event (e.g. Soltis and Kuzoff, 1995; Marhold and Lihová, 2006; Fehrer et al.,
2007). This approach led to the successful detection of hybrids in Rhinantheae and
Scrophularia (Article I-III; see the examples given below). When incongruence is not too
widespread, conflicting taxa can also be revealed using special software (Aberer et al.,
2013; Pérez Escobar et al., 2016). In cases where nuclear ribosomal DNA (ITS) is used
for reconstruction and concerted evolution of rDNA units (e.g. Arnheim et al., 1980) is
slowed down or non-operational, intra-individual polymorphisms in sequence data
(which might result from a hybridization event) can be extracted and interpreted in
order to detect possible parent sequences (Fuertes Aguilar and Nieto Feliner, 2003). In
Scrophularia, concerted evolution seems to be incomplete in some, but not all species,
resulting in both monomorphic and polymorphic ITS sequences (Article IV).
However, both tree incongruence and intra-individual nucleotide polymorphism
can result from a variety of sources other than hybridization / introgression. Long
branch attraction will lead to artificial relatedness of taxa which are subtended by long
branches in phylogenies (Felsenstein, 1978). This will ultimately cause gene tree
discordance; inappropriate sampling or model settings during calculations might also
generate incongruent trees. Both tree incongruence and intra-individual polymorphism
can be created by Incomplete Lineage Sorting (ILS; the persistence of ancient
polymorphism / deep coalescence; Degnan and Rosenberg, 2009), recombination, or the
presence of paralogous sequences (Álvarez and Wendel, 2003; Wolfe and Randle, 2004).
The latter two, together with processes of differential silencing and pseudogenization
are additionally intensified in the presence of hybridization (Álvarez and Wendel, 2003;
Volkov et al., 2007). The situation is particularly complex when the investigated lineages
are of recent divergence. Several authors have attempted to disentangle the different
processes leading to gene tree incongruence, especially to distinguish between ILS and
hybridization (Maureira Butler et al., 2008; van der Niet and Linder, 2008; Joly et al.,
2009; Konowalik et al., 2015). However, when a combination of different processes has
repeatedly influenced a phylogenetic history, single events might become difficult to
discern.
Furthermore, these phenomena can severely impact phylogenetic tree
reconstruction itself, e.g. by reduced node resolution in datasets containing intraindividual polymorphism. To overcome this situation, affected accessions have been
pruned from analyses, or polymorphisms were excluded or variously replaced (Fuertes
Aguilar and Nieto Feliner, 2003; Lorenz-Lemke et al., 2005; Scherson et al., 2008; Fehrer
et al., 2009). The method employed in Article IV, treating polymorphims as informative
(see Methodology, chapter 3.), proved to be most suitable as a maximum of data was
incorporated into the analyses; it resulted in considerably increased tree resolution
while only exceptionally contradicting the uncoded phylogeny (Fig. 2 of Article IV
compares both topologies). However, it must be stressed that any kind of reticulation
will fundamentally interfere with traditional approaches based on building
dichotomously branching trees; this problem of inherent conflict in the data cannot be
solved using coding techniques (Potts et al., 2014). Consequently, a large basal polytomy
remains in the nuclear tree in Fig. 10b (indicated by an arrow). In these cases,
examination of phylogenetic networks is a suitable alternative (Huson and Bryant,
2006); these again can be supported by incorporating information from polymorphisms
(see Article IV). Fig. 11 shows the consensus network of all trees leading to the
consensus tree in Fig. 10b. It illustrates the highly entangled relationships (resulting
from uncertainty and conflict) among and also within clades, which are concealed when
forced into a single bifurcating tree, and result in the aforementioned polytomy.
136
10000
17. Scariosa
1. Arguta
4d.
4a. California
13. Variegata
OG
2. Ningpoensis
4b. ENA
16. Striata
12. Libanotica
18.
4c.
14. Scoparia
3. China
7c.
7b.
15. Polyantha
6.
8. Scopolii
7a.
10. Peregrina
11. Canina
9. Vernalis
7. IPM
5. Nodosa
Fig. 11. Consensus network based on trees from both runs of the Bayesian analysis that resulted in the ITS
consensus tree shown in Fig. 10b. Clades are highlighted, colors correspond to those in Fig. 10b. The large
basal polytomy observed in the consensus tree can be explained in part by the highly networked part on
the left side (Anastomosantes group, see chapter 5.5.) of the consensus network
In the uniparentally inherited chloroplast, extensive reticulation (e.g. by repeated
introgression) can ultimately lead to a phylogenetic pattern that is no longer based on
taxonomic relationships but rather traces geographic proximity. Examples are found in
Antirrhinum L. (Plantaginaceae; Vargas et al., 2009), Phlomis L. (Lamiaceae; Albaladejo et
al., 2005), and many others. In Scrophularia, this effect is not too pronounced, with
taxonomic relationships still being visible in plastid trees (Articles II-IV). Some clades
("Nodosa", "NW (= New World) / Japan", "Orientalis", "Scopolii", "Polyantha") are
characterized by the occurrence of large diagnostic indels (see Fig. 7), which however
are not exclusive among clades (Article II, IV).
5.4.2. Hybrid speciation in Scrophularia - homoploid hybrid species
Detection of hybridization in Scrophularia is complicated by the fact that
corresponding patterns are often overlain by other processes. DNA sequences and
phylogenetic trees are likely influenced by hybridization, introgression, ILS, incipient
pseudogenization, and even recombination and paralogy among sequences cannot be
completely ruled out. To unravel all of these effects would require a different kind of
sampling and lies beyond the scope of this thesis. However, despite the abovementioned
difficulties, several examples of hybrid speciation within Scrophularia could be revealed
or confirmed in Articles II-IV. Hybridization is likely to have played a prominent role in
the evolution of the genus and has surely promoted its diversification, given the
occurrence of natural hybrids, its comparatively young age and its flower and
pollination biology (see Introduction, chapter 2).
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In the rather homogenous group of New World Scrophularia representatives,
most species have restricted distributions (see chapter 5.3.3.), with only S. marilandica
being more widespread in the eastern parts of North America. One species however, S.
lanceolata, the lanceleaf figwort, represents an exception, in being widely distributed
throughout the U.S.A. and Canada. I revealed this species to have a hybrid origin,
involving progenitors from the New Mexico clade (see Fig. 10) and S. marilandica from
the Eastern North America clade (Article II). Karyological evidence cannot confirm this
hypothesis (all species involved are high polyploids of variable chromosome numbers).
However, Scrophularia lanceolata is very similar to S. marilandica morphologically, and
also shares some characters with S. parviflora Wooton & Standl. from the New Mexico
clade, e.g. a similar pattern of corolla color and a spatulate staminode. Some
morphological traits (like the shape of the capsule) are intermediate between those of
the parental lineages; flowers and capsules are also larger than in the latter, which could
be interpreted as a sign of hybrid vigour. It seems possible that its reticulate origin is the
reason for the success of S. lanceolata, which has spread more widely than any other
Scrophularia in the New World.
The heterogeneous topography, different soil types, and the climatic history with
several glacial refugia across the Iberian Peninsula have resulted in several endemic
species as outlined above. Furthermore, we revealed that five Scrophularia Iberian
endemics are likely to be the result of homoploid hybrid speciation (Article III). This
mode of speciation, which does not involve a change in ploidy in the offspring, is still
regarded as unusual and rare, although several cases have been documented (Schumer
et al., 2014; Vallejo Marín and Hiscock, 2016). Scrophularia sublyrata Brot., S. oxyrhyncha
Coincy, S. reuteri Daveau, S. valdesii Ortega Oliv. & Devesa, and also the already
mentioned S. viciosoi from Málaga, Southern Spain, are morphologically related to the
Auriculata subclade, and, according to our analyses in Article III, originated from
hybridization of ancestors or members from the latter (acting as female parent in all but
S. viciosoi) and the Scorodonia subclade. As in the case of S. lanceolata, species from the
putative parental lineages can be successfully crossed, supporting the hypothesis of a
hybrid origin. In contrast to endemic species from the Strait of Gibraltar region (see
chapter 5.3.2.), S. sublyrata, S. oxyrhyncha, S. reuteri and S. valdesii are confined to
granite substrates; their distribution areas lie within the western floristic zone of the
Iberian Peninsula as defined by Moreno Saiz et al. (2013). The species are of very recent
divergence, having probably originated in the late Pleistocene (Navarro Pérez et al.,
2013). Diversification in the mountains of the Iberian Peninsula during Quaternary
climatic oscillations has been already discussed in chapter 5.3.2. In this context, range
shifts of species and secondary contact zones of course also provided ample
opportunities for hybridization. The resulting hybrid then could easily have been
isolated during subsequent periods of range contraction. Isolation from their parents is
regarded as essential for the establishment of newly formed homoploid hybrid species
(Rieseberg and Willis, 2007). Evidence for hybridization caused by Pleistocene climate
fluctuations coupled with heterogeneous topography has been found in many plant
lineages from the Mediterranean and the Iberian Peninsula, amongst others Armeria
Willd. (Plumbaginaceae; Gutiérrez Larena et al., 2002), Antirrhinum (Plantaginaceae;
Vargas et al., 2009) or Linaria Mill. (Blanco Pastor et al., 2012). It might have been
particularly frequent in mountainous areas of Southern Europe, where temperature
change effects were less serious and species could avoid them by altitudinal instead of
latitudinal migrations (Nieto Feliner, 2011), thus staying in relative proximity to other
populations. However, it has to be mentioned that the rapid formation of species
suffering secondary contacts should also have produced ILS (Blanco Pastor et al., 2012);
the latter authors tested for ILS as alternative explanation for phylogenetic
138
incongruence, which unfortunately was not possible with my Scrophularia dataset. Their
results supported the occurrence of both ILS and hybridization, and possibly also
homoploid hybrid speciation.
5.4.3. Allopolyploid hybrid species and hybrid lineages
In contrast to homoploid hybrid speciation, allopolyploidization is easier to
confirm: the respective hybrid taxa have a different ploidy level compared to their
parents, acquired by chromosome doubling after hybridization, by using a 'triploid
bridge' (Mallet, 2007), or resulting from the fusion of two unreduced gametes. This way,
chromosome numbers add additional evidence to that from tree incongruence and intraindividual polymorphism. The hypothesis of Ortega Olivencia and Devesa Alcaraz
(1990), of S. alpestris J.Gay ex Benth. (2n = 68) being an allopolyploid of S. scopolii Hoppe
ex Pers. (2n = 26; Scopolii clade) and S. bourgaeana Lange (2n = 42; Nodosa clade), was
confirmed by our phylogenetic reconstructions in Article III. The putative hybrid taxon
was incongruent in plastid and nuclear tree reconstructions, changing positions
between the two parent lineages. Scrophularia auriculata (2n = 84) was proposed to
have resulted from allopolyploid hybridization between (ancestors of) S. lyrata Willd.
(2n = 58; Auriculata subclade of the IPM clade) and S. umbrosa Dumort. (2n = 26, 52;
likewise incongruently placed within the IPM or Scopolii clade) by Grau (1979), based
on morphological traits. The respective results in Article III were ambiguous with
respect to S. lyrata; a relationship to S. umbrosa could not be found at all. In Article IV, a
specimen from S. auriculata with a confirmed chromosome number turned out to be
highly polymorphic in ITS; its clones revealed two ribotypes obtaining different
positions in the tree (Fig. 3 of Article IV). We concluded that hybridization of the
Algerian – Moroccan endemic S. hispida Desf. (2n = 58, Auriculata subclade, and
morphologically close to S. lyrata) and S. umbrosa or their ancestors likely have
generated the most widespread species within the Auriculata subclade, and probably
also S. racemosa Lowe, a Madeiran endemic with likewise 2n = 84 chromosomes. In
another accession from S. auriculata and one from S. lyrata, we found evidence for
introgression (chloroplast capture), by S. scorodonia and S. laxiflora (Scorodonia
subclade), respectively (Article IV).
Reticulation in Scrophularia is not limited to single taxa. Based on gene tree
discordance, it was revealed that the whole IPM clade, composed of Mediterranean and
Macaronesian representatives, likely originated by hybridization of members /
progenitors of two nowadays widespread lineages, S. umbrosa or the Scopolii lineage,
and the "Canina" lineage or allies, at around 5 mya (Articles III and IV). These ancestors
were inferred to have been distributed in Southwestern Asia and the Turkey-Caucasus
region, respectively, while the ancestor of the IPM clade had its range in the Western
Mediterranean. The exclusive chromosome number of 2n = 58, which is typical for the
latter, might have resulted from the merger of the two 2n = 26 chromosomes from both
parents (and subsequent chromosome doubling), with subsequent ascending
aneuploidy creating the number observed today. This does not seem unusual, as
aneuploidy is encountered in several species of the IPM clade, e.g. in S. sublyrata, S.
glabrata Aiton or S. viciosoi, and S. canina was exceptionally counted with 2n = 30
chromosomes (Ortega Olivencia and Devesa Alcaraz, 1990).
Even higher polyploidy is observed in the NW / Japan and Ningpoensis clades.
North American species have 2n = 86 to typically 2n = 96 chromosomes (excluding S.
montana Wooton which has 2n = 70-76), and one Japanese alongside some other
Eastern Asian taxa have been counted with up to 2n = 96, but also much lower numbers.
Generally, a high variability regarding chromosome counts is observed in Eastern Asian
139
and Southern Asian species, including diploid as well as polyploid chromosome
numbers. The origin of high polyploidy in these lineages remains unclear. Phylogenetic
reconstructions in Article IV suggest that hybridization might have been involved
regarding the New World and Japanese taxa. They are related to the Nodosa lineage as
well as the China clade, which is perfectly plausible given both their morphological
characteristics and their biogeographic ancestry. Tentative cloning of a Japanese and a
North American species however could not corroborate the hypothesis of a hybrid
origin, but as the event is expected to be more ancient, this does not necessarily refute it
either.
5.4.4. Combined effects of topography, hybridization and climate fluctuations
Altogether, it seems evident that a combination of geographic isolation, habitat
fragmentation and successful interspecific hybridization and polyploidization has been
the key factor in the diversification of Scrophularia. In general, reticulation and
polyploidization are now seen as major driving forces in the evolution and
diversification of plants (Soltis and Soltis, 2009; Abbott et al., 2013). A varied
topography could additionally support hybrid speciation, by stabilizing newly formed
hybrids through isolation from their parents; this might have been the case in the two
Iberian homoploid hybrid species S. oxyrhyncha and S. reuteri, now distributed in the
Cordillera Central and the Sierra Morena (Article III). Climatic changes however may
have triggered extensive gene flow in mountainous and other regions and resulted in
geographical structuring of haplotype relationships, as found in Armeria and also
Antirrhinum (Gutiérrez Larena et al., 2002; Vargas et al., 2009). However, Vargas et al.
(2009) and Blanco Pastor et al. (2012) also highlighted the role of geographical
speciation in addition to hybridization, evidenced by limited distributions of several
endemic, often endangered, species. This is also observed in Scrophularia; hybrid and
non-hybrid endemics with narrow distributions thereby are not limited to
Mediterranean taxa. On the other hand, the ability to generate polyploid lineages by
hybridization in Scrophularia seems to constitute a fitness benefit compared to its
closest sister genus Verbascum, where there is no evidence for hybrid speciation and
which lacks high polyploids. Interestingly, high polyploid lineages of Scrophularia have
colonized regions where few or no Verbascum species occur (China and the New World).
The reticulation processes which enabled this success are mirrored in the large amount
of incongruence and polymorphism encountered when reconstructing phylogenetic
relationships in the genus.
5.5. Taxonomy and morphological traits
All accessions of Scrophularia which were analyzed in this thesis formed a
monophyletic clade. Figworts therefore constitute a natural group, although the genetic
distance (nucleotide divergence) to Verbascum is comparatively low, given several clear
morphological differences (see pairwise distances and character differences in
Supplementary Table S1 of Article III). Surprisingly, the Himalayan - Tibetan endemic
genus Oreosolen was shown to be deeply nested within Scrophularia in all analyses of
Article IV (see arrows in Fig. 10). The large Tomiophyllum clade largely corresponds to,
but is not exactly identical with, Scrophularia sect. Tomiophyllum sensu Stiefelhagen
(1910). The remaining species mainly belong to Scrophularia sect. Anastomosantes; they
do not form a monophyletic clade, but one which is paraphyletic with respect to the
Tomiophyllum clade. This means that phylogenetic relationships to some point reflect
140
the morphology-based subgeneric classification proposed in Stiefelhagen's (1910)
monograph of the genus. Furthermore, they reveal S. sect. Tomiophyllum to be derived
from within S. sect. Anastomosantes, despite the putatively primitive traits of the former
(habit often subshrubby, xerophytic, general lack of polyploid chromosome numbers;
Carlbom, 1969). On the other hand, the mainly herbaceous, richly foliated, often mesoor hygrophytic members of S. sect. Anastomosantes (Stiefelhagen, 1910) are
characterized by a wide range in chromosome numbers, a wide ecological amplitude and
a geographic distribution exceeding that of S. sect. Tomiophyllum by far; this might
reflect a longer phylogenetic history.
The Tomiophyllum clade diverged from its sister clade at about 10.5 mya and
diversified from app. 8 mya (Article IV; Fig. 10a). Changes in aridity in the Middle East
during the second half of the Miocene may possibly have triggered these events (Ballato
et al., 2010). Ecological preferences of members of the two sections overlap, but their
habitats show a certain shift from moist sites on riverbanks and in forests (S. sect.
Anastomosantes) towards rock crevices and gravelly substrates with low humidity in S.
sect. Tomiophyllum; this indicates a greater tolerance of dry conditions. Furthermore,
Tomiophyllum species predominantly inhabit the dry parts of e.g. Iran and Turkey,
while not necessarily being absent from other areas. Interestingly, the first-branching
lineages of the Tomiophyllum clade, as supported by some of the nuclear phylogenetic
results (Fig. 10b, dashed lines; compare also Fig. 2b of Article IV), comprise some species
with morphological affinities to S. sect. Anastomosantes, e.g. S. nabataeorum Eig from the
Scariosa clade. On the other hand, the likewise basally branching Striata clade includes
some of the few species that have colonized truly arid environments, occurring in steppe
and desert habitats.
The lack of exclusive (albeit typical) morphological characteristics of
Scrophularia sects. Tomiophyllum and Anastomosantes illustrates a general problem in
the genus, which is encountered also in Scrophulariaceae and related families (see
chapters 2.1.1., 2.2.3., and 5.1.): morphological synapomorphies for phylogenetic clades
can hardly be found at all; many traits seem to have originated several times
independently and occur in several phylogenetically unrelated groups. In addition,
sequence divergence among Scrophularia species is often very low (Supplementary
Table S1 of Article III); both phenomena could result from recent divergence in some
clades, but are likely also due to hybridization processes. The occurrence of homoplastic
characters is a problem also shared with the sister genus Verbascum, as is the lack of
resolution in molecular phylogenetic trees (Ghahremaninejad et al., 2014).
Despite this apparent deficiency in morphological differentiation, several
examples of conspicuous floral morphology have emerged, often from clades with
otherwise 'ordinary' species. For example, the supposedly hummingbird-pollinated S.
macrantha Greene ex Stiefelh. (Shaw, 1962), endemic to New Mexico (Fig. 10, New
Mexico clade), possesses large, tubular, bright pink corollas, and was found to be sister
to S. laevis Wooton & Standl. (Article II), a species of similarly narrow endemic
distribution but with dull greenish corollas of about 12 mm (Martin and Hutchins,
1981), which was synonymized with S. montana by Shaw (1962). Similar flowers with
tubular but yellow corollas are found in some Southern and Eastern Asian species
(China clade); their pollinators are still unknown. The genus Oreosolen also features this
type of flower, and thus fits smoothly into its position within Scrophularia as suggested
by our molecular results in Article IV. Macaronesia has produced several unusual
Scrophularia flowers (Fig. 3d, g, i), including the Gran Canaria endemic S. calliantha
Webb. & Berthel. with its large, open, orange-red corollas of up to 23 mm. Passerine
birds are the main pollinators of this eye-catching species, alongside insects and even a
141
lizard (Ortega Olivencia et al., 2012). However, this conspicuous morphology does not
seem to be correlated with phylogenetic distinctness; the species remains unresolved
among other endemics within the Macaronesia subclade of the IPM clade (Fig. 1 of
Article III). On other archipelagoes, floral morphology seems to reflect the particular
insular conditions. The Caribbean species S. minutiflora Pennell (Eastern North America
clade) has very small, white corollas. These are typical for the flora of the Antilles and
are thought to be adapted to pollination by minute, endemic insects (Borhidi, 1996).
Altogether, pollinator shifts seem to be responsible for deviant corolla types in several
cases (see Navarro Pérez et al., 2013).
A greater reliance on self-fertilization apparently has influenced S. arguta and the
Arguta clade (Fig. 10). Flowers of the annual S. arguta are often self-pollinating and, in
addition to the chasmogamous flowers, the species possesses smaller cleistogamous
flowers, sometimes on particular shoots near the ground, and even subterranean
inflorescences (Dalgaard, 1979). This is likely to have supported the spread of the
species into areas otherwise unsuitable for Scrophularia, like the Sudan, Eritrea, Somalia
and Oman, where S. arguta is the only representative of the genus. The species from the
Arguta clade (which also includes S. lowei Dalgaard, Fig. 3g), occupy an isolated position
within the genus. The basal position of the clade as sister to all other species of
Scrophularia in the nuclear phylogeny (Fig. 10b) surely reflects its distinctness, but not
necessarily an annual ancestry of the genus as a whole. It is possible that the position of
S. arguta in ITS is artificially due to high substitution rates correlated with a mating
system that emphasizes selfing (Glémin et al., 2006).
5.6. Conclusions and Outlook
The studies and publications presented in this thesis have clarified the
fundamental phylogenetic relationships in the studied groups. Rhinantheae constitute a
predominantly European, or more generally Eurasian group, which dispersed into the
Southern Hemisphere with few representatives. The latter (Bellardia and Euphrasia)
have radiated in South America and Africa as well as New Guinea, New Zealand, and
Australia, respectively. The tribe now includes five main groups alongside five single
genera (see also McNeal et al., 2013; Pinto Carrasco et al., accepted). The genus
Scrophularia is divided into two main groups which largely correspond to those of the
latest taxonomic treatment. Its evolutionary history and biogeography were
substantially influenced by diversification in mountainous regions which constitute its
preferential distribution areas. Centers of species diversity are likewise often correlated
with global biodiversity hotspots, e.g. the Caucasus and the Irano-Anatolian hotspot,
Macaronesia and the Baetic–Rifan complex within the Mediterranean Basin hotspot, the
Mountains of Southwest China hotspot or the Caribbean Islands hotspot (Mittermeier et
al., 2004; Médail and Diadema, 2009). While montane habitats have promoted allopatric
speciation and in several cases preserve species integrity by providing spatial isolation,
the genus' potential for widespread interspecific hybridization (leading to homoploid
hybrid speciation and allopolyploidy), also in the context of historical climatic changes,
has contributed essentially to the diversity observed today, with species distributed
over most of the Northern Hemisphere.
The delimitation of phylogenetic lineages based on molecular data in
Scrophularia is hampered by considerable incongruence and ambiguity in the data,
accompanied by a variable morphology which lacks distinctive shared traits in many
cases. The problem of convergence in morphological characters is encountered also
within Rhinantheae, but while in the latter, the exclusion of incongruent specimens leads
142
to a reliable phylogenetic hypothesis, relationships are excessively interwoven in
Scrophularia. Incongruence and ITS polymorphism are caused by various processes
including hybridization, introgression and ILS, and often their effects overlap, making it
impossible to distinguish single events. Simply analyzing a greater number of molecular
markers does not solve the problem of conflict due to reticulation. Instead in this thesis,
I have chosen to focus on methods which allow a maximum of information to be drawn
from plastid markers and, more importantly, ITS, which as a biparentally inherited
marker is particularly valuable to trace reticulation. Using these approaches, a
phylogenetic framework of the genus has been built and is now available for further,
more detailed study, which might aim at several different aspects:
Complex relationships in certain clades require studies involving multiple
populations, a comprehensive geographic sampling and the according methodical
approaches, including those intended to distinguish hybridization / introgression from
ILS. Furthermore, extensive cloning could provide valuable insights into reticulation
processes within and between clades and species; highly polymorphic markers like
AFLPs or microsatellites might be helpful to assess intraspecific variation. Apart from
that, whole genome analyses by next-generation sequencing as now widely applied,
should be able to add to the understanding of Scrophularia evolution. Another important
focus lies on chromosome evolution within the genus. Providing chromosome counts for
poorly investigated groups, and assessing ploidy levels and genome sizes will help to
further elucidate the role of allopolyploidization.
Ongoing research in Scrophularia concentrates on morphological evolution. The
considerable variability of morphological traits is likely linked to its complex
evolutionary history. In this regard, all previous taxonomic concepts are re-evaluated,
relevant morphological characters extracted, and, together with karyological analyses,
linked to the phylogenetic results presented in this thesis (Scheunert and Heubl, in
prep.). Together with a formal assessment of the taxonomic status of the Himalayan Tibetan endemic Oreosolen, this should provide the basis for a thorough taxonomic
revision of the genus Scrophularia.
143
144
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7. Acknowledgements
First of all I would like to thank my supervisor Günther Heubl, for giving me the
opportunity to conduct my Ph.D. thesis in his lab group, for his continuing support and
being available at any time for helpful discussions. I also want to thank him for the
relaxed working atmosphere and for the great liberty regarding the conceptual design of
my work. The herbaria of the Botanische Staatssammlung (M) and LMU Munich (MSB)
and their curators are greatly acknowledged for providing me with an extensive
collection of Scrophularia specimens, which were the basis of my research. Furthermore,
the herbaria and curators of A, B, E, GH, HAL, KUN, MA, MJG, W, WU are thanked for
providing loans, for access to the specimens for study and for help in obtaining leaf
material. In addition, I owe thanks to the many people who at various times provided
plants, leaf material, fruits and seeds, and photos for my studies. Susanne Renner is
acknowledged for help in obtaining Scrophularia specimens from China and for help in
dating analyses. The Universität Bayern e.V. is thanked for providing graduate funding
for this thesis by means of the Bayerisches Eliteförderungsgesetz (BayEFG). Thanks are
also due to Jochen Heinrichs, Herwig Stibor, Gisela Grupe, Jörg Nickelsen and Martin Heß
for being referees of my Ph.D. thesis.
I am much obliged to Dirk Albach, for his constant willingness to give valuable
advice on a variety of topics, especially with interpreting incongruence in plant
phylogenies. Guido Grimm is greatly acknowledged for his introduction to the pitfalls
and possibilities of phylogenetic reconstruction using ITS data in reticulate lineages. I
also want to thank Dieter Podlech, for his continuing interest in my studies and for
helpful advice. Furthermore, I want to thank Jakub Těšitel and Andreas Fleischmann for
kindly providing photographs for this thesis.
I would like to express my gratitude to Tanja Ernst, for her invaluable, neverending help in the lab (I could not have done this without you!), but also for her friendly
company during many mornings and afternoons. Thanks are also due to Andrea Brandl,
who watered my plants at all times and ever-so-often enlighted our working days with
her bakery mastership. Furthermore, I want to thank all of my Ph.D. colleagues at the AG
Heubl and beyond, especially Andreas Fleischmann, Basti Gardt, Christian Bräuchler, Flo
Turini, Natalie Cusimano, Norbert Holstein, Stefan Kattari, Sylvia Söhner and Yasaman
Salmaki, for their friendship and their support, and for sharing with me some of the best
years of my life. In particular, I want to thank Andreas Fleischmann, for being the
uniquely bright and friendly person he is, for readily sharing his tremendous knowledge,
for his generous help at any time; and for being the one who stayed.
I am deeply indebted to my best friends, Ingrid Zeilinger, Karin Friedrich and Susi
Pfeffer, for helping me through the difficult times of my Ph.D., for readily listening to all
of my often-repeated cant and for being interested in my work. I am grateful to my
beloved Ehefreund Frank Arnoldi, for your very-long-time support, for your being there
all the time, and for living through all of the ups and downs together. And thanks for all
those cups of tea! Finally, I want to thank my parents. Amongst a million other things,
also for conveying to me a deep love for science and nature, without which this task
would have been impossible to accomplish. I am deeply thankful to my dad, for his
continuing financial and emotional support, and for not one second losing his faith in
me. Thank you!
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