DISSERTATION
Titel der Dissertation
Molecular studies in Bromeliaceae:
Implications of plastid and nuclear DNA markers
for phylogeny, biogeography, and character evolution
with emphasis on a new classification of Tillandsioideae
Verfasser
Mag.rer.nat. Michael Harald Johannes Barfuss
angestrebter akademischer Grad
Doktor der Naturwissenschaften (Dr.rer.nat.)
Wien, 2012
Studienkennzahl lt. Studienblatt:
A 091 438
Dissertationsgebiet lt. Studienblatt:
Botanik
Betreuerin:
Ao. Univ.-Prof. Dr. Rosabelle Samuel
Acknowledgements
Warm thanks go to my supervisor, Rosabelle Samuel, for her support and guidance through
the years, and to Walter Till for his long-standing collaboration and sharing his taxonomic
expertise on Bromeliaceae without which the research would not have been successful.
Thanks go to Tod F. Stuessy, emeritus head of the department of Systematic and
Evolutionary Botany at the University of Vienna, for making the laboratory facilities available.
Above all, I would like to thank my colleagues from the molecular laboratory, Elfriede
Grasserbauer, Verena Klejna and Gudrun Kohl, for their patience and support that enabled me
to complete this work.
I would like to extend my gratitude to people who provided plant material, especially
Renate Ehlers, Liselotte and Helmut Hromadnik, Elton M.C. Leme, Jose M. Manzanares, and
Juan P. Pinzón.
Many thanks go to all co-authors, especially to Thomas J. Givnish, Georg Zizka and
Katharina Schulte for helpful discussions, and also to Félix Forest and Mark W. Chase at the
Royal Botanic Gardens, Kew, U.K., with whom I shared useful discussions on low-copy nuclear
genes during my stay at the Jodrell Laboratory.
Special thanks go to Ovidiu Paun, Anton Russell, and Hermann Voglmayr for valuable
discussions, critical review of some parts of this thesis and technical support.
Several members of the department of Systematic and Evolutionary Botany in Vienna
have supported me intellectually and morally and this is gratefully acknowledged: AnneCaroline Cosendai, Kathere Emadzade, Patricio López Sepúlveda, Eva Temsch, Barbara Turner,
and former students, Cordula Blöch, Sutee Duangjai, Eva Maria Mayr, Carolin A. Rebernig,
Stefan Safer, Reinhold Stockenhuber.
Finally I want to thank my family and all friends all over the world who have helped and
supported me in various ways, and anybody else I may have forgotten to mention.
Financial support provided by the University of Vienna, the Commission for
Interdisciplinary Ecological Studies (KIÖS, grant no. 2007-02) at the Austrian Academy of
Sciences (ÖAW), the SYNTHESYS (Synthesis of Systematic Resources) program funded by
European Union, and the Austrian Science Foundation (FWF; grant no. P13690) is greatly
acknowledged.
3
Danksagung
Besonderer Dank geht an meine Betreuerin, Rosabelle Samuel, für ihre langjährige
Unterstützung und Begleitung, und an Walter Till für seine fortwährende Zusammenarbeit und
Einbringung seiner taxonomischen Bromelien-Expertise, ohne die diese Forschungsarbeit nicht
erfolgreich gewesen wäre.
Dank geht an Tod F. Stuessy, emeritierter Leiter des Departments für Botanische
Systematik und Evolutionsforschung der Universität Wien, für die Bereitstellung der
Laboreinrichtungen.
Vor allem möchte ich meinen Kolleginnen aus dem Molekularlabor, Elfriede Grasserbauer,
Verena Klejna und Gudrun Kohl, für ihre Geduld und Unterstützung danken, die es mir
ermöglicht haben, diese Arbeit fertig zu stellen.
Herzlicher Dank geht an mehrere Personen, die Pflanzenmaterial zur Verfügung gestellt
haben, insbesondere Renate Ehlers, Helmut und Liselotte Hromadnik, Elton M.C. Leme, Jose
M. Manzanares, und Juan P. Pinzón.
Dank geht an alle Mitautoren, vor allem an Thomas J. Givnish, Georg Zizka und Katharina
Schulte für hilfreiche Diskussionen und auch an Félix Forest und Mark W. Chase der „Royal
Botanic Gardens, Kew“ (U.K.) für wertvolle Diskussionen über nukleäre Gene während meines
Forschungsaufenthaltes im „Jodrell Laboratory“.
Besonderer Dank geht an Ovidiu Paun, Anton Russell und Hermann Voglmayr für
wertvolle Diskussionen, kritische Überprüfung einiger Teile dieser Arbeit und technische
Unterstützung.
Mehrere Kollegen des Departments für Botanische Systematik und Evolutionsforschung
der Universität Wien haben mich intellektuell und moralisch unterstützt, wofür ich mich aufs
Herzlichste bedanken möchte: Anne-Caroline Cosendai, Kathere Emadzade, Patricio López
Sepúlveda, Eva Temsch, Barbara Turner; und ehemalige Studenten, Cordula Blöch, Sutee
Duangjai, Eva Maria Mayr, Carolin A. Rebernig, Stefan Safer, Reinhold Stockenhuber.
Abschließend möchte ich meiner Familie und allen Freunden auf der ganzen Welt, die mir
geholfen und mich in verschiedenster Weise unterstützt haben, und all jene ich vergessen
habe zu erwähnen, danken.
Finanzielle Unterstützung wurde von der Universität Wien, der Kommission für
Interdisziplinäre Ökologische Studien (KIÖS, Projektnr. 2007-02) an der Österreichischen
Akademie der Wissenschaften (ÖAW), dem von der Europäischen Union geförderte
SYNTHESYS-Programm („Synthesis of Systematic Resources“), und den Fonds zur Förderung
der Wissenschaftlichen Forschung (FWF; Projektnr. P13690) bereit gestellt.
4
Contents
Acknowledgements.............................................................................................................................. 3
Danksagung
....................................................................................................................................... 4
Abstract ....................................................................................................................................... 7
Kurzfassung................................................................................................................................. 9
Introduction .............................................................................................................................. 11
Molecular markers for plant phylogenetics ............................................................................. 11
Characteristics, taxonomic history, and previous molecular studies in Bromeliaceae ............ 12
Taxonomic history and previous molecular studies in Tillandsioideae .................................... 17
Aims of the present study......................................................................................................... 19
References ................................................................................................................................ 19
Part 1.
Application of plastid DNA markers in phylogenetic
reconstructions of Bromeliaceae............................................................. 27
Chapter 1. Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae:
Insights from an eight-locus plastid phylogeny .................................................. 29
Part 2.
Optimization of nuclear DNA markers and their application
in phylogenetic reconstructions of subfamilies Bromelioideae
and Tillandsioideae (Bromeliaceae) ....................................................... 55
Chapter 2. Phylogeny of Bromelioideae (Bromeliaceae) inferred from nuclear and plastid
DNA loci reveals the evolution of the tank habit within the subfamily .............. 57
Chapter 3. Optimizing eight nuclear DNA markers for phylogenetic studies in recently diverged angiosperms: A case study in Bromeliaceae subfamily Tillandsioideae . 73
Part 3.
Classification of Bromeliaceae subfamily Tillandsioideae............. 117
Chapter 4. Progress towards a new classification of Tillandsioideae ................................ 119
Chapter 5. A new classification of Bromeliaceae subfamily Tillandsioideae inferred from
DNA sequences data of two genomes and morphology .................................. 129
Appendix ................................................................................................................................. 207
Conclusions ............................................................................................................................. 233
Curriculum Vitae ................................................................................................................... 235
5
Abstract
The monocot family Bromeliaceae comprises approximately 3,140 species distributed in tropical and subtropical regions of the New World from the southern United States to southern
Argentina. The family is subdivided into eight subfamilies; the most species-rich are Tillandsioideae and Bromelioideae. Taxonomic concepts within Bromeliaceae are highly problematic,
since discriminating morphological characters have been shown to be homoplastic or plesiomorphic. The present study aims to provide a robust phylogenetic framework for Bromeliaceae, especially for the most diverse and complicated subfamilies Bromelioideae and Tillandsioideae. Resulting phylogenies provide a basis to estimate the usefulness of morphological characters and to propose or strengthen hypotheses concerning evolutionary traits, biogeography,
age and origins of bromeliads. The main questions raised are:
(1) Do additional sequence data from the plastid genome and a wider sampling within
Bromeliaceae provide a better resolved, robust phylogenetic framework? What are the
reasons for the low DNA sequence divergence observed up to now?
(2) Can nuclear DNA sequences be successfully implemented for phylogenetic reconstruction? What are the challenges to optimize nuclear markers and do they perform better
than plastid loci?
(3) Can the resulting phylogeny based on plastid and nuclear DNA sequences together
with the re-evaluated morphological characters provide a reasonable, stable classification?
To provide a more robust phylogenetic hypothesis for the classification of Bromeliaceae, eight
rapidly evolving plastid DNA markers (atpB-rbcL, matK, ndhF, psbA-trnH, rpl32-trnL, rps16, trnL
intron, and trnL-trnF) and 90 bromeliad species were included in the current study and analysed using maximum-parsimony, maximum-likelihood, and Bayesian approaches. Results support the formerly proposed eight-subfamily classification based on the single plastid gene
ndhF. Support values for five of the subfamilies increase, but that for Lindmanioideae, Puyoideae, and Bromelioideae decrease as a result of expanded taxon sampling, including several
more divergent species. The initially proposed monophyletic origin of Puyoideae cannot be
unambiguously confirmed. Calibration of the resulting phylogeny against time and biogeographic analysis reveals that Bromeliaceae originated in the Guayana Shield about 100 million
years ago (Ma) and spread radially into adjacent areas ca. 16—13 Ma. Extant lineages arose
between 20 and 5 Ma. Andean uplift facilitated diversification of core Tillandsioideae about
14.2 Ma and Bromelioideae 10.1 Ma, the latter having their greatest diversity in the Brazilian
Shield due to dispersal from the Andes. The most species-rich genera did not appear before
8.7 My with a high diversification between 5 and 4 Ma, which is most likely the reason for the
comparatively low sequence divergence.
To test the usefulness of nuclear regions for phylogenetic reconstruction in Bromelioideae, DNA sequences of part of the low-copy nuclear gene phosphoribulokinase (PRK) and five
7
plastid loci (matK, 3'trnK intron, trnL intron, trnL-trnF, and atpB-rbcL) were investigated. Phylogenetic trees obtained from analyses of the PRK sequences do not contradict trees obtained
from plastid markers. The PRK matrix shows a significantly higher number of potentially PICs
(phylogenetically informative characters) than the plastid dataset (16.9% vs. 3.1%), which improves resolution and support in the resulting trees. Although PRK is not able to resolve relationships completely, the combined analysis with plastid markers yields good support for several uncertain relationships observed previously. The early diverging lineages can be identified
(“basal bromelioids”) and the remainder of the subfamily clustered into a highly supported
clade (“eu-bromelioids”). Results indicate that taxonomic circumscriptions within “core bromeliads” are still insufficient, and relationships complex and difficult to solve. Several genera appear polyphyletic, and Aechmea as well as Quesnelia remain the most complicated genera of
the subfamily. Most-parsimonious character state reconstructions for two evolutionary traits
(tank habit, sepal symmetry) indicate that both characters have undergone few transitions
within the subfamily and thus are not as homoplasious as previously assumed.
The comparative study of nuclear DNA sequences within Tillandsioideae shows that some
nuclear markers are able to provide more information and a higher degree of resolution in
phylogenetic trees than plastid markers. However, their utility does not depend only on sequence variability, but also on methodological challenges in using traditional Sangersequencing. The internal transcribed spacer of nuclear ribosomal DNA (ITS nrDNA) is not recommended as a suitable marker for phylogenetic investigations of Bromeliaceae due to the
presence of strong secondary structures which create problems in amplification and sequencing as well as its low number of PICs for resolving deeper nodes. Amplified fragments of the
genes malate synthase (MS) and RNA polymerase II, beta subunit (RPB2) are not helpful due to
their small size and limited number of PICs. Glucose-6-phosphate isomerase (PGIC), nitrate
reductase (NIA), and xanthine dehydrogenase (XDH) need to be further investigated. Phosphoribulokinase (PRK) and phytochrome C (PHYC) are useful nuclear markers and able to provide considerable resolution in phylogenetic trees, but some relationships are poorly supported.
The combined analysis of nuclear DNA sequence data (PRK, PHYC) with the already existing plastid DNA sequence data (atpB-rbcL, matK, rbcL, partial rbcL-accD, rps16 intron, partial
trnK intron, trnL intron and trnL-trnF) shows a significant increase of resolution within phylogenetic trees of Tillandsioideae. Nine accepted genera can be re-circumscribed and three new
genera are described taxonomically based on morphology. For morphologically distinct species
groups within Racinaea and Tillandsia, two new subgenera are erected. Viridantha has been
downgraded to subgeneric rank. Poor sampling within the Cipuropsis-Mezobromelia clade and
missing support for clades within Tillandsia prevent the recognition of further taxonomic
groups.
8
Kurzfassung
Die Ananasgewächse (Bromeliaceae) sind mit etwa 3.140 Arten eine der artenreichsten Familien der einkeimblättrigen Blütenpflanzen, die in tropischen und subtropischen Regionen der
Neuen Welt vom Süden der Vereinigten Staaten bis in den Süden Argentiniens vorkommen.
Die Familie ist in acht Unterfamilien unterteilt, deren artenreichste die Tillandsioideae und
Bromelioideae sind. Taxonomische Konzepte innerhalb der Bromeliaceae sind sehr problematisch, da zur Unterscheidung herangezogene morphologische Merkmale homoplastisch oder
plesiomorph sind. Die vorliegende Studie zielt darauf ab, eine gut gestützte Stammbaumrekonstruktion für die Bromeliaceae zu erstellen, insbesondere für die komplexen Unterfamilien
Bromelioideae und Tillandsioideae. Die resultierenden Phylogenien sollen eine Grundlage bieten, die taxonomische Verwendbarkeit von morphologischen Merkmalen zu bewerten und
Hypothesen zur Merkmalsevolution, Biogeographie, Alter und Herkunft der Bromelien aufzustellen oder zu untermauern. Die wichtigsten Fragenstellungen sind:
(4) Zeigen eine breitere Probennahme innerhalb der Bromeliaceae sowie zusätzliche Sequenz-Daten aus dem Plastiden-Genom einen besser aufgelösten und gut gestützten
Stammbaum? Was sind Gründe für die bisher beobachtete geringe DNS-SequenzDivergenz?
(5) Können nukleäre DNS-Sequenzen erfolgreich zur Erstellung von Stammbaumrekonstruktion verwendet werden? Was sind die Herausforderungen um nukleäre Marker zu
optimieren und sind sie besser geeignet als Plastiden-Marker?
(6) Kann man mit Hilfe der auf Plastiden- und Kern-DNS-Sequenzen basierenden Phylogenie zusammen mit neu bewerteten morphologischen Merkmalen eine vernünftige,
stabile Klassifikation erreichen?
Um eine besser abgestützte phylogenetische Hypothese für die Klassifizierung der Bromeliaceae zu erreichen, wurden in der aktuellen Studie acht schnell evoluierende Plastiden-DNSMarker (atpB-rbcL, matK, ndhF, psbA-trnH, rpl32-trnL, rps16, trnL Intron und trnL-trnF) und 90
Bromelienarten ausgewählt und mit Maximum-Parsimony-, Maximum-Likelihood- und Bayesian-Methoden analysiert. Die Ergebnisse unterstützen die schon früher vorgeschlagene Einteilung in acht Unterfamilien, die auf einem einzigen Plastiden-Gen (ndhF) basiert. Unterstützungswerte für fünf der Unterfamilien haben sich erhöht, jedoch die für Lindmanioideae,
Puyoideae und Bromelioideae sind infolge der erweiternden Taxon-Auswahl gesunken. Die
ursprünglich vorgeschlagene Monophylie der Puyoideae kann nicht eindeutig bestätigt werden. Eine kalibrierte zeitliche Zuordnung der resultierenden Phylogenie und biogeographische
Analysen zeigen, dass die Bromeliaceae vor rund 100 Millionen Jahren (My) am Guayana-Schild
entstanden sind und sich radial in die angrenzenden Gebiete vor ca. 16-13 My ausgebreitet
haben. Heute noch lebende Entwicklungslinien entstanden vor 20-5 My. Die Andenhebung
begünstigte die Artbildung innerhalb der Kern-Tillandsioideae vor etwa 14,2 My und die der
Bromelioideae vor 10,1 My, wobei letztere ihre größte Vielfalt am brasilianischen Schild aufgrund der Ausbreitung von den Anden her hat. Die heute artenreichsten Gattungen mit einer
9
erhöhten Artbildungsrate vor 5-4 Ma sind nicht vor 8,7 My erschienen, was höchstwahrscheinlich der Grund für die vergleichsweise niedrige Sequenzdivergenz ist.
Um die Verwendbarkeit von nukleären Regionen für phylogenetische Untersuchungen in
den Bromelioideae zu testen, wurden DNS-Sequenzen von einem Abschnitt des in geringer
Kopienzahl vorliegenden nukleären Gens Phosphoribulokinase (PRK) und fünf PlastidenMarker (matK, 3'trnK Intron, trnL Intron, trnL-trnF und atpB-rbcL) untersucht. Stammbäume
aus Analysen der PRK-Daten widersprechen nicht den Ergebnissen, die mit Hilfe von PlastidenMarkern erhalten werden. Die PRK-Matrix zeigt eine deutlich höhere Anzahl an potenziellen
PICs (phylogenetisch informative Merkmale), die die Auflösung und die Absicherung der resultierenden Stammbäume verbessert, als der Plastiden-Datensatz (16,9% vs. 3,1%). Obwohl PRK
nicht in der Lage ist die Verwandtschaftsbeziehungen vollständig aufzulösen, liefert die kombinierte Analyse mit den Plastiden-Markern eine gute Unterstützung für mehrere zuvor unsichere Verwandtschaftsverhältnisse. Die früh abzweigenden Entwicklungslinien konnten identifiziert werden („basal bromelioids“) und der Rest der Unterfamilie ist in einem hoch unterstützten Ast vereinigt („eu-bromelioids“). Die Ergebnisse zeigen, dass die taxonomischen Beziehungen von Gattungen innerhalb der „core bromelioids“ verflochten und schwierig zu lösen sind.
Mehrere Gattungen erscheinen polyphyletisch und Aechmea sowie Quesnelia bleiben die
komplexesten Gattungen der Unterfamilie. Most-Parsimonious-Rekonstruktionen von Merkmalszuständen für Trichterhabitus und Kelchblattsymmetrie zeigen, dass beide Merkmale innerhalb der Unterfamilie nur wenigen morphologischen Veränderungen unterworfen waren
und daher nicht so stark homoplastisch sind wie bisher angenommen.
Die vergleichende Studie von nukleären DNS-Sequenzen innerhalb der Tillandsioideae
zeigt, dass einige nukleäre Marker in der Lage sind, mehr Informationen und eine höhere Auflösung in den Stammbäumen zu erzielen als Plastiden-Marker. Allerdings ist ihre Verwertbarkeit nicht nur von der Sequenzvariabilität, sondern auch von methodischen Herausforderungen der traditionellen Sanger-Sequenzierung abhängig. Der „internal transcribed spacer“ der
nukleären ribosomalen DNS (ITS nrDNS) ist aufgrund von vorhandenen starken Sekundärstrukturen, die Probleme bei der Amplifikation und Sequenzierung bereiten, sowie seiner geringen
Anzahl an PICs kein geeigneter Marker zum Auflösen tieferer Knoten in phylogenetischen Untersuchungen bei den Bromeliaceae. Amplifizierte Fragmente der Gene Malat-Synthase (MS)
und RNA-Polymerase-II, Beta-Untereinheit (RPB2) sind aufgrund ihrer geringen Länge und der
begrenzten Anzahl an PICs nicht sonderlich hilfreich. Glucose-6-Phosphat-Isomerase (PGIC),
Nitrat-Reduktase (NIA) und Xanthin-Dehydrogenase (XDH) müssen weiter untersucht werden.
Phosphoribulokinase (PRK) und Phytochrom C (PHYC) sind nützliche nukleäre Marker und bringen eine gute Auflösung innerhalb vieler Bereiche der Stammbäumen, jedoch sind einige Verwandtschaftsverhältnisse nicht aufgelöst oder nur schlecht unterstützt.
Die kombinierte Analyse der Kern-DNS-Sequenzdaten (PRK, PHYC) mit den bereits vorhandenen Plastiden-DNS-Sequenzdaten (atpB-rbcL, matK, rbcL, einem Teilstück des rbcL-accD,
rps16 intron, einem Teilstück des trnK intron, trnL intron und trnL-trnF) zeigt eine deutliche
Steigerung der Auflösung innerhalb der Stammbäume der Tillandsioideae. Basierend auf morphologischen Merkmalen konnten neun bisher akzeptierte Gattungen neu definiert und drei
Gattungen neu beschrieben werden. Für morphologisch abweichende Artengruppen innerhalb
von Racinaea und Tillandsia werden zwei neue Untergattungen aufgestellt. Viridantha wird auf
Untergattungsniveau herabgestuft. Geringe Probenahme im Cipuropsis-Mezobromelia-Ast und
fehlende Unterstützung für Verwandtschaftsgruppen innerhalb von Tillandsia verhindern eine
taxonomische Beschreibung weiterer Artengruppen.
10
Introduction
Molecular markers for plant phylogenetics
Since the first extensive publication of an angiosperm phylogeny based on plastid rbcL sequence data (Chase & al., 1993), molecular studies in plants using DNA sequences have become popular. Advancements in sequencing technologies in both hardware and software have
facilitated the addition of vast amounts of sequence information to public databases (e.g.,
GenBank) and a great number of published phylogenetic studies over the last 20 years. Milestones in sequencing technology have included the development of the chain-termination
method (Sanger-sequencing) by Sanger & al. (1977) and the release of affordable automated
plate (ABI PRISM 377 Genetic Analyzer, Life Technologies) and, later, capillary sequencers (e.g.,
Applied Biosystems 3730 DNA analyser, Life Technologies; Amersham Bioscience MegaBACE
4000, GE Healthcare) for detection of fluorescently labelled fragments. Preparation, handling,
and throughput of sequences improved greatly, and generating sequence data became a routine exercise. Since 2005, next generation sequencing (NGS) technologies have enabled highthroughput sequencing using fluorescence (454, Roche; Solexa, Illumina; SOLiD, Life Technologies) or pH change measurements (Ion Torrent, Life Technologies) for detection of incorporated nucleotides. Although different applications have recently been developed, high costs,
handling and analysing the huge amounts of sequence data, and the rather uncontrolled sequencing of genomic fragments are still limiting factors for general application of NGS methodologies to plant phylogenetics.
During the period 1993–2000 the most important sources of data for reconstructing plant
phylogenies were a small number of plastid DNA loci and the multi-copy 18S-5.8S-26S nuclear
ribosomal DNA (nrDNA) repeat unit, e.g.,
(1) plastid DNA markers: e.g., atpB (e.g., Hoot & al., 1995); atpB-rbcL (e.g., Manen & al.,
1994); matK (e.g., Johnson & Solitis, 1994, 1995; Liang & Hilu, 1996); ndhF (e.g.,
Olmstead & Reeves, 1995; Olmstead & al., 2000); psbA-trnH (e.g., Sang & al., 1997);
rbcL (e.g., Nickrent & Soltis, 1995); rps16 intron (e.g., Oxelman & al., 1997); trnT-trnLtrnF (e.g., Taberlet & al., 1991; Gielly & Taberlet, 1996; Sang & al., 1997);
(2) nuclear ribosomal DNA markers: e.g., 18S nrDNA (e.g., Nickrent & Soltis, 1995); 26S
nrDNA (e.g., Bult & al., 1995; Kuzoff & al., 1998); external transcribed spacer (ETS nrDNA) (e.g., Baldwin & Markos, 1999); internal transcribed spacer (ITS nrDNA) (e.g.,
White & al., 1990; Baldwin, 1992; Sun & al., 1994; Baldwin & al., 1995; Sang & al.,
1995; Blattner, 1999; Douzery & al., 1999).
More conserved gene regions (atpB, rbcL, 18S nrDNA, 26S nrDNA) were applied at higher taxonomic levels, faster evolving genes (matK, ndhF) at intermediate levels, and more variable
introns and spacer regions (atpB-rbcL, psbA-trnH, rps16 intron, trnT-trnL-trnF, ETS nrDNA, ITS
nrDNA) at interspecific levels. Although new primers for novel markers had been published
(e.g., Demesure & al., 1995; Dumolin-Lapegue & al., 1997; Small & al., 1998), most studies
11
BARFUSS, M.H.J.
MOLECULAR STUDIES
continued to use these frequently sequenced plastid loci. Comparative studies on several plastid DNA markers including information on existing or new universal primers (Shaw & al., 2005,
2007) have increased the number of applicable plastid DNA markers and primers, but also
promoted the further use of plastid DNA sequences solely. Additional published nrDNA primers (e.g., Gruenstaeudl & al, 2009) continue to facilitate the usage of the ITS nrDNA (e.g.,
Russell & al., 2010a; Hřibová & al., 2011).
The first low-copy nuclear DNA genes investigated for their phylogenetic utility belonged
to the phytochrome gene family (PHYA, PHYB, PHYC, PHYD, PHYE; e.g., Mathews & al., 1995;
Mathews & Sharrock, 1996, 1997; Mathews & Donoghue, 1999; Mathews & al., 2000). Only a
few additional studies had utilized other low-copy nuclear DNA loci (e.g., ADH: Morton & al.,
1996; Sang & al., 1997; Sang & Zhang, 1999; ncpGS: Emshwiller & Doyle, 1999; NIA: Howarth &
Baum, 2002; PEPC: Gehrig & al., 1998, 2001; RPB2: Denton & al., 1998). It has been shown that
low-copy nuclear DNA sequences are more variable and phylogenetically informative markers
than plastid DNA regions (e.g., Strand & al., 1997; Sang, 2002; Mort & Crawford, 2004; Small &
al., 2004), but direct sequencing is unfeasible in many cases because of their biparental inheritance and high a frequency of heterozygous individuals carrying alleles of different lengths. In
the case of multi-copy genes, differentiating between paralogous and orthologous sequences
becomes problematic. The need for cloning, and difficulties in interpreting results due to evolutionary processes like hybridization (e.g., Sang, 2004; Ma & al., 2010) or incomplete lineage
sorting (e.g., Piñeiro & al., 2009; Willyard & al., 2009) have limited their usage. However, lowcopy nuclear DNA sequences have been employed with success for some plant groups, e.g.,
angiosperms (RPB2: Oxelman & al., 2004; XDH: Morton & al., 2011), Arecaceae (MS: Lewis &
Doyle, 2001, 2002; Thomas & al., 2006; PRK: Lewis & Doyle, 2002; RPB2: Thomas & al., 2006),
Asteraceae (PGIC: Ford & al., 2006; Stuessy & al., 2011; GADPH: Vaezi & Brouillet, 2009), Orchidaceae (XDH: Górniak & al., 2010), Paeoniaceae (G3PAT: Tank & Sang, 2001), and Passifloraceae (ncpGS: Yockteng & Nadot, 2004; Clarkson & al., 2010). Nevertheless, most phylogenetic studies in flowering plants are still based on maternally inherited, single-copy plastid DNA
regions and the ITS nrDNA, which is usually well homogenized due to the phenomenon of concerted evolution. The absence of universal primers for low-copy nuclear genes has limited their
amplification in a wider range of taxonomic levels in angiosperms.
In certain plant groups the study of few plastid DNA markers together with the ITS nrDNA
has been shown to provide insufficient information for reconstructing phylogenies at lower
taxonomic levels (e.g., Barfuss & al., 2005; Lukas, 2010; Safer, 2011). Two hypotheses can be
formulated, either that in certain plant groups the plastid genome and nrDNA regions evolve
at a much slower rate (e.g., Gaut & al., 1992, 1997), or that extant species have undergone a
rapid diversification process in their recent history (e.g., Cronn & al., 2002; Hughes & Eastwood, 2006), which facilitated great morphological diversity, but comparatively little sequence
divergence. In both cases a combined analysis of several plastid DNA markers (e.g., Chase & al.,
2006; Russell & al., 2010b) or the study of faster-evolving low-copy nuclear loci (e.g., Álvarez &
al., 2008; Russell & al., 2010a) are possible solutions.
Characteristics, taxonomic history, and previous molecular studies in Bromeliaceae
Bromeliaceae is among the most conspicuous and species-rich groups of monocot angiosperms, constituting an early-diverging lineage within the order Poales (Givnish & al., 2007,
2010, see Figure 1; APG III, 2009). They are distributed in various, ecologically diverse habitats
in tropical and subtropical regions of the New World ranging from the southern United States
12
IN BROMELIACEAE
INTRODUCTION
Figure 1. Phylogram of the single most parsimonious tree resulting from analysis of the plastome data showing the
phylogenetic position of Bromeliaceae as the earliest-diverging lineage of Poales. Branch lengths are proportional to
the number of inferred substitutions along each branch. Bootstrap support for each node is shown above the corresponding branch. Monocots are highlighted with magenta branches; cyperids, xyrids, restiids, and graminids, with
colored boxes. (Taken from Givnish & al., 2010).
(Virginia) to Patagonia in southern Argentina (Smith & Downs, 1974; Givnish & al., 1997; Benzing 2000). Their greatest species diversity is found in mountainous regions at elevations between 1000 and 2500 m.a.s.l. throughout the entire distributional range. Only one species,
Pitcairnia feliciana, is found outside this area in Western Africa, which has been shown to be a
result of long-distance dispersal about 10 Ma (Givnish & al., 2007). This supports the hypothesis that extant bromeliads have developed much later than the Gondwanan continent breakup, similarly to other flowering plant families (e.g., Cactaceae).
13
BARFUSS, M.H.J.
MOLECULAR STUDIES
Bromeliads are morphologically very distinctive, hence their inclusion in a separate order Bromeliales. However, a relationship to Velloziaceae has been proposed by cladistic analyses of
morphological characters (Gilmartin & Brown, 1987) and the early molecular studies supported genetic affinities to Rapateaceae or Mayacaceae (Clark & al., 1993; Duvall & al., 1993). The
family is characterized by typical monocot flowers (K3, C3, A3+3, G(3)) arranged in often showy
inflorescences, distinctive leaf rosettes that are often water-impounding (“tanks”), and leaves
that bear absorptive trichomes for water and nutrient uptake. The latter two features have
provided key innovations to favour colonization of habitats of alternating water and nutrient
supply as epiphytes (e.g., Benzing, 2000; Crayn & al., 2004, see Figure 2). Additional adaptions
to drought stress, like succulence and the evolution of the CAM photosynthetic pathway, have
also enabled their establishment in less competitive, extreme and rocky environments as lithophytes (e.g., McWilliams, 1974; Givnish & al., 2007). Several species of the family are of important horticultural value and many botanical gardens, amateur botanists, and private collectors host a large number of species ex situ in living plant collections.
In Bromeliaceae, ca. 3,140 species are classified into 58 genera, which have traditionally
been assigned to three subfamilies (Smith & Downs, 1974, 1977, 1979; Smith & Till, 1998):
(1) Bromelioideae: inferior ovarys, baccate fruits, and serrate or rarely entire leave margins;
(2) Pitcairnioideae: superior to partly inferior ovarys, capsular fruits, bicaudate, winged or
rarely naked seeds, and serrate or rarely entire leave margins;
(3) Tillandsioideae: superior to semi- inferior ovarys, capsular fruits, plumose seed appendages, and exclusively entire leave margins.
Recent molecular studies have revealed that both Bromelioideae and Tillandsioideae are monophyletic and well characterized by their traditional morphology (e.g., Terry & al., 1997a, b;
Crayn & al., 2004; Barfuss & al., 2005; Givnish & al., 2004, 2007; Schulte & al., 2005). Pitcairnioideae is paraphyletic and morphological characters used to define this subfamily are plesiomorphic. Therefore Givnish & al. (2007, see Figure 3) established five additional subfamilies
based on DNA sequence data and a re-evaluation of morphological characters to circumscribe
natural, monophyletic lineages: Brocchinioideae, Lindmanioideae, Hechtioideae, Navioideae,
and Puyoideae.
The most species-rich and taxonomically complicated subfamilies of Bromeliaceae are
Bromelioideae and Tillandsioideae. Subfamily Bromelioideae is the second largest with 32 genera and ca. 800 species (Smith & Till, 1998; Luther 2010). Although distributed over nearly the
whole range of the family, the centre of diversity is found in the Atlantic rain forest of southeastern Brazil (Smith & Downs, 1979). The largest and taxonomically most complex genus of
Bromelioideae is Aechmea (260 spp.). Subfamily Tillandsioideae is the largest subfamily with
nine generally accepted genera and ca. 1,300 species (Smith & Downs, 1977; Smith & Till,
1998; Luther, 2010; Luther & Rabinowitz, 2010) found throughout the distribution range of
Bromeliaceae, indicating the greatest potential to adapt to extreme environments. Centres of
diversity are the northern Andes in Peru, Ecuador and Colombia, the Atlantic rain forest of
south-eastern Brazil, and Central America (Smith & Downs, 1977). It is considered the most
morphologically diverse subfamily, hosting the three large genera Guzmania (210 spp.),
Vriesea (266 spp.), and the most species-rich genus of Bromeliaceae, Tillandsia (626 spp.).
Generic and subgeneric delimitations within subfamilies based on morphology have been
shown to be problematic and in need of urgent revision (e.g., Smith & Kress, 1989, 1990;
14
IN BROMELIACEAE
INTRODUCTION
Figure 2. Strict consensus tree and most-parsimonious reconstructions of the combined matK and rps16 intron data
for 51 species of Bromeliaceae showing the evolution of life-form and photosynthetic pathway. Bootstrap values
13
are indicated above the corresponding branches. Carbon-isotope ratios (δ C values in ‰) are shown for the taxa
analysed. The derived character-states “epiphytic” and “CAM” are highlighted in blue and red, respectively. (Taken
from Crayn & al., 2004).
Brown & Terry, 1992; Grant, 1993b, 1995a; Read & Baensch, 1994; Betancur & MirandaEsquivel, 1999; Brown & Leme, 2005; Betancur & Salinas, 2006; de Sousa & Wendt, 2008).
Several studies investigating distinctive morphological features, which were not yet applied to
the taxonomy, have provided evidence that many characters are plesiomorphic or homoplastic
and hence in their current application fail to delimit natural groups within Bromeliaceae. Interand infrageneric relationships, especially of Bromelioideae and Tillandsioideae, still remained
poorly understood and taxonomic circumscriptions inadequate, because studies have relied on
a very restricted number of species in comparison to the species richness of the subfamilies
and, often, morphological characters are missing from herbarium specimens. Repeated changes of taxonomic boundaries by transferring or excluding species, changes in taxonomic ranks,
and newly proposed segregates since the last comprehensive monograph of the family (Smith
15
BARFUSS, M.H.J.
MOLECULAR STUDIES
Figure 3. Ultrametric tree for Bromeliaceae based on cross-verified penalized likelihood, showing inferred chronology of cladogenesis over the past 20 My. The new subfamilial classification is highlighted by brackets; membership of
subfamilies in the three traditional subfamilies is indicated by shaded bars. Hollow bar = Bromelioideae; gray bar =
Tillandsioideae; solid bars = Pitcairnioideae. (Taken from Givnish & al., 2007).
& Downs, 1974, 1977, 1979), as well as the greatly increased number of newly described species (Luther, 2010) indicate an urgent need for modern taxonomic revision based on reliable
morphological characters.
The first molecular studies on Bromeliaceae used restriction site variation in plastid DNA
(Ranker & al., 1990) to infer phylogenetic relationships within bromeliads. Early studies that
included sequence data used the rbcL gene to clarify either subfamilial relationships or familial,
ordinal and superordinal relationships of Bromeliaceae and related monocot families (Clark &
al., 1993). Their results confirmed a monophyletic origin of Bromeliaceae, but relationships of
the three subfamilies could not be clearly resolved, due to limited taxon sampling. Two studies
on the ndhF gene from the small single-copy region of the plastid genome were published by
Terry & al. (1997a, b). These studies focused on subfamilial relationships (1997a, 30 bromeliad
species) and relationships below the subfamilial level of Tillandsioideae (1997b, 25 tillandsioid
species). Since the resolution of trees obtained from analyses of ndhF sequences was rather
16
IN BROMELIACEAE
INTRODUCTION
low, subsequent publications used the more rapidly evolving trnL intron (Horres & al., 2000; 64
bromeliad species), matK, and rps16 intron sequences (Crayn & al., 2000, 2004; 51 bromeliad
species) to construct phylogenies and infer evolution of certain traits of bromeliads (e.g., CAM
photosynthesis, epiphytic habit). However, these results also showed low sequence divergence
among taxa and it was initially speculated that the plastid genome of Bromeliaceae evolves at
a much slower rate than in other angiosperm lineages (Gaut & al., 1992, 1997; Givnish & al.,
2004). The greatest drawbacks of early bromeliad phylogenetic publications were their reliance on only a few sequences of the plastid genome and the inclusion of a very limited set of
species. Therefore subsequent studies focused on the large subfamilies Tillandsioideae
(Barfuss & al., 2005) and Bromelioideae (Schulte & Zizka, 2008) with a greater selection of taxa
and four to seven plastid markers in concatenated data matrices for analyses. Although it was
possible to resolve major phylogenetic units within the family and subfamilies, the results still
showed low resolution and low support for some deeper nodes. Sequence divergence at generic and interspecific levels was still comparatively low.
Nuclear DNA markers were only recently included in phylogenetic studies of bromeliads.
Studies using limited sets of taxa were undertaken (1) for Bromelioideae using partial sequences of the gene phosphoribulokinase (PRK) (Schulte & al., 2009; see chapter 2); (2) for
Tillandsia subg. Tillandsia using sequences of the internal transcribed spacer 2 (ITS2) and the
ETS nrDNA (Chew & al., 2010); (3) for Aechmea sequencing parts of the genes RNA polymerase
II, beta subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and ETS nrDNA
(Sass & Specht, 2010); (4) for Puya using parts of exon 1 of phytochrome C (PHYC) (Jabaily &
Sytsma, 2010); and (5) for Alcantarea investigating FLORICAULA/LEAFY (FLO/LFY) (Versieux &
al., 2012). Despite the increased usage of nuclear regions, relationships within Bromelioideae
and Tillandsioideae still remain inadequately resolved, particularly within complicated and
species-rich genera such as Aechmea, Tillandsia, and Vriesea.
Taxonomic history and previous molecular studies in Tillandsioideae
In Tillandsioideae more than 1,300 species are classified into nine accepted genera: Alcantarea
(28 spp.), Catopsis (18 spp.), Glomeropitcairnia (2 spp.), Guzmania (210 spp.), Mezobromelia (9
spp.), Racinaea (65 spp.), Tillandsia (626 spp.), Vriesea (266 spp.), and Werauhia (87 spp.). The
large genera Tillandsia and Vriesea as well as Werauhia are further divided into subgenera or
sections (Till, 2000a, b). Since the last monograph for Tillandsioideae by Smith & Downs (1977)
there have been changes in taxonomic limits (Tillandsia, Vriesea), establishment of new genera
(Alcantarea: Grant, 1995; Werauhia: Grant, 1995; Racinaea: Spencer & Smith, 1993; Viridantha: Espejo-Serna, 2002), and transfers of species to other genera (e.g., xerophytic Vriesea
species: Grant, 1993b, 2005). These changes have demonstrated the unreliability of previous
taxonomies based on traditional morphological characters (e.g., flower arrangement, corolla
tube connations, stamen and style position, presence vs. absence of petal appendages). Additional studies on gross morphology of flowers have supported this view (e.g., Brown & Gilmartin, 1984, 1989; Böhme, 1988; Gross, 1988; Gortan, 1991; Halbritter, 1992) and shown that the
three largest genera (Guzmania, Tillandsia, Vriesea) and Mezobromelia are paraphyletic. The
new description and resurrection, respectively, of the genera Viridantha (Espejo-Serna, 2002)
and Sodiroa (Betancur & Miranda-Esquivel, 1999) are not generally accepted (e.g., Barfuss &
al., 2005; Luther 2010). The remaining five currently accepted genera seem to be well circumscribed, with Catopsis and Glomeropitcairnia being the most distinctive genera of the subfamily (Smith & Till, 1998; Till, 2000a).
17
BARFUSS, M.H.J.
MOLECULAR STUDIES
Figure 4. Phylogram of one most parsimonious tree of 110 Tillandsioideae and 12 outgroup taxa obtained from
combined analysis of seven plastid markers. Branch length is given above the branches. Gray bars indicate the tribal
classification of Tillandsioideae, black ones the other subfamilies and the super-outgroup (Rapateaceae). (Taken
from Barfuss & al., 2005).
The two earliest molecular studies focusing on Tillandsioideae were restricted to a single
marker and a limited sample set (ndhF: Terry & al., 1997b; trnL intron: Horres & al., 2000). The
outcomes were correspondingly poor: although they supported the monophyly of Tillandsioideae and the distinct positions of Catopsis and Glomeropitcairnia, relationships of the remaining genera were mostly unresolved. A more recent study with an increased taxon sampling and
seven plastid DNA markers (Barfuss & al., 2005; see Figure 4) confirmed these results but was
18
IN BROMELIACEAE
INTRODUCTION
also able to resolve major taxonomic units and clarify relationships of additional genera (Alcantarea, Guzmania, Werauhia). The remaining genera turned out to be paraphyletic (Vriesea,
Tillandsia) and/or poorly genetically differentiated (Racinaea, Tillandsia) as evident from short
and weakly- or unsupported branches. This demonstrated the need for more rapidly evolving
markers, preferably from the nuclear genome. Two recently published studies utilized either
nrDNA regions (ITS2 and ETS nrDNA for Tillandsia subg. Tillandsia: Chew & al., 2010) or a single
low-copy nuclear gene (FLO/LFY for Alcantarea: Versieux & al., 2012), but were restricted to
small subgroups of Tillandsioideae and a poor sample selection.
Aims of the present study
The scope of the present investigation was defined by three main questions:
(4) Do additional sequence data from the plastid genome and a wider sampling within
Bromeliaceae provide a better resolved, robust phylogenetic framework? What are the
reasons for the low DNA sequence divergence observed up to now?
(5) Can nuclear DNA sequences be successfully implemented for phylogenetic reconstruction? What are the challenges to optimize nuclear markers, and do they perform better than plastid loci?
(6) Can the resulting phylogenies based on plastid and nuclear DNA sequences together
with the re-evaluated morphological characters provide a reasonable, stable classification?
To answer these questions the work was divided into three parts, which correspond to the
three chapters of the present thesis: (1) Application of additional plastid DNA sequence data to
phylogenetic reconstruction in Bromeliaceae to test the eight-subfamily classification and to
strengthen hypotheses concerning biogeography, origin, age, and key trait evolution of extant
bromeliad lineages, pp. 27–54; (2) Optimization of nuclear DNA markers and their application
to phylogenetic reconstructions within Bromeliaceae (in comparison to plastid DNA markers)
to answer questions of relationships and character evolution within Bromelioideae and Tillandsioideae, pp. 55–116; (3) Implementation of phylogenetic results in the classification and
taxonomy of Tillandsioideae, pp. 117–206.
References
Álvarez, I., Costa, A. & Feliner, G.N. 2008. Selecting single-copy nuclear genes for plant phylogenetics: A preliminary analysis for the Senecioneae (Asteraceae). J. Molec. Evol. 66: 276–
291.
APG III (The angiosperm phylogeny group). 2009. An update of the Angiosperm Phylogeny
Group classification for the orders and families of flowering plants: APG III. Bot. J. Linn.
Soc. 161: 105–121.
Baldwin, B.G. 1992. Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: an example from the Compositae. Molec. Phylog. Evol. 1: 3–16.
Baldwin, B.G. & Markos, S. 1999. Phylogenetic utility of the external transcribed spacer (ETS)
of 18S-26S rDNA: Congruence of ETS and ITS Trees of Calycadenia (Compositae). Molec.
Phylog. Evol. 10: 449–463.
Baldwin, B.G., Sanderson, M.J., Porter, J.M., Wojciechowski, M.F., Campbell, C.S. & Donoghue, M.J. 1995. The ITS region of nuclear ribosomal DNA: A valuable source of evidence on angiosperm phylogeny. Ann. Missouri Bot. Gard. 82: 247–277.
19
BARFUSS, M.H.J.
MOLECULAR STUDIES
Barfuss, M.H.J., Samuel, R., Till, W. & Stuessy, T.F. 2005. Phylogenetic relationships in subfamily Tillandsioideae (Bromeliaceae) based on DNA sequence data from seven plastid regions. Amer. J. Bot. 92: 337–351.
Benzing, D.H. 2000. Bromeliaceae: profile of an adaptive radiation. Cambridge University
Press, Cambridge, UK.
Betancur, J. & Miranda-Esquivel, D.R. 1999. ¿Existe Sodiroa? Revista de la Academia Colombiana de Ciencias Exactas, Físicas y Naturales 23: 189–194.
Betancur, J. & Salinas, N.R. 2006. El ocaso de Pseudaechmea (Bromeliaceae: Bromelioideae):
the Pseudaechmea (Bromeliaceae: Bromelioideae) twilight. Caldasia 28: 157–164.
Blattner, F.R. 1999. Direct amplification of the entire ITS region from poorly preserved plant
material using recombinant PCR. BioTechniques 27: 1180–1186.
Böhme, S. 1988. Bromelienstudien III. Vergleichende Untersuchungen zu Bau, Lage und systematischer Verwertbarkeit der Septalnektarien von Bromelien. Tropische und subtropische Pflanzenwelt 62: 125–274.
Brown, G.K. & Gilmartin, A.J. 1984. Stigma structure and variation in Bromeliaceae - neglected
taxonomic characters. Brittonia 36: 364–374.
Brown, G.K. & Gilmartin, A.J. 1989. Stigma types in Bromeliaceae. A systematic survey. Syst.
Bot. 14: 110–132.
Brown, G.K. & Leme, E.M.C. 2005. The re-establishment of Andrea (Bromeliaceae: Bromelioideae), a monotypic genus from Southeastern Brazil threatened with extinction. Taxon 54:
63–70.
Brown, G.K. & Terry, R.G. 1992. Petal appendages in Bromeliaceae. Amer. J. Bot. 79: 1051–
1071.
Bult, C.J., Sweere, J.A. & Zimmer, E.A. 1995. Cryptic sequence simplicity, nucleotide composition bias, and molecular coevolution in the large subunit of ribosomal DNA in plants: implications for phylogenetic analyses. Ann. Missouri Bot. Gard. 82: 235–246.
Chase, M.W. & al. 1993. Phylogenetics of seed plants: an analysis of nucleotide-sequences
from the plastid gene rbcL. Ann. Missouri Bot. Gard. 80: 528–580.
Chase, M.W., Fay, M.F., Devey, D.S., Maurin, O., Rønsted, N., Davies, T.J., Pillon, Y. & al.
2006. Multigene analyses of monocot relationships: A summary. Aliso 22: 63–75.
Chew, T., De Luna, E. & González, D. 2010. Phylogenetic relationships of the pseudobulbous
Tillandsia species (Bromeliaceae) inferred from cladistic analyses of ITS 2, 5.8S ribosomal
RNA gene, and ETS sequences. Syst. Bot. 35: 86–95.
Clark, W.D., Gaut, B.S., Duvall, M.R. & Clegg, M.T. 1993. Phylogenetic relationships of the
Bromeliiflorae-Commeliniflorae-Zingiberiflorae complex of monocots based on rbcL sequence comparisons. Ann. Missouri Bot. Gard. 80: 987–998.
Clarkson, J.J., Kelly, L.J., Leitch, A.R., Knapp, S. & Chase, M.W. 2010. Nuclear glutamine synthetase evolution in Nicotiana: Phylogenetics and the origins of allotetraploid and homoploid (diploid) hybrids. Molec. Phylog. Evol. 55: 99–112.
Crayn, D.M., Terry, R.G., Smith, J.A.C. & Winter, K. 2000. Molecular systematic investigations
in Pitcairnioideae (Bromeliaceae) as a basis for understanding the evolution of crassulacean acid metabolism (CAM). In K. L. Wilson and D. A. Morrison [eds.], Monocots: systematics and evolution, 569–579. CSIRO Publishing, Melbourne, Australia.
Crayn, D.M., Winter, K. & Smith, J.A.C. 2004. Multiple origins of crassulacean acid metabolism
and the epiphytic habit in the Neotropical family Bromeliaceae. Proc. Natl. Acad. Sci.
U.S.A. 101: 3703–3708.
20
IN BROMELIACEAE
INTRODUCTION
Cronn, R.C., Small, R.L., Haselkorn, T. & Wendel, J.F. 2002. Rapid diversification of the cotton
genus (Gossypium: Malvaceae) revealed by analysis of sixteen nuclear and chloroplast
genes. Amer. J. Bot. 89: 707–725.
Demesure, B., Sodzi, N. & Petit R.J. 1995. A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Molec. Ecol.
4: 129–131.
Denton, A.L., McConaughy, B.L. & Hall, B.D. 1998. Usefulness of RNA polymerase II coding
sequences for estimation of green plant phylogeny. Molec. Biol. Evol. 15: 1082–1085.
De Sousa, L.D.F. & Wendt, T. 2008. Taxonomy and conservation of the genus Lymania (Bromeliaceae) in the southern Bahian Atlantic Forest of Brazil. Bot. J. Linn. Soc. 157: 47–66.
Douzery, E.J.P., Pridgeon, A.M., Kores, P., Linder, H.P., Kurzweil, H. & Chase, M.W. 1999.
Molecular phylogenetics of Diseae (Orchidaceae): A contribution from nuclear ribosomal
ITS sequences. Amer. J. Bot. 86: 887–899.
Dumolin-Lapegue, S., Pemonge, M.H. & Petit, R.J. 1997. An enlarged set of consensus primers
for the study of organelle DNA in plants. Molec. Ecol. 6: 393–397.
Duvall, M.R., Clegg, M.T., Chase, M.W., Clark, W.D., Kress, W.J., Hills, H.G., Eguiarte, L.E.,
Smith, J.F., Gaut, B.S., Zimmer, E.A. & Learn Jr., G.H. 1993. Phylogenetic hypotheses for
the monocotyledons constructed from rbcL sequence data. Ann. Missouri Bot. Gard. 80:
607–619.
Emshwiller, E. & Doyle, J.J. 1999. Chloroplast-expressed glutamine synthetase (ncpGS): Potential utility for phylogenetic studies with an example from Oxalis (Oxalidaceae). Molec. Phylog. Evol. 12: 310–319.
Espejo-Serna, A. 2002. Viridantha, un género nuevo de Bromeliaceae (Tillandsioideae) endémico de México. Acta Botánica Mexicana 60: 25–35.
Ford, V.S., Lee, J., Baldwin, B.G. & Gottlieb, L.D. 2006. Species divergence and relationships in
Stephanomeria (Compositae): PGIC phylogeny compared to prior biosystematic studies.
Amer. J. Bot. 93: 480–490.
Gaut, B.S., Clark, L.G., Wendel, J.F. & Muse, S.V. 1997. Comparisons of the molecular evolutionary process at rbcL and ndhF in the grass family (Poaceae). Molec. Biol. Evol. 14: 769–
777.
Gaut, B.S., Muse, S.V., Clark, W.D. & Clegg, M.T. 1992. Relative rates of nucleotide substitution at the rbcL locus in monocotyledonous plants. J. Molec. Evol. 35: 292–303.
Gehrig, H.H., Heute, V. & Kluge, M. 1998. Toward a better knowledge of the molecular evolution of phosphoenolpyruvate carboxylase by comparison of partial cDNA sequences. J.
Mol. Evol. 46: 107–114.
Gehrig, H.H., Heute, V. & Kluge, M. 2001. New partial sequences of phosphoenolpyruvate
carboxylase as molecular phylogenetic markers. Molec. Phylog. Evol. 20: 262–274.
Gielly, L. & Taberlet, P. 1996. A phylogeny of the European gentians inferred from chloroplast
trnL (UAA) intron sequences. Bot. J. Linn. Soc. 120: 57–75.
Gilmartin, A.J. & Brown, G.K. 1987. Bromeliales, related monocots, and resolution of relationships among Bromeliaceae subfamilies. Syst. Bot. 12: 493–500.
Givnish, T.J., Ames, M., McNeal, J.R., McKain, M.R., Steele, P.R., dePamphilis, C.W., Graham,
S.W. & al. 2010. Assembling the tree of the monocotyledons: Plastome sequence phylogeny and evolution of Poales. Ann. Missouri Bot. Gard. 97: 584–616.
Givnish, T.J., Millam, K.C., Berry, P.E. & Sytsma, K.J. 2007. Phylogeny, adaptive radiation, and
historical biogeography of Bromeliaceae inferred from ndhF sequence data. Pp. 3–26 In:
21
BARFUSS, M.H.J.
MOLECULAR STUDIES
Columbus, J.T., Friar, E.A., Porter, J.M., Prince, L.M. & Simpson M.G. (eds.), Monocots:
Comparative Biology and Evolution – Poales. Claremont: Rancho Santa Ana Botanic Garden (RSABG).
Givnish, T.J., Milliam, T.M., Evans, T.M., Hall, J.C., Berry, P.E. & Terry, R.G. 2004. Ancient vicariance or long-distance dispersal? Inferences about phylogeny and South AmericanAfrican disjunctions in Rapateaceae and Bromeliaceae based on ndhF sequence data. Int.
J. Plant Sci. 165 (4, Suppl.), 35–54.
Givnish, T.J., Sytsma, K.J., Smith, J.F., Hohn, W.J., Benzing, D.H. & Burkhardt, E.M. 1997. Molecular evolution and adaptive radiation in Brocchinia (Bromeliaceae: Pitcairnioideae) atop
tepuis of the Guayana shield. Pp. 259–311. In: Givnish, T.J., Sytsma, K.J. (eds.), Molecular
Evolution and Adaptive Radiation. Cambridge University Press, New York.
Górniak, M., Paun, O. & Chase, M.W. 2010. Phylogenetic relationships within Orchidaceae
based on a low-copy nuclear coding gene, XDH: Congruence with organellar and nuclear
ribosomal DNA results. Molec. Phylog. Evol. 56: 784–795.
Gortan, G. 1991. Narbenformen bei Bromeliaceen: Variationsmöglichkeiten und Überlegungen
zu systematisch-taxonomischen Korrelationen. M.Sc. thesis, Universität Wien, Vienna,
Austria.
Grant, J.R. 1993b. True Tillandsias misplaced in Vriesea (Bromeliaceae: Tillandsioideae). Phytologia 75: 170–175.
Grant, J.R. 1995a. Bromelienstudien. The resurrection of Alcantarea and Werauhia, a new
genus. Tropische und subtropische Pflanzenwelt 91: 1–57.
Grant, J.R. 2005 ("2004"). New Combinations and Names in Andean Pitcairnia, Tillandsia, and
Werauhia (Bromeliaceae). Vidalia 2: 23–25.
Gross, E. 1988. Bromelienstudien IV. Zur Morphologie der Bromeliaceen-Samen unter
Berücksichtigung systematisch-taxonomischer Aspekte. Tropische und subtropische Pflanzenwelt 64: 415–625.
Gruenstaeudl, M., Urtubey, E., Jansen, R.K., Samuel, R., Barfuss, M.H.J. & Stuessy, T.F. 2009.
Phylogeny of Barnadesioideae (Asteraceae) inferred from DNA sequence data and morphology. Molec. Phylog. Evol. 51: 572–587.
Hřibová, E., Čížková, J., Christelová, P., Taudien, S., de Langhe, E. & Doležel, J. 2011. The ITS15.8S-ITS2 sequence region in the Musaceae: Structure, diversity and use in molecular phylogeny. PLoS one 6: e17863 (1–11), doi: 10.1371/journal.pone.0017863.
Halbritter, H. 1992. Morphologie und systematische Bedeutung des Pollens der Bromeliaceae.
Grana 31: 197–212.
Horres, R., Zizka, G., Kahl, G. & Weising, K. 2000. Molecular phylogenetics of Bromeliaceae:
Evidence from trnL (UAA) intron sequences of the chloroplast genome. Pl. Biol. 2: 306–
315.
Hoot, S.B. & Douglas, A.W. 1998. Phylogeny of the Proteaceae based on atpB and atpB-rbcL
intergenic spacer region sequences. Australian Syst. Bot. 11: 301–320.
Howarth, D.G. & Baum, D.A. 2002. Phylogenetic utility of a nuclear intron from nitrate reductase for the study of closely related plant species. Molec. Phylog. Evol. 23: 525–528.
Hughes, Colin & Eastwood, R. 2006. Island radiation on a continental scale: Exceptional rates
of plant diversification after uplift of the Andes. Proc. Natl. Acad. Sci. U.S.A. 93: 10334–
10339.
Jabaily, R.S. & Sytsma, K.J. 2010. Phylogenetics of Puya (Bromeliaceae): Placement, major
lineages, and evolution of Chilean species. Amer. J. Bot. 97: 337–356.
22
IN BROMELIACEAE
INTRODUCTION
Johnson, L.A. & Soltis, D.E. 1994. matK DNA sequence and phylogenetic reconstruction in Saxifragaceae s.s. Syst. Bot. 19: 143–156.
Johnson, L.A. & Soltis, D.E. 1995. Phylogenetic inference in Saxifragaceae sensu stricto and
Gilia (Polemoniaceae) using matK sequences. Ann. Missouri Bot. Gard. 82: 149–175.
Kuzoff, R.K., Sweere, J.A., Soltis, D.E., Soltis, P.S. & Zimmer, E.A. 1998. The phylogenetic potential of entire 26S rDNA sequences in plants. Molec. Biol. Evol. 15: 251–263.
Lewis, C.E. & Doyle, J.J. 2001. Phylogenetic Utility of the Nuclear Gene Malate Synthase in the
Palm Family (Arecaceae). Molec. Phylogen. Evol. 19: 409–420.
Lewis, C.E. & Doyle, J.J. 2002. A phylogenetic analysis of tribe Areceae (Arecaceae) using two
low-copy nuclear genes. Pl. Syst. Evol. 236: 1–17.
Liang, H. & Hilu, K.W. 1996. Application of the matK gene sequences to grass systematics. Can.
J. Bot. 74: 125–134.
Lukas, B. 2010. Molekulare Phylogenie und Phytochemie der Gattung Origanum. Dissertation,
University of Vienna.
Luther, H.E. 2010. An alphabetical list of bromeliad binomials. Pp. I-IV, 1–45 In: Rabinowitz, L.
& Holst, B.K. (eds.), An alphabetical list of bromeliad binomials, 12th edition. Sarasota: The
Sarasota Bromeliad Society & Marie Selby Botanical Gardens (MSBG).
Luther, H.E. & Rabinowitz, L. 2010. De Rebus Bromeliacearum IV. Selbyana 30: 147–189.
Ma, J.X., Li, Y.N., Vogl, C., Ehrendorfer, F. & Guo, Y.P. 2010. Allopolyploid speciation and ongoing backcrossing between diploid progenitor and tetraploid progeny lineages in the
Achillea millefolium species complex: Analyses of single-copy nuclear genes and genomic
AFLP. B.M.C. Evol. Biol. 10: 100 (1–11), doi:10.1186/1471-2148-10-100.
Manen, J.-F., Natali, A. & Ehrendorfer, F. 1994. Phylogeny of the Rubiaceae-Rubieae inferred
from the sequence of a cpDNA intergene region. Pl. Syst. Evol. 190: 195–211.
Mathews, S. & Donoghue, M.J. 1999. The root of angiosperm phylogeny inferred from duplicate phytochrome genes. Science 286: 947–950.
Mathews, S., Lavin, M. & Sharrock, R.A. 1995. Evolution of the phytochrome gene family and
its utility for phylogenetic analyses of angiosperms. Ann. Missouri Bot. Gard. 82:.296–321.
Mathews, S. & Sharrock, R.A. 1996. The phytochrome gene family in grasses (Poaceae): a phylogeny and evidence that grasses have a subset of the loci found in dicot angiosperms.
Molec. Biol. Evol. 13: 1141–1150.
Mathews, S. & Sharrock, R.A. 1997. Phytochrome gene diversity. Plant Cell Environ. 20: 666–
671.
Mathews, S., Tsai, R.C. & Kellogg, E.A. 2000. Phylogenetic structure in the grass family (Poaceae): evidence from the nuclear gene phytochrome B. Amer. J. Bot. 87: 96–107.
McWilliams, E.L. 1974. Evolutionary ecology. Pp 40–55 In L. B. Smith & R. J. Downs, Pitcairnioideae (Bromeliaceae). Flora Neotropica Monongraph 14, part 1. Hafner Press, New York,
New York, USA.
Mort, M.E. & Crawford, D.J. 2004. The continuing search: low-copy nuclear sequences for
lower-level plant molecular phylogenetic studies. Taxon 53: 257–261.
Morton, C.M. 2011. Newly sequenced nuclear gene (XDH) for inferring angiosperm phylogeny.
Ann. Missouri Bot. Gard. 98: 63–89.
Morton, B.R., Gaut, B. & Clegg, M.T. 1996. Evolution of alcohol dehydrogenase genes in the
palm and grass families. Proc. Natl. Acad. Sci. U.S.A. 93: 11735–11739.
Nickrent, D.L. & Soltis, D.E. 1995. A comparison of angiosperm phylogenies from nuclear 18S
rDNA and rbcL sequences. Ann. Missouri Bot. Gard. 82: 208–234.
23
BARFUSS, M.H.J.
MOLECULAR STUDIES
Olmstead, R.G. & Reeves, P.A. 1995. Evidence for the polyphyly of the Scrophulariaceae based
on chloroplast rbcL and ndhF sequences. Ann. Missouri Bot. Gard. 82: 176–193.
Olmstead, R.G., Jansen R.K., Kim K.J. & Wagstaff, S.J. 2000. The phylogeny of the Asteridae s.
l. based on chloroplast ndhF sequences. Molec. Phylog. Evol. 16: 96–112.
Oxelman, B., Lidén, M. & Berglund, D. 1997. Chloroplast rps16 intron phylogeny of the tribe
Sileneae (Caryophyllaceae). Pl. Syst. Evol. 206: 393–410.
Oxelman, B., Yoshikawa, N., McConaughy, B.L., Luo, J., Denton, A.L. & Hall, B.D. 2004. RPB2
gene phylogeny in flowering plants, with particular emphasis on asterids. Molec. Phylog.
Evol. 32: 462–479.
Piñeiro, R., Costa, A., Aguilar, J.F. & Feliner, G.N. 2009. Overcoming paralogy and incomplete
lineage sorting to detect a phylogeographic signal: A GapC study of Armeria pungens.
Botany-Botanique 87: 164–177.
Ranker, T.A., Soltis, D.E., Soltis, P.S. & Gilmartin, A.J. 1990. Subfamilial phylogenetic relationships of the Bromeliaceae: evidence from chloroplast DNA restriction site variation. Syst.
Bot. 15: 425–434.
Read, R.W. & Baensch, H.U. 1994. Ursulaea; a new genus of Mexican Bromeliads. J. Bromeliad
Soc. 44: 205–211.
Russell, A., Samuel, R., Klejna, V., Barfuss, M.H.J., Rupp, B. & Chase, M.W. 2010a. Reticulate
evolution in diploid and tetraploid species of Polystachya (Orchidaceae) as shown by plastid DNA sequences and low-copy nuclear genes. Ann. Bot. (Oxford) 106: 37–56.
Russell, A., Samuel, R., Rupp, B., Barfuss, M.H.J., Safran, M., Besendorfer, V. & Chase, M.W.
2010b. Phylogenetics and cytology of a pantropical orchid genus Polystachya (Polystachyinae; Vandeae; Orchidaceae): Evidence from plastid DNA sequence data. Taxon 59: 389–
404.
Safer, S. (2011). Molecular and phytochemical investigations on the genus Leontopodium. Dissertation, University of Innsbruck.
Small, R.L., Cronn, R.C. & Wendel, J.F. 2004. Use of nuclear genes for phylogeny reconstruction in plants. Aust. Syst. Bot. 17: 145–170.
Samuel, R., Kathriarachchi, H., Hoffmann, P., Barfuss, M.H.J., Wurdack, K.J., Davis, C.C. &
Chase, M.W. 2005. Molecular phylogenetics of Phyllanthaceae: Evidence from plastid
matK and nuclear PHYC sequences. Amer. J. Bot. 92: 132–141.
Sang, T. 2002. Utility of low-copy nuclear gene sequences in plant phylogenetics. Critical Reviews in Biochemistry and Molecular Biology 37: 121–147.
Sang, T., Crawford, D.J. & Stuessy, T.F. 1995. Documentation of reticulate evolution in peonies
(Paeonia) using sequences of internal transcribed spacer of nuclear ribosomal DNA: implications for biogeography and concerted evolution. Proc. Natl. Acad. Sci. U.S.A. 92: 6813–
6817.
Sang, T., Crawford, D.J. & Stuessy, T.F. 1997. Chloroplast DNA phylogeny, reticulate evolution,
and biogeography of Paeonia (Paeoniaceae). Amer. J. Bot. 84: 1120–1136.
Sang, T., Donoghue, M.J. & Zhang, D. 1997. Evolution of alcohol dehydrogenase genes in peonies (Paeonia): phylogenetic relationships of putative non-hybrid species. Molec. Biol.
Evol. 14: 994–1007.
Sang, T., Pan, J., Zhang, D., Ferguson, D., Wang, C., Pan, K.-Y. & Hong, D.-Y. 2004. Origins of
polyploids: an example from peonies (Paeonia) and a model for angiosperms. Biol. J. Linn.
Soc.. 82: 561–571.
24
IN BROMELIACEAE
INTRODUCTION
Sang, T. & Zhang, D. 1999. Reconstructing hybrid speciation using sequences of low-copy nuclear genes: Hybrid origins of five Paeonia species based on Adh gene phylogenies. Syst.
Bot. 24: 148–163.
Sanger F, Nicklen, S & Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. U.S.A. 74: 5463–5467.
Sass, C. & Specht, C.D. 2010. Phylogenetic estimation of the core bromelioids with an emphasis on the genus Aechmea (Bromeliaceae). Molec. Phylog. Evol. 55: 559–571.
Schulte, K., Barfuss, M.H.J. & Zizka, G. 2009. Phylogeny of Bromelioideae (Bromeliaceae) inferred from nuclear and plastid DNA loci reveals the evolution of the tank habit within the
subfamily. Molec. Phylogen. Evol. 51: 327–339.
Schulte, K. & Zizka, G. 2008. Multi locus plastid phylogeny of Bromelioideae (Bromeliaceae)
and the taxonomic utility of petal appendages and pollen characters. Candollea 63: 209–
225.
Shaw, J., Lickey, E.B., Beck, J.T., Farmer, S.B., Liu, W.S., Miller, J., Siripun, K.C., Winder, C.T.,
Schilling, E.E. & Small, R.L. 2005. The tortoise and the hare II: Relative utility of 21
noncoding chloroplast DNA sequences for phylogenetic analysis. Amer. J. Bot. 92: 142–
166.
Shaw, J., Lickey, E.B., Schilling, E.E. & Small, R.L. 2007. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms:
The tortoise and the hare III. Amer. J. Bot. 94: 275–288.
Small, R.L., Ryburn, J.A., Cronn, R.C., Seelanan, T. & Wendel, J.F. 1998. The tortoise and the
hare: choosing between noncoding plastome and nuclear Adh sequences for phylogenetic
reconstruction in a recently diverged plant group. Amer. J. Bot. 85: 1301–1315.
Smith, L.B. & Downs, R.J. 1974. Pitcairnioideae (Bromeliaceae). In Flora Neotropica. Monograph 14, vol. 1, 1–660. Hafner Press, New York, USA.
Smith, L.B. & Downs, R.J. 1977. Tillandsioideae (Bromeliaceae). In Flora Neotropica. Monograph 14, vol. 2, 661–1492. Hafner Press, New York, USA.
Smith, L.B. & Downs, R.J. 1979. Bromelioideae (Bromeliaceae). In Flora Neotropica. Monograph 14, vol. 3, 1493–2142. New York Botanical Garden, Bronx, New York, USA.
Smith, L.B. & Kress, W.J. 1989. New or restored genera of Bromeliaceae. Phytologia 66, 70–79.
Smith, L.B. & Kress, W.J. 1990. New genera of Bromeliaceae. Phytologia 69, 271–274.
Smith, L.B. & Till, W. 1998. Bromeliaceae. In K. Kubitzki [ed.], The Families and Genera of Vascular Plants, vol. 4, 74–99. Spriger, Berlin, Germany.
Spencer, M.A. & Smith, L.B. 1993. Racinaea, a new genus of Bromeliaceae (Tillandsioideae).
Phytologia 74: 151–160.
Strand, A.E., LeebensMack, J. & Milligan, B.G. 1997. Nuclear DNA-based markers for plant
evolutionary biology. Molec. Ecol. 6: 113–118.
Stuessy, T.F., Blöch, C., Villaseñor, J.L., Rebernig, C.A. & Weiss-Schneeweiss, H. 2011. Phylogenetic analyses of DNA sequences with chromosomal and morphological data confirm
and refine sectional and series classification within Melampodium (Asteraceae, Millerieae). Taxon 60: 436–449.
Sun, Y., Skinner, D.Z., Liang, G.H. & Hulbert, S.H. 1994. Phylogenetic analysis of Sorghum and
related taxa using Internal Transcribed Spacers of nuclear ribosomal DNA. Theor. Appl.
Genet. 89: 26–32.
Taberlet, P., Gielly, L., Pautou G. & Bouvet, J. 1991. Universal primers for amplification of
three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105–1109.
25
Tank, D.C. & Sang, T. 2001. Phylogenetic utility of the glycerol-3-phosphate acyltransferase
gene: Evolution and implications in Paeonia (Paeoniaceae). Molec. Phylog. Evol. 19: 421–
429.
Terry, R.G., Brown, G.K. & Olmstead, R.G. 1997a. Examination of subfamilial phylogeny in
Bromeliaceae using comparative sequencing of the plastid locus ndhF. Amer. J. Bot. 84:
664–670.
Terry, R.G., Brown, G.K. & Olmstead, R.G. 1997b. Phylogenetic relationships in subfamily Tillandsioideae (Bromeliaceae) using ndhF sequences. Syst. Bot. 22: 333–345.
Thomas, M.M., Garwood, N.C., Baker, W.J., Henderson, S.A., Russell, S.J., Hodel, D.R. &
Bateman, R.M. 2006. Molecular phylogeny of the palm genus Chamaedorea, based on the
low-copy nuclear genes PRK and RPB2. Molec. Phylogen. Evol. 38: 398–415.
Till, W. 2000a. Tillandsioideae. In D. H. Benzing [ed.], Bromeliaceae: profile of an adaptive radiation, 555–572. Cambridge University Press, Cambridge, UK.
Till, W. 2000b. Tillandsia and Racinaea. In D. H. Benzing [ed.], Bromeliaceae: profile of an
adaptive radiation, 573–586. Cambridge University Press, Cambridge, UK.
Vaezi, J. & Brouillet, L. 2009. Phylogenetic relationships among diploid species of Symphyotrichum (Asteraceae: Astereae) based on two nuclear markers, ITS and GAPDH. Molec.
Phylog. Evol. 51: 540–553.
Versieux, L.M., Barbará, T., Wanderley, M.d.G.L., Calvente, A., Fay, M.F. & Lexer, C. 2012.
Molecular phylogenetics of the Brazilian giant bromeliads (Alcantarea, Bromeliaceae): implications for morphological evolution and biogeography. Molec. Phylog. Evol. 64: 177–
189.
White, T.J., Bruns, T., Lee, S. & Taylor, J. 1990. Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics, Pp. 315–322 in Innis, M.A., Gelfand, D.H., Sninsky,
J.J. & White, T.J. (eds.), PCR protocols: A guide to methods and applications. San Diego:
Academic Press.
Willyard, A., Cronn. R. & Liston, A. 2009. Reticulate evolution and incomplete lineage sorting
among the ponderosa pines. Molec. Phylog. Evol. 52: 498–511.
Yockteng, R. & Nadot, S. 2004. Phylogenetic relationships among Passiflora species based on
the glutamine synthetase nuclear gene expressed in chloroplast (ncpGS). Molec. Phylog.
Evol. 31: 379–396.
26
Part 1
Application of plastid DNA markers in
phylogenetic reconstructions of Bromeliaceae
Chapter 1
Phylogeny, adaptive radiation, and historical
biogeography in Bromeliaceae: Insights from
an eight-locus plastid phylogeny
Thomas J. Givnish, Michael H.J. Barfuss, Benjamin Van Ee,
Ricarda Riina, Katharina Schulte, Ralf Horres, Philip A. Gonsiska,
Rachel S. Jabaily, Darren M. Crayn, J. Andrew C. Smith,
Klaus Winter, Gregory K. Brown, Timothy M. Evans,
Bruce K. Holst, Harry Luther, Walter Till, Georg Zizka,
Paul E. Berry & Kenneth J. Sytsma
Status: published, American Journal of Botany 98 (5): 872–895. 2011.
Contribution: data collection, preliminary data analyses, manuscript writing/editing.
31
BARFUSS, M.H.J.
32
BROMELIAD PHYLOGENY
AND EVOLUTION
PART 1, CHAPTER 1
33
BARFUSS, M.H.J.
34
BROMELIAD PHYLOGENY
AND EVOLUTION
PART 1, CHAPTER 1
35
BARFUSS, M.H.J.
36
BROMELIAD PHYLOGENY
AND EVOLUTION
PART 1, CHAPTER 1
37
BARFUSS, M.H.J.
38
BROMELIAD PHYLOGENY
AND EVOLUTION
PART 1, CHAPTER 1
39
BARFUSS, M.H.J.
40
BROMELIAD PHYLOGENY
AND EVOLUTION
PART 1, CHAPTER 1
41
BARFUSS, M.H.J.
42
BROMELIAD PHYLOGENY
AND EVOLUTION
PART 1, CHAPTER 1
43
BARFUSS, M.H.J.
44
BROMELIAD PHYLOGENY
AND EVOLUTION
PART 1, CHAPTER 1
45
BARFUSS, M.H.J.
46
BROMELIAD PHYLOGENY
AND EVOLUTION
PART 1, CHAPTER 1
47
BARFUSS, M.H.J.
48
BROMELIAD PHYLOGENY
AND EVOLUTION
PART 1, CHAPTER 1
49
BARFUSS, M.H.J.
50
BROMELIAD PHYLOGENY
AND EVOLUTION
PART 1, CHAPTER 1
51
BARFUSS, M.H.J.
52
BROMELIAD PHYLOGENY
AND EVOLUTION
PART 1, CHAPTER 1
53
54
Part 2
Optimization of nuclear DNA markers and their
application in phylogenetic reconstructions of
subfamilies Bromelioideae and Tillandsioideae
(Bromeliaceae)
Chapter 2
Phylogeny of Bromelioideae (Bromeliaceae)
inferred from nuclear and plastid DNA loci
reveals the evolution of the tank habit
within the subfamily
Katharina Schulte, Michael H.J. Barfuss & Georg Zizka
Status: published, Molecular Phylogenetics and Evolution 51 (2): 327–339. 2009.
Contribution: primer design, data collection, preliminary data analyses, manuscript writing/editing.
59
BARFUSS, M.H.J.
60
PHYLOGENY OF
BROMELIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 2
61
BARFUSS, M.H.J.
62
PHYLOGENY OF
BROMELIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 2
63
BARFUSS, M.H.J.
64
PHYLOGENY OF
BROMELIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 2
65
BARFUSS, M.H.J.
66
PHYLOGENY OF
BROMELIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 2
67
BARFUSS, M.H.J.
68
PHYLOGENY OF
BROMELIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 2
69
BARFUSS, M.H.J.
70
PHYLOGENY OF
BROMELIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 2
71
Chapter 3
Optimizing eight nuclear DNA markers for
phylogenetic studies in recently diverged angiosperms:
A case study in Bromeliaceae subfamily Tillandsioideae
Michael H.J. Barfuss 1, Rosabelle Samuel 1 & Félix Forest 2
1
University of Vienna, Faculty of Life Sciences, Department of Systematic and Evolutionary
Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria
2
Royal Botanic Gardens, Kew, Jodrell Laboratory, Richmond, Surrey, TW9 3DS, U.K.
Author for correspondence: Michael H.J. Barfuss, michael.h.j.barfuss@univie.ac.at
Keywords: glucose-6-phosphate isomerase, cytosolic; malate synthase; low-copy nuclear DNA;
nitrate reductase 1, [NADH]; ITS nuclear ribosomal DNA; phosphoribulokinase; phytochrome C;
RNA polymerase II, beta subunit; xanthine dehydrogenase
Running Title: Nuclear DNA markers in Tillandsioideae (Bromeliaceae)
Status: in preparation, intended to be submitted to American Journal of Botany
Contribution: data collection, data analyses, manuscript writing
Abstract
Comparative studies of nuclear DNA sequences within Tillandsioideae show that several nuclear markers are able to provide more information and a higher degree of resolution in phylogenetic trees than previously used plastid markers. However, their utility depends not only on
sequence variability, but also on methodological challenges, especially when traditional Sanger-sequencing is used. The presence of strong secondary structures in ITS nrDNA creates problems in amplification and sequencing. Therefore it is not recommended as a suitable marker
for phylogenetic investigations of Bromeliaceae, also because of low resolution at deeper
nodes. However, ITS nrDNA regions might be useful for delimiting species. Amplified fragments
of the genes malate synthase (MS) and RNA polymerase II, beta subunit (RPB2) are not helpful
due to their relatively short length and restricted number of PICs. Glucose-6-phosphate isomerase (PGIC) needs to be further investigated, but sequence length and variation found in the
two species sequenced suggests it will be suitable at generic and higher taxonomic levels.
Likewise, nitrate reductase 1, [NADH] (NIA) and xanthine dehydrogenase (XDH) need further
investigations; the first might be suitable for lower taxonomical levels, while the second would
be better suited for higher levels. Phosphoribulokinase (PRK) seems to be one of the most
promising markers, but length differences between bromeliad taxa and indels within alleles of
heterozygous accessions made it the most difficult to edit and align and thus time-consuming.
For phytochrome C (PHYC) generating sequences and the editing/alignment have been straight
forward. PRK and PHYC are useful nuclear markers and able to provide considerable resolution
in phylogenetic trees, though some relationships remain uncertain.
75
Introduction
Despite rapid technical progress in molecular systematics, the goal of reconstructing fully resolved and well-supported phylogenetic trees in many species-rich and/or recently diverged
angiosperm groups remains challenging (e.g., Lukas, 2010; Safer, 2011). Many molecular studies still analyze only plastid DNA markers or the multi-copy internal transcribed spacer nuclear
ribosomal DNA (ITS nrDNA) of the 18S-5.8S-26S nrDNA repeat unit (e.g., Gruenstaudl & al.,
2009; Russell & al., 2010). Availability of universal primers for recently characterized plastid
markers (Shaw & al., 2005; 2007) and the ITS nrDNA region (White & al., 1990; Sun & al., 1994;
Blattner, 1999; Gruenstaeudl & al., 2009) as well as the most advantageous single-copy nature
of plastid DNA and the usually well-homogenized ITS nrDNA (concerted evolution) made them
more suitable for phylogenetic studies. Usually these markers are simple to amplify, directly
sequence, and align across lower taxonomic levels. Also, design of new primers became much
easier due to the steadily increasing number of available sequences and complete plastid genomes publically available. However, a major challenge of commonly used plastid DNA and
nrDNA markers is resolution of phylogenetic relationships at all taxonomic levels, even when
many markers are combined. A common strategy to solve these problems is the use of lowcopy nuclear DNA (e.g., Strand & al., 1997; Sang, 2002; Chapman 2007; Álvarez & al., 2008),
but these markers are often avoided for several reasons. Inheritance of plastid DNA is predominantly uniparental and maternal in plants, whereas nuclear DNA is biparentally inherited, although due to conversion nrDNA can become uniparental. Processes like incomplete lineage
sorting, hybridization, coalescence times longer than species ages, and introgression may
complicate interpretation of results (Piñeiro & al., 2009; Willyard & al., 2009). More difficulties
arise when polyploid taxa or multi-copy nuclear genes and gene families are investigated (e.g.,
Sang & al., 2004). Analyzing and interpreting such data can be problematic because multiple
alleles and paralogs are present. However, for particular evolutionary questions and certain
plant groups with low variation in plastid DNA or nrDNA sequences, more rapidly evolving lowcopy nuclear DNA markers are a promising solution. An increasing number of studies published
over the recent years have utilized such sequences to investigate phylogenetic relationships
(e.g., Emshwiller & Doyle, 1999; Cronn & al., 2002; Yockteng & Nadot, 2004; Vaezi & Brouillet,
2009), reticulate evolution (e.g., Ma & al., 2010; Russell & al., 2010) and hybrid speciation (e.g.,
Sang & Zhang, 1999; Tank & Sang, 2001; Clarkson & al., 2010), or to uncover genome evolution
(e.g., Wendel, 2000).
The pineapple family (Bromeliaceae), with more than 3,100 species assigned to 58 genera,
represents a large and ecologically important group of monocots for which infrafamilial classification and phylogenetic relationships have been problematic for many years. Several earlier
studies have tried to create well-resolved trees using one or a few plastid markers with limited
success (Terry & al., 1997a, b; Horres & al., 2000; Crayn & al., 2004). Accompanying studies
focusing on the largest subfamilies Tillandsioideae (Barfuss & al., 2005) and Bromelioideae
(Schulte & Zizka, 2008) as well as Bromeliaceae in general (Givnish & al., 2011), both including
a wider selection of taxa and plastid markers, were able to resolve the main phylogenetic units
within the family but still showed low support for some deeper nodes and comparatively little
sequence divergence between genera and species. Efforts to sequence and combine many
plastid spacer regions that are supposed to evolve more rapidly (Shaw & al., 2005, 2007) than
previously sequenced markers for Bromeliaceae (Barfuss & al., 2005; Schulte & Zizka, 2008;
77
BARFUSS, M.H.J.
NUCLEAR DNA MARKERS
Givnish & al., 2011: atpB-rbcL, trnK-matK, ndhF, psbA-trnH, rpL32-trnL, rps16 intron, and
5'trnL-3'trnL-trnF) are of limited success in gathering sufficient phylogenetic information at
lower taxonomic levels (M. H. J. Barfuss, unpublished data: atpI-atpH, psbD-trnT, psbJ-petA,
rpoB-trnC, trnD-trnT, 3'trnV-ndhC,and trnQ-5'rps16). Low sequence divergence associated with
plastid DNA sequences in phylogenetic studies of bromeliads were initially mainly attributed to
generally low substitution rates in the bromeliad plastid genome (e.g., Gaut & al., 1992;
Givnish & al., 2004, 2007). Insights from the recently published eight-locus plastid analysis of
Bromeliaceae have shown that the family itself is old (100 My), but extant lineages of bromeliads arose less than 20 My ago (Givnish & al., 2011). The most species-rich clades are less than
5 My old. These results support the hypothesis that extant lineages have undergone recent
and rapid radiations and subsequent diversification in newly formed ecological niches and not
that substitution rates are lower than in other angiosperm lineages. The first molecular studies
of bromeliads that include low-copy nuclear DNA sequences were recently published by
Schulte & al. (2009) for Bromelioideae (phosphoribulokinase, PRK), Chew & al. (2010) for Tillandsia subgenus Tillandsia (internal transcribed spacer 2, ITS 2, external transcribed spacer,
ETS nrDNA), Sass & Specht (2010) for Aechmea (RNA polymerase II, beta subunit, RPB2, glyceraldehyde-3-phosphate dehydrogenase, G3PDH, ETS nrDNA), Jabaily & Sytsma (2010) for Puya
(phytochrome C, PHYC), and Versieux & al. (2012) for Alcantarea (FLORICAULA/LEAFY,
FLO/LFY). However, relationships within Bromeliaceae still remain inadequately understood
because these markers were applied only to restricted sets of taxa.
In this study we aim (1) to investigate primers for several nuclear DNA markers of angiosperms with focus on monocots and Bromeliaceae especially; (2) to resolve lower taxonomic
units of subfamily Tillandsioideae down to species groups; (3) to evaluate relationships of recently diverged species; and (4) to detect hybrids, possible hybrid speciation, and reticulate
evolution. We also selected the ITS nrDNA since the complete region was never used in a phylogenetic study for a large set of samples above the subgeneric level.
Materials and methods
Selection of taxa
For initial primer trials, three sets of few individuals of Tillandsioideae (2, 8, or 13) were selected. Larger sets of samples (64, 72, or 114) with a minimum of two accessions per major clade
in other analyses were chosen based on analyses of seven plastid markers (Barfuss & al., 2005)
to demonstrate variability of lineages. The complete set of samples (444) was finally analyzed
for the most promising nuclear DNA markers. A sample list including botanical authors is given
in Supplementary Data of chapter 5; accession details can be obtained from the first author
upon request.
Extraction of high-quality DNA
Initially the CTAB procedure established by Doyle and Doyle (1987) modified for minipreps (1.5
or 2 mL tubes) was used. Due to the high content of polysaccharides, polyphenols, and other
secondary metabolites in more than ⅔ of the Tillandsioideae species studied, this method did
not yield sufficient amounts of high quality DNA, which is necessary to amplify suitable quantities of low-copy nuclear DNA. Therefore total genomic DNA was extracted from fresh or silicagel dried leaf tissue using the altered CTAB procedure of Tel-Zur & al. (1999) with further
78
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
reagent modifications, and with adjustments within the protocol, mainly implemented to allow
the samples to be processed as minipreps. Most important chemical changes from the original
protocol include (1) the addition of 1% PVP-40 into the Sorbitol buffer, (2) the increase of the
CTAB concentration to 3% and the NaCl concentration to 3 M in the high-salt CTAB buffer, and
(3) the addition of PVP-40 into the extraction buffer. In most cases this method yielded superior quantities of high molecular weight DNA which could be used immediately for PCR. When
good DNA was visualized on 1% TAE agarose gels, but initial PCR trials were not successful,
DNA was further purified or newly extracted using a modified CTAB/DNeasy Plant Mini Kit (QIAGEN) procedure. Protocols have been developed either starting from DNA dissolved in TE
buffer or after CTAB extraction before (and therefore not doing a) precipitation with isopropanol, to avoid precipitating of any unwanted contaminants (mainly polyphenolic compounds)
which could inhibit Taq polymerase activity. This combined method yielded much better DNA
extracts than using the QIAGEN kit alone. Detailed protocols of all modifications of the extraction procedures can be obtained from the first author upon request.
Selection of nuclear markers
The ITS nrDNA region (e.g., White & al., 1990; Sun & al., 1994; Gruenstaeudl & al., 2009; Chew
& al., 2010) and seven low-copy nuclear DNA markers were selected and tested: malate synthase (MS; e.g., Lewis & Doyle, 2001, 2002), RNA polymerase II, beta subunit (RPB2; e.g., Denton & al., 1998; Oxelman & al., 2004; Thomas & al., 2006; Sass & Specht, 2010), glucose-6phosphate isomerase, cytosolic (G6PIC = phosphoglucose isomerase C, PGIC; Terauchi & al.,
1997; Ford & al., 2006), nitrate reductase 1, [NADH] (NR1 = nitrate reductase apoenzyme, NIA :
e.g., Howarth & Baum, 2002), xanthine dehydrogenase (XDH; Górniak & al., 2010; Morton,
2011), phosphoribulokinase (PRK; e.g., Lewis & Doyle, 2002; Thomas & al., 2006; Schulte & al.,
2009), and phytochrome C (PHYC; e.g., Mathews & Donoghue, 1999; Samuel & al., 2005; Russell & al., 2010). Regions were selected based on the availability of primers and/or DNA sequences available in GenBank to be able to design primers for Bromeliaceae. The primary target was to obtain one or more genomic markers that are either homogenized (ITS nrDNA) or
can be treated as effectively single-copy (other nuclear markers) to be sequenced directly.
Other parameters for the selection of markers were (1) performance of primers and reagents
for both amplification (e.g., primer annealing, primer-dimer formation) and sequencing (e.g.,
homogeneity), (2) occurrence of homopolymers and microsatellites, which can give problems
in sequencing, (3) amplicon length, and (4) number of potential phylogenetically informative
characters (PICs). The genetic structure of each low-copy nuclear DNA region was determined
from the assembled RefSeq genome of Oryza sativa and is summarized in Table 1. Positions of
primers selected finally are given in Figure 1, A–H. All sequences generated for this study will
be deposited in GenBank (http://www.ncbi.nlm.nih.gov/genbank).
Nomenclature of primers
Positions of primers on chromosomes and within the coding sequence (CDS) of the genes were
determined by BLAST searches, both within the entire database and the annotated genes of
the assembled RefSeq genome of Oryza sativa (Tables 3–10). This was possible because all
primers are located in exons at least for ¾ of their length. Names of previously published primers were taken from their primary publication, and those of newly generated or modified
ones were derived from the CDS position of the last primer base at the 3' end. If a modified
primer ended at the same position as the original one, a number after a hyphen was attached.
79
BARFUSS, M.H.J.
NUCLEAR DNA MARKERS
Letter extensions after a hyphen indicate primers specific to a particular plant group (an = angiosperms, mo = monocots, br = bromeliads).
Table 1. Attributes of the low-copy nuclear genes based on GenBank information from the assembled RefSeq genome of Oryza sativa. Amplified regions are highlighted in red. Annotation of malate synthase and RNA polymerase
II, beta subunit was incomplete or wrong and therefore done manually aided with reference sequences of Arabidopsis thaliana (RPB2) or Zea mays (MS).
exon 1
intron 1
exon 2
intron 2
exon 3
intron 3
exon 4
intron 4
exon 5
intron 5
exon 6
intron 6
exon 7
intron 7
exon 8
intron 8
exon 9
intron 9
exon 10
intron 10
exon 11
intron 11
exon 12
intron 12
exon 13
intron 13
exon 14
intron 14
exon 15
intron 15
exon 16
intron 16
exon 17
intron 17
exon 18
intron 18
exon 19
intron 19
exon 20
intron 20
exon 21
intron 21
exon 22
intron 22
exon 23
intron 23
exon 24
intron 24
exon 25
80
malate synthase
(MS)
RNA polymerase II,
beta subunit (RPB2)
glucose-6-phosphate isomerase,
cytosolic (G6PIC, PGIC)
Chromosome: 4 (NC_008397)
Gene: MS (Os04g0486950)
mRNA: NM_001187064
Total range: 24,724,583-24,726,722
total length: 2131 (complement)
Processed length: 1704
Protein product length: 567
Copy number: 1
Chromosome: 3 (NC_008396)
Gene: RPB2 (Os03g0646800)
mRNA: NM_001057305
Total range: 25,794,125-25,801,820
Total length: 7,696
Processed length: 3,555
Protein product length: 1184
Copy number: 1
Chromosome: 6 (NC_008399)
Gene: PGIC-b (Os06g0256500)
mRNA: NM_001063851
Total range: 8,151,838-8,158,107
Total length: 6,270
Processed length: 1,704
Protein product length: 567
Copy number: 2
Duplicate gene: PGIC-a (Os03g0776000)
Incomplete annotation in GenBank!
Incomplete annotation in GenBank!
1-375
376-459
460-785
786-1048
1049-1379
1380-1468
1469-2140
1-234
235-1792
1793-2014
2015-2085
2086-2376
2377-2582
2583-2780
2781-2907
2908-2972
2973-3054
3055-3141
3142-3254
3255-3375
3376-3466
3467-3511
3512-3587
3588-3725
3726-3837
3838-3910
3911-3989
3990-4066
4067-4148
4149-4256
4257-4374
4375-4491
4492-4790
4791-4946
4947-5046
5047-5148
5149-5302
5303-5428
5429-5503
5504-5581
5582-5656
5657-5752
5753-5858
5859-5974
5975-6047
6048-6117
6118-6196
6197-6250
6251-6328
6329-6472
6473-6563
6564-6863
6864-7046
7047-7469
7470-7582
7583-7696
375
84
326
263
331
89
672
234
1558
222
71
291
206
198
127
65
82
87
113
121
91
45
76
138
112
73
79
77
82
108
118
117
299
156
100
102
154
126
75
78
75
96
106
116
73
70
79
54
78
144
91
300
183
423
113
114
1-51
52-205
206-282
283-375
358-439
440-512
513-560
561-862
863-1018
1019-1093
1094-1190
1191-1288
1289-1331
1332-1721
1722-1789
1790-1879
1880-1964
1965-2044
2045-2093
2094-2314
2315-2387
2388-2465
2466-2526
2527-3460
3461-3527
3528-3596
3597-3653
3654-3746
3747-3800
3801-3883
3884-4012
4013-4538
4539-4602
4603-4712
4713-4786
4787-4869
4870-4923
4924-5042
5043-5111
5112-5740
5741-5830
5831-6002
6003-6110
6111-6222
6223-6270
51
154
77
75
82
73
48
302
156
75
97
98
43
390
68
90
85
80
49
221
73
78
61
934
67
69
57
93
54
83
129
526
64
110
74
83
54
119
69
629
90
172
108
112
48
IN TILLANDSIOIDEAE (BROMELIACEAE)
exon 1
intron 1
exon 2
intron 2
exon 3
intron 3
exon 4
intron 4
exon 5
intron 5
exon 6
intron 6
exon 7
intron 7
exon 8
intron 8
exon 9
intron 9
exon 10
intron 10
exon 11
intron 11
exon 12
intron 12
exon 13
intron 13
exon 14
intron 14
exon 15
intron 15
exon 16
intron 16
exon 17
intron 17
exon 18
intron 18
exon 19
intron 19
exon 20
intron 20
exon 21
intron 21
exon 22
intron 22
exon 23
intron 23
exon 24
intron 24
exon 25
PART 2, CHAPTER 3
nitrate reductase 1,
[NADH] (NR1, NIA)
xanthine dehydrogenase
(XDH)
phosphoribulokinase
(PRK)
phytochrome C
(PHYC)
Chromosome: 8 (NC_008401)
Gene: NIA-a (Os08g0468100)
mRNA: NM_001068541
Total range: 23,121,355-23,126,236
Total length: 4,882
Processed length: 2,751
Protein product length: 916
Copy number: 3
Duplicate genes: NIA-b (Os02g0770800)
NIA-c (Os08g0468700)
Chromosome: 3 (NC_008396)
Gene: XDH (Os03g0429800)
mRNA: NM_001056955
Total range: 18,621,710-18,634,104
Total length: 12,395
Processed length: 4,110
Protein product length: 1,369
Copy number: 1
Chromosome: 2 (NC_008395)
Gene: PRK-a (Os02g0698000)
mRNA: NM_001054360
Total range: 29,588,876-29,590,869
Total length: 1,994
Processed length: 1,212
Protein product length: 403
Copy number: 2
Duplicate genes: PRK-b (Os04g0595700)
complement: Os02g0697900 (partial)
Chromosome: 3 (NC_008396)
Gene: PHYC (Os03g0752100)
mRNA: NM_001057831
Total range: 31,768,179-31,772,647
Total length: 4,469
Processed length: 3,414
Protein product length: 1,137
Copy number: 1
Similar genes: PHYA (Os03g0719800)
PHYB (Os03g0309200)
1-1039
1040-1147
1148-1288
1289-3226
3227-3459
3460-3544
3545-4882
1-142
143-1269
1270-1369
1370-1846
1847-1955
1956-2595
2596-4098
4099-4938
4939-5181
5182-5291
5292-5606
5607-6859
6860-7099
8000-7435
7436-7720
7721-7795
7796-8017
8018-8565
8566-8727
8728-8871
8872-9006
9007-10912
10913-11149
11150-11247
11248-11421
11422-12152
12153-12395
1-545
546-838
839-923
924-1012
1013-1097
1098-1267
1268-1512
1513-1742
1743-1994
1-2065
2066-2169
2170-2986
2987-3379
3380-3673
3674-4231
4232-4469
1039
108
141
1938
233
84
1338
142
1127
100
477
109
640
1503
840
243
110
315
1253
240
336
285
75
222
548
162
144
135
1906
237
98
174
731
243
545
293
85
89
85
170
245
230
252
2065
104
817
393
294
558
238
81
BARFUSS, M.H.J.
NUCLEAR DNA MARKERS
Figure 1. Genetic structure of each nuclear DNA region based on GenBank information from the assembled RefSeq
genome of Oryza sativa. Approximate positions of primers finally used are indicated by arrow heads. Annotation of
malate synthase and RNA polymerase II, beta subunit was incomplete or wrong and therefore done manually aided
with reference sequences of Arabidopsis thaliana (RPB2) or Zea mays (MS).
82
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
83
BARFUSS, M.H.J.
84
Table 2. Amplified markers, number of taxa studied, and PCR conditions of each marker.
marker
no. of taxa studied
PCR conditions
ITS nrDNA
114
1× 97°C for 2 min; 15× 99°C for 20 s, 64°C for 15 s, 72°C for 2 min; 20× 99°C for 15 s, 64°C for 10 s, 72°C for 2 min + 5 s/cycle; 1× 72°C for 7 min; 1× 4°C for ∞.
MS
13
1× 95°C for 2 min; 5× 95°C for 30 s, 60°C for 30 s, 72°C for 2 min; 30× 95°C for 30 s, 54°C for 30 s, 72°C for 2 min; 1× 72°C for 7 min; 1× 4°C for ∞.
RPB2
8
1× 95°C for 2 min; 40× 95°C for 30 s, 55°C for 30 s, 72°C for 1 min; 1× 72°C for 7 min; 1× 4°C for ∞.
PGIC
2
1× 95°C for 2 min; 35× 95°C for 30 s, 50°C for 30 s, 70°C for 2 min; 1× 70°C for 7 min; 1× 4°C for ∞.
NIA
71
1× 95°C for 2 min; 10× 95°C for 30 s, 65°C - 1°C/cycle for 30 s, 70°C for 2 min; 30× 95°C for 30 s, 58°C for 30 s, 70°C for 2 min; 1× 70°C for 7 min; 1× 4°C for ∞.
XDH
65
1× 95°C for 2 min; 10× 95°C for 30 s, 65°C - 1°C/cycle for 30 s, 70°C for 2 min; 30× 95°C for 30 s, 58°C for 30 s, 70°C for 2 min; 1× 70°C for 7 min; 1× 4°C for ∞.
PRK
444
1× 95°C for 2 min; 15× 95°C for 30 s, 62°C for 30 s, 70°C for 1 min 30 s; 20× 95°C for 30 s, 62°C for 30 s, 70°C for 1 min 30 s + 5 s/cycle; 1× 70°C for 7 min; 1× 4°C for ∞.
PHYC
444
1× 95°C for 2 min; 20× 95°C for 30 s, 59°C for 30 s, 70°C for 1 min 30 s; 20× 95°C for 30 s, 59°C for 30 s, 70°C for 1 min 30 s + 5 s/cycle; 1× 70°C for 7 min; 1× 4°C for ∞.
NUCLEAR DNA MARKERS
name
sequence (5'–3')
position
length
direction
use/success
specificity
reference
primers initially aivailable
17SE (AB101)
ACGAATTCATGGTCCGGTGAAGTGTTCG
18S nrDNA, 3' end
28 bp
forward
PCR unsuccessful
angiosperms
Sun & al., 1994
ITS-A
GGAAGGAGAAGTCGTAACAAGG
18S nrDNA, 3' end
22 bp
forward
primer design
angiosperms
Blattner, 1999
ITS5
GGAAGTAAAAGTCGTAACAAGG
18S nrDNA, 3' end
22 bp
forward
PCR unsuccessful
universal
White & al., 1990
ITS1
TCCGTAGGTGAACCTGCGG
18S nrDNA, 3' end
19 bp
forward
not used
universal
White & al., 1990
ITS-D
CTCTCGGCAACGGATATCTCG
5.8S nrDNA, 5' end
21 bp
forward
primer design
angiosperms
Blattner, 1999
ITS3
GCATCGATGAAGAACGCAGC
5.8S nrDNA
20 bp
forward
not used
universal
White & al., 1990
ITS2
GCTGCGTTCTTCATCGATGC
5.8S nrDNA
20 bp
reverse
PCR unsuccessful
universal
White & al., 1990
ITS-C
GCAATTCACACCAAGTATCGC
5.8S nrDNA
21 bp
reverse
PCR unsuccessful
angiosperms
Blattner, 1999
ITS-B
CTTTTCCTCCGCTTATTGATATG
26S nrDNA, 5' end
23 bp
reverse
not used
angiosperms
Blattner, 1999
ITS4
TCCTCCGCTTATTGATATGC
26S nrDNA, 5' end
20 bp
reverse
PCR unsuccessful
universal
White & al., 1990
26SE (AB102)
TAGAATTCCCCGGTTCGCTCGCCGTTAC
26S nrDNA, 5' end
28 bp
reverse
PCR unsuccessful
angiosperms
Sun & al., 1994
IN TILLANDSIOIDEAE (BROMELIACEAE)
Table 3. Primers tested for the internal transcribed spacer of nuclear ribosomal DNA (ITS nrDNA). Highlighted primers performed best and were selected for PCR or sequencing.
bp = base pairs.
primers subsequently tested
ITS18Sf
ACCGATTGAATGGTCCGGTGAAGTGTTCG
18S nrDNA, 3' end
29 bp
forward
PCR
angiosperms
Gruenstaeudl & al., 2009
ITS18Scsf
GAATGGTCCGGTGAAGTGTTCG
18S nrDNA, 3' end
22 bp
forward
sequencing
angiosperms
this study
ITS5c
AGAGGAAGGAGAAGTCGTAACAAGGT
18S nrDNA, 3' end
26 bp
forward
sequencing
angiosperms
this study; modified from “ITS5” & “ITS-A”
ITS5.8Sf
ACTCTCGGCAACGGATATCTCGGCTC
5.8S nrDNA, 5' end
26 bp
forward
PCR
angiosperms
Gruenstaeudl & al., 2009
ITS5.8Scsf
GACTCTCGGCAACGGATATCTCG
5.8S nrDNA, 5' end
23 bp
forward
sequencing
angiosperms
this study; modified from “ITS-D”
ITS-E
CGGCAACGGATATCTCGGCTC
5.8S nrDNA, 5' end
21 bp
forward
sequencing
angiosperms
Blattner 1999
ITS5.8Scsr
GATGCGTGACGCCCAGGCAG
5.8S nrDNA, 3' end
20 bp
reverse
sequencing
angiosperms
this study
ITS5.8Sr
ATGCGTGACGCCCAGGCAGACGTG
5.8S nrDNA, 3' end
24 bp
reverse
PCR
angiosperms
Gruenstaeudl & al., 2009
ATGCGTGACGCCCAGGCAGRCGTG
5.8S nrDNA, 3' end
24 bp
reverse
PCR
angiosperms
this study; modified from “ITS5.8Sr”
TGACGCCCAGGCAGRCGTGC
5.8S nrDNA, 3' end
20 bp
reverse
sequencing
angiosperms
this study
26SE-2
CGGTTCGCTCGCCGTTACTA
26S nrDNA, 5' end
20 bp
reverse
sequencing
angiosperms
this study
ITS26Scsr
GGACGCTTCTCCAGACTACAATTCG
26S nrDNA, 5' end
25 bp
reverse
sequencing
angiosperms
this study
ITS26Sr
CTGAGGACGCTTCTCCAGACTACAATTCG
26S nrDNA, 5' end
29 bp
reverse
PCR
angiosperms
Gruenstaeudl & al., 2009
85
PART 2, CHAPTER 3
ITS5.8Sr-an
ITS5.8Scsr-2
BARFUSS, M.H.J.
86
Table 4. Primers tested for malate synthase (MS). Only highlighted primers worked and were selected for PCR and sequencing. Position of primers was determined from the
assembled RefSeq genome of Oryza sativa. bp = base pairs. CDS = coding sequence. Chr = chromosome.
name
sequence (5'–3')
CDS position (bp)
Chr position (bp)
length
direction
use/success
specificity
reference
primers initially tested
ms400f
GGAAGATGRTCATCAAYGCNCTYAAYTC
exon 1, 329–356
24,726,367–24,726,394
28 bp
forward
unsuccessful
angiosperms
Lewis & Doyle 2001
ms356f
GGAAGATGRTMATCAAYGCRCTKAAYTC
exon 1, 329–356
24,726,367–24,726,394
28 bp
forward
unsuccessful
angiosperms
D. Springate, RBG Kew, unpublished;
modified from “ms400f”
ms526f
GGACTATAAGCTTCCATGACCTC
exon 2, 455–477
24,726,162–24,726,184
23 bp
forward
unsuccessful
palms,
some other monocots
Lewis & Doyle 2001
ms943r
GTCTTNACRTAGCTGAADATRTARTCCC
exon 3, 872–899
24,725,504–24,725,477
28 bp
reverse
unsuccessful
angiosperms
Lewis & Doyle 2001
ms1408r
CCARTTCTGVACBCKGCTGATCTCCGC
exon 4, 1,408–1,434
24,724,853–24,724,879
27 bp
reverse
unsuccessful
angiosperms
D. Springate, RBG Kew, unpublished
ms1488r
TTCRTAYYTNAKCCAYTGCCAGTTYTG
exon 4, 1,426–1,452
24,724,835–24,724,861
27 bp
reverse
unsuccessful
angiosperms
Lewis & Doyle 2001
primers subsequently tested
GGAAGATGRTCATCAAYGCNCTBAAYTC
exon 1, 329–356
24,726,367–24,726,394
28 bp
forward
unsuccessful
monocots
this study;
modified from “ms400f”
ms428f (MSe2f)
GCTGGGARAACCTGATGARVGGCCA
exon 2, 404–428
24,726,211–24,726,235
25 bp
forward
PCR/sequencing
monocots
this study
ms553r (MSe2r)
CCGTCGAYGAGRATRTGGGCCTC
exon 2, 553–575
24,726,064–24,726,086
23 bp
reverse
unsuccessful
monocots
this study
ms837f (MSe3f)
CARATGRAYGAGATVCTNTACGAGCTG
exon 3, 811–837
24,725,539–24,725,565
27 bp
forward
unsuccessful
monocots
this study
ms960r (MSe3r)
GGARRTCGGAGTAGCTSCKCATGAAG
exon 3, 960–985
24,725,391–24,725,416
26 bp
reverse
PCR/sequencing
monocots
this study
ms1411r (MSe4r)
TGCCAGTTCTGVACBCKGCTGATCTC
exon 4, 1,411–1,436
24,724,851–24,724,876
26 bp
reverse
unsuccessful
monocots
this study;
modified from “ms1488r”
NUCLEAR DNA MARKERS
ms356f-2
name
sequence (5'–3')
CDS position (bp)
Chr position (bp)
length
direction
use/success
specificity
reference
P6F
TGGGGAATGATGTGTCCTGC
exon 11, 1,513–1,532
25,798,152–25,798,171
20 bp
forward
unsuccessful
angiosperms
Denton & al., 1998
P7F
CCYCGTAATACWTAYCARTCWGC
exon 17, 2,164–2,186
25,799,631–25,799,653
23 bp
forward
unsuccessful
angiosperms
Denton & al., 1998
P7R
CCCATGGCTTGCTTCCCCAT
exon 17 2,188–2,207
25,799,655–25,799,674
20 bp
reverse
unsuccessful
angiosperms
Denton & al., 1998
P10F
CARGARGATATGCCATGGAC
exon 23, 2,851–2,870
25,800,820–25,800,839
20 bp
forward
PCR/sequencing
angiosperms
Denton & al., 1998
P10R
CCCATRATACACTCAATGAGYTG
exon 23, 2,938–2,960
25,800,907–25,800,929
23 bp
reverse
unsuccessful
angiosperms
Denton & al., 1998
P11aR
GTGAATCTTGTCATCMACCATATGC
exon 24, 3,153–3,177
25,801,305–25,801,329
25 bp
reverse
PCR/sequencing
angiosperms
Denton & al., 1998
IN TILLANDSIOIDEAE (BROMELIACEAE)
Table 5. Primers tested for RNA polymerase II, beta subunit (RPB2). Only highlighted primers worked and were selected for PCR and sequencing. Position of primers was determined from the assembled RefSeq genome of Oryza sativa. bp = base pairs. CDS = coding sequence. Chr = chromosome.
Table 6. Primers tested for glucose-6-phosphate isomerase, cytosolic (PGIC). Only highlighted primers worked and were used for PCR and sequencing. Position of primers was
determined from the assembled RefSeq genome of Oryza sativa. bp = base pairs. CDS = coding sequence. Chr = chromosome.
name
sequence (5'–3')
CDS position (bp)
Chr position (bp)
length
direction
use/success
specificity
reference
AA11F (93.4)
TTYGCNTTYTGGGAYTGGGT
exon 11, 793–812
8,154,188–8,154,207
20 bp
forward
PCR/sequencing
universal
Ford & al., 2006 (Terauchi & al., 1997)
AA16F
ATGGARAGYAAYGGNAARGG
exon 16, 1,075–1,094
8,155,727–8,155,746
20 bp
forward
unsuccessful
angiosperms
Ford & al., 2006
AA16R
CCYTTNCCRTTRCTYTCCAT
exon 16, 1,075–1,094
8,155,727–8,155,746
20 bp
reverse
PCR/sequencing
angiosperms
Ford & al., 2006
CCCCAYTGRTCRAAIGARTTDATICCCCA
exon 21, 1,504–1,532
8,157,623–8,157,651
29 bp
reverse
unsuccessful
angiosperms
Ford & al., 2006
CCCCAYTGRTCRAANGARTTDATNCCCCA
exon 21, 1,504–1,532
8,157,623–8,157,651
29 bp
reverse
unsuccessful
angiosperms
this study; modified from “AA21RM”
93.9H
TCIACICCCCAITGRTCTAAIGARTTIAT
exon 21, 1,510–1,538
8,157,629–8,157,657
29 bp
reverse
unsuccessful
universal
Terauchi & al., 1997
yamv
TCIACICCCCAITGRTCAAAIGARTTIAT
exon 21, 1,510–1,538
8,157,629–8,157,657
29 bp
reverse
unsuccessful
angiosperms
Ford & al., 2006; modified from “93.9H”
pgic1510r
TCNACNCCCCANTGRTCAAANGARTTNAT
exon 21, 1,510–1,538
8,157,629–8,157,657
29 bp
reverse
unsuccessful
angiosperms
this study; modified from “yamv”
87
PART 2, CHAPTER 3
AA21RM
pgic1504r
BARFUSS, M.H.J.
88
Table 7. Primers tested for nitrate reductase 1, [NADH] (NIA). Highlighted primers performed best and were selected for PCR and/or sequencing. Position of primers was determined from the assembled RefSeq genome of Oryza sativa. bp = base pairs. CDS = coding sequence. Chr = chromosome.
name
sequence (5'–3')
CDS position (bp)
Chr position (bp)
length
direction
use/success
specificity
reference
primers initially tested
NIA2F
TCBGTGATTACGACGCCGTGTCATGA
exon 2, 1,087–1,112
23,122,549–23,122,574
26 bp
forward
unsuccessful
angiosperms
Howarth & Baum 2002
NIA2R
GACCARAARCACCARCACCARTAYT
exon 3, 1,283–1,307
23,124,683–23,124,707
25 bp
reverse
unsuccessful
angiosperms
Howarth & Baum 2002
NIA3F
AARTAYTGGTGYTGGTGYTTYTGGTC
exon 3, 1,282–1,307
23,124,682–23,124,707
26 bp
forward
PCR/sequencing
angiosperms
Howarth & Baum 2002
NIA3R
GAACCARCARTTGTTCATCATDCC
exon 4, 1,414–1,437
23,124,899–23,124,922
24 bp
reverse
PCR/sequencing
angiosperms
Howarth & Baum 2002
primers subsequently tested
nia410f (nia1f)
CTNATGCACCACGGNTTCATCAC
exon 1, 388–410
23,121,742–23,121,764
23 bp
forward
PCR/sequencing
angiosperms
this study
nia413f (nia1f-2)
CTNATGCACCACGGNTTYATCACCCC
exon 1, 388–413
23,121,742–23,121,767
26 bp
forward
PCR
angiosperms
this study
nia1042r (nia2r-3)
TGATKATRTAYTCYGGCTTRTACCACCA
exon 2, 1,042–1,069
23,122,504–23,122,531
28 bp
reverse
PCR/sequencing
angiosperms
this study
nia1073f (nia2f-2)
GGTACAAGCCRGARTAYATMATYAACGA
exon 2, 1,046–1,073
23,122,508–23,122,535
28 bp
forward
unsuccessful
angiosperms
this study
nia1108r (nia2r-2)
GTTGATGGGYARRATCTCSTCGTG
exon 2, 1,108–1,131
23,122,570–23,122,593
24 bp
reverse
unsuccessful
angiosperms
this study
nia1390r 1 (nia3r-2)
ACCATGACGTTCCAGATGAGCTTCTC
exon 3, 1,390–1,413 (-1,415)
23,124,790–23,124,815
26 bp
reverse
unsuccessful
angiosperms
this study
1
italicized numbers indicate base positions that partly lie within introns (GT motive of intron start).
NUCLEAR DNA MARKERS
note:
name
sequence (5'–3')
CDS position (bp)
Chr position (bp)
length
direction
use/success
specificity
reference
xdh422f (xdh4f-1)
CCYGGTTTYRTBATGTCVATGTATGC
exon 4, 397–422
18,624,350–18,624,375
26 bp
forward
PCR
monocots
this study
xdh437f (xdh4f-2)
GTCVATGTATGCVTTRYTRMGRTCAAG
exon 4, 411–437
18,624,364–18,624,390
27 bp
forward
PCR
monocots
this study
xdh479f (xdh4f-3)
TGARGARCARATYGAAGAWWGCCTTGC
exon 4, 453–479
18,624,406–18,624,432
27 bp
forward
PCR/sequencing
monocots
this study
xdh974f (xdh4intf-2)
GTTACCCATGTGGCGGAGCTTAATGC
exon 4, 949–974
18,624,902–18,624,927
26 bp
forward
sequencing
bromeliads
this study
xdh1033f-br (xdh4intf-1)
GTGCTTCTGTGAGACTGACACAGCTCC
exon 4, 1,007–1,033
18,624,960–18,624,986
27 bp
forward
sequencing
bromeliads
this study
xdh1165r-br (xdh4intr-1)
TAGGTCAGATATTGGACTAGCAGTACA
exon 4, 1,165–1,191
18,625,118–18,625,144
27 bp
reverse
sequencing
bromeliads
this study
xdh1169r-br (xdh4intr-2)
GGGTTTAGGTCAGATATTGGACTAGCAG
exon 4, 1,169–1,196
18,625,122–18,625,149
28 bp
reverse
sequencing
bromeliads
this study
xdh1611r (xdh4r-1)
CGRAAYTCHACCATYCCMCCWGGWGCA
exon 4, 1,611–1,637
18,625,564–18,625,590
27 bp
reverse
PCR/sequencing
monocots
this study
xdh1612r (xdh4r-2)
CGRAAYTCHACCATYCCMCCWGGWGC
exon 4, 1,612–1,637
18,625,565–18,625,590
26 bp
reverse
PCR
monocots
this study
xdh1880f (xdh5f-1)
GGTYACWGGDGARGCDGAATATRCTGA
exon 5, 1,854–1,880
18,626,647–18,626,673
27 bp
forward
unsuccessful
monocots
this study
xdh1883f (xdh5f-2)
ACWGGDGARGCDGAATATRCTGAYGA
exon 5, 1,858–1,883
18,626,651–18,626,676
26 bp
forward
unsuccessful
monocots
this study
ACYTGDGTRGATGAWATCATRTGAAYTTC
exon 6, 2,386–2,412 (–2,414)
18,627,289–18,627,317
29 bp
reverse
unsuccessful
monocots
this study
ACYTGDGTRGATGAWATCATRTGAA
exon 6, 2,390–2,412 (–2,414)
18,627,293–18,627,317
25 bp
reverse
unsuccessful
monocots
this study
xdh2386r1 (xdh6r-1)
1
xdh2390r (xdh6r-2)
note:
1
IN TILLANDSIOIDEAE (BROMELIACEAE)
Table 8. Primers tested for xanthine dehydrogenase (XDH). Highlighted primers performed best and were selected for PCR and/or sequencing. Position of primers was determined from the assembled RefSeq genome of Oryza sativa. bp = base pairs. CDS = coding sequence. Chr = chromosome.
italicized numbers indicate base positions that partly lie within introns (GT motive of intron start).
PART 2, CHAPTER 3
89
name
sequence (5'–3')
CDS position (bp)
Chr position (bp)
length
direction
use/success
specificity
AAYGAYTTTGAYCTYATGTATGARCARGT
exon 1, 394–422
29,589,269–29,589,297
29 bp
forward
unsuccessful
angiosperms
Lewis & Doyle 2002
prk596f (prk663f)
GAYTTCAGYATYTAYTTRGACAT
exon 2, 574–596
29,589,742–29,589,764
23 bp
forward
PCR/sequencing
angiosperms
D. Springate, RBG Kew, unpublished
prK973r (prk1040r)
TCTRTCAAATTGYCCRTCCATYTC
exon 5, 973–996
29,590,630–29,590,653
24 bp
reverse
PCR/sequencing
angiosperms
D. Springate, RBG Kew, unpublished
prk1167r
ATGGTYTGRAANARACCNGTNCCRTTGTTGC
exon 5, 1,100–1,130
29,590,757–29,590,787
31 bp
reverse
PCR/sequencing
angiosperms
Lewis & Doyle 2002
26 bp
forward
PCR
bromeliads
this study;
modified from “prk622f“
BARFUSS, M.H.J.
90
Table 9. Primers tested for phosphoribulokinase (PRK). Highlighted primers performed best and were selected for PCR or sequencing. Position of primers was determined from
the assembled RefSeq genome of Oryza sativa. bp = base pairs. CDS = coding sequence. Chr = chromosome.
reference
primers initially tested
prk488f 1
primers subsequently tested
TCAGCAATGAGGTTAAATTTGCATGG
exon 2, 596–621
29,589,764–29,589,789
prk622f
CAGCAATGAGGTTAAATTTGCATGGA
exon 2, 597–622
29,589,765–29,589,790
26 bp
forward
PCR/sequencing
bromeliads
Schulte, Barfuss & Zizka 2009
prk630f2
AAATTTGCATGGAAAATTCAGGTC
exon 2, 610-630 (–633)
29,589,778–29,589,801
24 bp
forward
sequencing
bromeliads
this study
CTGCAGATCCGCAGAAGAAATATGC
exon 4, (710–) 716–734
29,590,137–29,590,161
25 bp
forward
sequencing
bromeliads
Schulte, Barfuss & Zizka 2009
CCGCAGATCCGCAGAAGAAATTTTC
exon 4, (710–) 716–734
29,590,137–29,590,161
25 bp
forward
sequencing
Brocchinia
this study;
modified from “prk734f”
prk889r
GGGTATGAGCATGTCAATTTCCTCCC
exon 4, 889–914
29,590,316–29,590,341
26 bp
reverse
sequencing
bromeliads
Schulte, Barfuss & Zizka 2009
prk890r
GGGTATGAGCATGTCAATTTCCTCC
exon 4, 890–914
29,590,317–29,590,341
25 bp
reverse
sequencing
bromeliads
this study;
modified from “prk889r”
prk1057r
CTTCAGCATTTGTTGTGTCACCTC
exon 5, 1,057–1,080
29,590,714–29,590,737
24 bp
reverse
sequencing
bromeliads
this study
prk1069r
GAAAATCTGCRTGCTTCAGCATTTG
exon 5, 1,069–1,093
29,590,726–29,590,750
25 bp
reverse
PCR/sequencing
bromeliads
Schulte, Barfuss & Zizka 2009
prk1069r-2
GGAAAATCTGCRTGCTTCAGCATTTG
exon 5, 1,069–1,094
29,590,726–29,590,750
26 bp
reverse
PCR
bromeliads
this study;
modified from “prk1069r”
prk734f (prk735f)
prk734f-2
notes:
2,4
1
2,3
unlike given in Lewis & Doyle (2002) primer position of “prk488f” is in exon 1 but not in exon 3.
italicized numbers indicate base positions that partly lie within introns.
3
position of “prk735f” was initially wrongly determined (Schulte & al, 2009) and therefor corrected from “prk734f”.
4
for the genus Brocchinia primer “prk734f” had to be modified because of mismatches that give problems in sequencing.
2
NUCLEAR DNA MARKERS
prk621f
name
sequence (5'–3')
CDS position (bp)
Chr position (bp)
length
direction
use
specificity
reference
primers used initially for primer design
PHYupstream1
TCWGGNAARCCNTTYTAYGC
exon 1, 484–503
31,768,662–31,768,681
20 bp
forward
primer design
angiosperms
Mathews & Donoghue 1999
PHYupstream2
CCITTYTAYGSIATHYTICAYMG
exon 1, 493–515
31,768,671–31,768,693
23 bp
forward
primer design
angiosperms
Mathews & Donoghue 1999
PhyCdownstream
GRATKGCATCCATYTCMAYRTC
exon 1, 1,705–1,726
31,769,883–31,769,904
22 bp
reverse
primer design
angiosperms
Mathews & Donoghue 1999
primers subsequently tested
Russell & al., 2010b,
modified from “PHYupstream1”
this study,
modified from “PHYupstream2”
this study,
modified from “PHYupstream2”
phyc503f-mo
TCVGGGAAGCCSTTYTAYGC
exon 1, 484–503
31,768,662–31,768,681
20 bp
forward
PCR/sequencing
monocots
phyc515f-mo
AAGCCCTTYTACGCVATMATGCACCG
exon 1, 490–515
31,768,668–31,768,693
26 bp
forward
PCR/sequencing
monocots
phyc515f-br
AAGCCCTTYTACGCTATCCTGCACCG
exon 1, 490–515
31,768,668–31,768,693
26 bp
forward
PCR
bromeliads
phyc524f-br
GCTATCCTGCACCGGATCGAYGT
exon 1, 502–524
31,768,680–31,768,702
23 bp
forward
sequencing
bromeliads
this study
this study
this study
phyc974f-mo
GCTCCTCAYGGCTGYCAYGCTCA
exon 1, 952–974
31,769,130–31,769,152
23 bp
forward
sequencing
some monocots:
bromeliads, orchids
phyc974f-br
GCTCCTCACGGCTGCCACGCTCA
exon 1, 952–974
31,769,130–31,769,152
23 bp
forward
sequencing
bromeliads
some monocots:
bromeliads, orchids
some monocots:
bromeliads, orchids
some monocots:
bromeliads, orchids
some monocots:
bromeliads, orchids
IN TILLANDSIOIDEAE (BROMELIACEAE)
Table 10. Primers used/tested for phytochrome C (PHYC). Highlighted primers performed best and were selected for PCR or sequencing. Position of primers was determined
from the assembled RefSeq genome of Oryza sativa. bp = base pairs. CDS = coding sequence. Chr = chromosome.
this study
phyc991f-mo
CCAYGCTCARTAYATGGCTAATATGG
exon 1, 966–991
31,769,144–31,769,169
26 bp
forward
sequencing
phyc1145r-mo
CCTGMARCARGAACTCACAAGCATATC
exon 1, 1,145–1,171
31,769,323–31,769,349
27 bp
reverse
sequencing
phyc1145r2-mo
CAACAGGAACTCACAAGCATATC
exon 1, 1,145–1,167
31,769,323–31,769,345
23 bp
reverse
sequencing
phyc1210r-mo
GGATATGCTTCTCCTTTGYTTGAGC
exon 1, 1,210–1,234
31,769,388–31,769,412
25 bp
reverse
sequencing
phyc1690r-br
TCAACATCTTCCCAYGGGAGGCT
exon 1, 1,690–1,712
31,769,868–31,769,890
23 bp
reverse
sequencing
bromeliads
this study
phyc1699r-mo
ATWGCATCCATTTCAACATCYTCCCA
exon 1, 1,699–1,724
31,769,877–31,769,902
26 bp
reverse
PCR/sequencing
monocots
this study
phyc1699r-br
ATWGCATCCATTTCAACATCTTCCCA
exon 1, 1,699–1,724
31,769,877–31,769,902
26 bp
reverse
PCR
bromeliads
this study
phyc1705r-mo
GRATWGCATCCATYTCAACATC
exon 1, 1,705–1,726
31,769,883–31,769,904
22 bp
reverse
PCR/sequencing
monocots
Russell & al., 2010b,
modified from “PhyCdownstream”
Russell & al., 2010b
this study;
modified from “phyc1145r-mo”
this study
PART 2, CHAPTER 3
91
BARFUSS, M.H.J.
92
Table 11. Attributes of analyzed matrices and parsimony scores of equally most parsimonious trees after analysis using PAUP*. ITS nrDNA was analysed with (incl.) and without
(excl.) the problematic region of ITS1. Data of plastid DNA was taken from Barfuss & al., (2005). The sequence length is given for individual markers only. bp = base pairs, var.
char. = variable characters, PUIC = parsimony uninformative characters, PIC = parsimony informative characters, CI = consistency index, RI = retention index, RC = rescaled
consistency index, HI = homoplasy index.
marker
no. of taxa studied
amplicon lenght [bp]
no. of ch.
no. of constant ch.
no. of variable ch.
no. of PUIC.
no. of PICs
no. of trees
tree length
CI
RI
RC
HI
ITS nrDNA (incl.)
111
884–1,017
1,291
899 (69.6%)
392 (30.4%)
173 (13.4%)
219 (17.0%)
>10,000
936
0.590
0.748
0.441
0.410
ITS nrDNA (excl.)
111
884–1,017
1,057
788 (74.5%)
269 (25.5%)
120 (11.4%)
149 (14.1%)
>10,000
634
0.588
0.779
0.458
0.412
MS
13
659–707
710
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
RPB2
8
520–522
523
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
PGIC
2
983
983
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
NIA
72
649–800
1,022
734 (71.8%)
288 (28.2%)
164 (16.1%)
124 (12.1%)
>10,000
527
0.691
0.784
0.542
0.309
XDH
64
1,110
1,110
860 (77.5%)
250 (22.5%)
138 (12.4%)
112 (10.1%)
>10,000
369
0.743
0.740
0.549
0.257
PRK
444
831–1,692
3291
2,554 (77.6%)
737 (22.4 %)
203 (6.2%)
534 (16.2%)
>10,000
2,232
0.478
0.870
0.416
0.522
PHYC
444
1159–1,192
1,228
743 (60.5%)
485 (39.5%)
116 (9.4%)
369 (30.1%)
>10,000
1,398
0.473
0.867
0.410
0.527
plastid DNA
120
∑ of 7 markers
6,277
5,294 (84,3 %)
983 (15,7 %)
440 (7.0%)
543 (8.7%)
>10,000
1,713
0.654
0.830
0.543
0.346
NUCLEAR DNA MARKERS
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
Amplification, sequencing, and cloning of low-copy nuclear DNA markers
Gradient PCR conditions were optimized with either a Mastercycler gradient (Eppendorf) or a
Veriti 96-Well Thermal Cycler (Applied Biosystems, Life Technologies) using their gradient function or independent temperature blocks, respectively. Gradient PCR was usually done in two
successive steps, (1) running gradients for the annealing temperature, and (2) running gradients for the extension temperature.
The first step is necessary to find the best annealing temperature for a given primer pair,
which can differ greatly from calculated melting temperatures using different programs for
oligonucleotides (e.g., NetPrimer; http://www.premierbiosoft.com/netprimer/index.html).
General PCR conditions for the first step were: 1× 95°C for 2 min; 35× 95°C for 30 s, X°C for 30
s, 72°C for Y min; 1× 72°C for 7 min; and 4°C for ∞; where X stands for the annealing temperature variation and Y for the extension time used. The latter was dependent on the expected
fragment length and ranged between one and three minutes according to the general rule that
Taq amplifies at a rate of 1 kb per minute. Usually two rounds of annealing temperature gradients were performed, initially with a wide temperature variation of 48–68°C and later a narrower range determined based on the results of the first round.
The second step was done because the extension temperature depends not only on the
working optimum of Taq polymerase but also on the base composition (GC content) in certain
areas of the amplified region (Su & al., 1996). AT-rich sections might need a reduced extension
temperature, since these segments start to denature sometimes even below 72°C and Taq
polymerase is not able to function. The online program POLAND (Steger, 1994;
http://www.biophys.uni-duesseldorf.de/html/local/POLAND/poland.html) can be used to
check for 50% temperature probabilities of each base in a given sequence, which is a theoretical calculation using different formulas for how strongly bases are bound to each other. This
presents a problem with this approach because at least one sequence of the targeted region
for a given study group is required a priori to conduct this analysis. The general PCR conditions
for the second step were: 1× 95°C for 2 min; 35× 95°C for 30 s, X°C for 30 s, Z°C for Y min; 1×
Z°C for 7 min; and 4°C for ∞; where Z refers to a temperature variation of 60–72°C. Usually
one gradient run was sufficient to verify whether a denaturing temperature of 72°C is too high.
A third gradient step to determine the denaturing temperature was necessary only for ITS
nrDNA, because Taq is not able to pass through GC-rich regions, which are still bound at 72°C
because they were not denatured at 95°C completely. If necessary, further PCR optimization
steps were undertaken and/or specific PCR conditions established for each marker (Table 2).
Occasionally other parameters for markers such as two-step PCR, inclusion of a touchdown
cycle, or an increase of the extension time by 5 s at later cycles has been shown to produce
greater amounts of PCR product. Each gradient PCR was done in 0.2 mL 12-strip PCR tubes (or
96-well PCR plates) using 10 µL reactions including 9 µL 1.1× ReddyMix PCR Master Mix containing 2.5 mM MgCl2 (AB-0619; Thermofisher, ABgene), 0.4 µM of each primer (0.2 µL at 20
µM = 20 pmol/µL), 4% (0.4 µL) dimethysulfoxide (DMSO), and 0.2 µL (approximately 25 ng/µL)
template DNA.
Amplifications of each marker using optimized conditions were usually carried out one a
Mastercycler gradient in 25 µL reactions using 22.5 µL 1.1× ReddyMix PCR Master Mix containing 2.5 mM MgCl2, 0.5 µL (0.4 µM) of each primer at 20 µM, 1 µL (4%) DMSO, and 0.5 µL template DNA. Volumes were scaled up proportionally when more PCR product was necessary to
perform cycle sequencing with additional primers. When single bands were present on agarose
93
BARFUSS, M.H.J.
NUCLEAR DNA MARKERS
gels, PCR products were purified either with a 1:2 mixture of exonuclease I (20 units/µL; Fermentas) and alkaline phosphatase (either shrimp or FastAP thermosensitive alkaline phosphatase, 1 unit/µL; Fermentas) or ExoSAP-IT (Amersham, GE Healthcare) to degrade single stranded DNA fragments and dNTPs (Werle & al., 1994); 2.5 µL of the enzyme mixture (scaled up
proportionally for higher volumes) was added to each 25 µL PCR reaction and incubated at
37°C for 45 min, followed by deactivating the enzymes at 85°C for 15 min.
Cycle sequencing reactions were usually performed on a 96-Well GeneAmp PCR System
9700 (Applied Biosystems, Life Technologies) using a modified reaction protocol: 0.5 µL BigDye
Terminator v3.1 (Applied Biosystems, Life Technologies), 1 µL (0.4 µM) primer at 4 µM, 1.75 µL
5× sequencing buffer, 3–6.75 µL purified PCR product, and 0–3.75 µL PCR-grade water. The
general temperature profile (except for ITS nrDNA) was: 1× 96°C for 1 min; 35× 96°C for 10 s,
50°C for 5 s; 60°C for 3 min; 4°C for ∞. Modifications to the original profile are increased number of cycles (orig. 25 cycles) and reduced extension time (orig. 4 min). Cycle sequencing products were purified by gel filtration using MultiScreen filter plates (MAHVN4550; Milipore) and
the cross-linked dextran gel Sephadex G-50 Superfine or Fine (GE Healthcare) according to the
Millipore protocol (Tech Note TN053) with few modifications (more details provided upon
request from the first author). Sequences were run on either a 16-capillary sequencer (3130xl
Genetic Analyzer, Applied Biosystems, Life Technologies) or a 48-capillary sequencer (3730
DNA Analyzer; Applied Biosystems, Life Technologies) following manufacturer’s instructions.
When cloning was required, the proof-reading Phusion High-Fidelity DNA polymerase (F530S; Finnzymes) was used to reduce the accumulation of polymerase errors in the cloned
sequences and the formation of artificial chimeric PCR products. PCR reactions were 50 µL,
including 34.7 µL purified water, 10 µL 5× Phusion High Fidelity PCR buffer, 0.8 µL MgCl2 at a
concentration of 50 mM, 1 µL dNTPs at 10 mM each, 0.5 µL Phusion DNA polymerase (2U/µL),
1 µL of each primer at 20 mM, and 1 µL template DNA. Thermocycling was performed according to manufacturer’s instructions with slight modifications to fit the optimize PCR conditions
for each marker. Amplified DNA was verified and purified on 1% TAE agarose gels using the
Invisorb Spin DNA Extraction Kit (Invitek) following the manufacturer’s instructions. Since the
proof reading DNA polymerase is not able to create 3' A-overhangs, which is necessary to proceed with standard TA cloning, purified PCR products were incubated at 72°C for 20 min using
a mixture of 0.5 µL dATPs at 10 mM, 2.5 µL 10× Taq PCR buffer containing (NH4)2SO4, 0.1 µL
Taq polymerase (5U/µL), and 1.5 µL MgCl2 at 25 mM per 25 µL purified PCR product (all products from Fermentas). Cloning was performed using the pGEM-T Easy Vector System
(Promega) following manufacturer’s instructions. Successfully transformed colonies were suspended in 1× TE buffer, denatured for 10 min at 95°C, placed on ice, centrifuged to collect cell
debris, and used as a template for PCR. Subsequent amplification and sequencing were performed as described above.
Amplification and sequencing of ITS nrDNA
PCR of the ITS nrDNA (Figure 1, A) was initially attempted using the universal primers “ITS2”,
“ITS3”, “ITS4” and “ITS5” (White & al., 1990; Baldwin & al., 1995) and the angiosperm-specific
primers “17SE” and “26SE” (Sun & al., 1994; Douzery & al., 1999). No useful PCR products were
obtained at the first step of gradient PCR using standard reactions. Therefore the new angiosperm-specific PCR primers “ITS18Sf”, “ITS5.8Sf”, “ITS5.8Sr”, and “ITS26Sr” with a significantly
higher annealing temperature were employed (Gruenstaeudl & al., 2009). These primers have
been developed using large-unit ribosomal sequences of angiosperms from GenBank plus a
94
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
sequences of Bromeliaceae. The new internal 5.8S nrDNA primers, “ITS5.8Sf” and “ITS5.8Sr”,
were used because “ITS3” and “ITS2” of White & al. (1990) do not produce overlapping reads
and have a low annealing temperature. For further PCR trials, standard reactions were altered
by adding DMSO in final concentrations of up to 10% and/or betaine up to 1 M; these additives
are thought to aid in amplification by reducing GC binding during extension. Betaine had no
obvious effect, but DMSO at a final concentration of 3% produced a PCR product of the expected length in at least some samples. Therefore, in a final optimization step a gradient run
for the denaturing temperature was performed using a temperature range of 94–99°C. Already
at 96°C more samples showed the expected PCR product and at a denaturing temperature of
99°C consistently all samples had comparatively strong bands. Additionally an extension time
twice as high as the general rule (1 kb/m) increased the amount significantly. The optimized
PCR conditions are given in Table 2.
For the initial phase of cycle sequencing for ITS nrDNA, all four newly generated primers
and the standard reaction protocol and PCR conditions were used. Two problems in these sequences were detected. Firstly electropherograms had n-1 patterns because of high primer
melting temperatures (around 70 °C), which was solved by using the primers from Blattner
(1999) and/or several newly designed sequencing primers that have annealing temperatures
around 65°C. The second problem was that only internal primers sequencing towards the 26S
nrDNA gave complete reads. In all other cases signal intensity dropped and was mostly completely lost. In such cases, the dGTP BigDye Terminator v3.0 Cycle Sequencing Kit (Applied Biosystems, Life Technologies) procedure has been modified. To be able to run this chemistry on
the capillary sequencers it was mixed in a ratio of 1:4 (dGTP v3.0:v3.1) with the BigDye Terminator v3.1 to avoid strong banding compression patterns that are visible in capillary electrophoresis by using the dGTP BigDye Terminator v3.0 alone. Also the effect of DMSO and betaine
was tested. The general sequencing protocol was modified by using 1 µL per reaction of the
BigDye Terminator mix, 1 µL (0.4 µM) primer at 4 µM, 1 µL 5× sequencing buffer, 1.6 µL (0.8
M) betaine at 5 M, 0.4 µL (4%) DMSO, 4 µL purified PCR product, and 1 µL PCR-grade water.
Cycling conditions were changed to: 1× 96°C for 1 min, 99°C for 30 s, and 60°C for 3 min; 35×
96°C for 20 s, 60°C for 3 min; and 4°C for ∞. No annealing step was necessary since all primers
used for sequencing have annealing temperatures higher than 60°C. Details of all PCR and sequencing primers tested and finally used for ITS nrDNA are given in Table 3.
Analyses of nuclear DNA sequences
Raw sequences were initially analyzed and edited using the Sequencing Analysis Software v5.3
(Applied Biosystems, Life Technologies). Forward and reverse DNA strands from both PCR
and/or internal sequencing primers were assembled with the SeqMan Pro module of the Lasergene v8.1 software package (DNASTAR). Contigs were edited and allelic consensus sequences were exported as text file in fasta format. Fasta files were pooled and aligned with
MUSCLE v3.8 (Edgar, 2004a, b) leaving default parameters unchanged and then adjusted by
eye with BioEdit v7.0.5 (Hall, 1999) following the guidelines of Kelchner (2000) and Borsch &
Quandt (2009). In polymorphic DNA regions containing numerous insertion/deletion (indel)
events related taxa were aligned first. Later these blocks of aligned taxa were aligned using the
profile alignment function of MUSCLE and adjusted by eye using BioEdit.
95
BARFUSS, M.H.J.
NUCLEAR DNA MARKERS
Figure 2. Secondary structure (hairpins) for eight selected taxa of Tillandsioideae of the same problematic region
of the ITS1 obtained from RNA folding on the mfold web sever.
96
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
97
Figure 3. Alignment of a section of the ITS1 region showing the problematic region that has an extremely stable secondary structure, which gives problems in PCR and sequencing.
BARFUSS, M.H.J.
NUCLEAR DNA MARKERS
The Alignment of ITS nrDNA was guided by their secondary structures (Figures 2, 3). Stem-loop
regions (hairpins) were identified either visually or on the mfold web server (Zuker, 2003). A
certain GC rich region within ITS1 could not be unambiguously aligned (Figure 3). Aligned ITS
nrDNA sequences were checked for the presence of the conserved angiosperm motifs -GGCRY[4–7 N]-GYGYCAAGGAA- in ITS1 (Liu & Schardl, 1994), -GAATTGCAGAATCC- in the 5.8S nrDNA
region (Jobes & Thien, 1997), and the conserved (C1–C6) and variable (V1–V6) domains in ITS2
(Hershkovitz & Zimmer, 1996) to exclude pseudogenes. Alignments of low-copy nuclear DNA
markers were adjusted by checking the reading frame, by determining the angiosperm-specific
intron-flanking GT-AG motive in intron regions, and by using annotated genes of the assembled RefSeq genome of Oryza sativa and other annotated sequences from GenBank. Aligned
matrices can be obtained from the first author upon request.
Since PCR products can contain two or more alleles and were mostly sequenced directly in
this study, heterozygous individuals contain two types of polymorphisms: (1) single nucleotide
polymorphisms (SNPs) that are visible as double peaks at certain positions, and (2) length polymorphisms caused by insertion/deletion (indels) events, which result in polymorphisms at
every subsequent position in the electropherogram. Unambiguous SNPs were coded using the
IUPAC-IUB symbols for nucleotide nomenclature. Indels made assembly of contigs difficult. To
be able to assemble such contigs, the type of indel was identified by visual inspection and usually the shorter allele was ignored and manually overwritten by the sequence of the longer.
Assembled contigs were edited with special care to correct for handling mistakes and the initially ignored sequence of the shorter allele was checked not to miss any mutations. If two or
more indels between priming sites were observed, editing was highly problematic and errorprone; in such cases, these individuals were excluded or the PCR products cloned and sequenced.
Nuclear DNA alignments were inspected prior to phylogenetic analyses for the impact of
SNPs and indels within allelic consensus sequences of a given individual (Jabaily & Sytsma,
2010). The presence of similar alleles in more distantly related taxa may indicate possible hybridization events including reticulations and introgressions, polyploidization, and occurrence
of incomplete lineage sorting. When no significant conflict was detected (homoplasious SNPs
within accessions of distantly related taxa were ignored), sequences were included and differences were assumed to be most likely due to allelic variation within a single species or within a
species complex. Otherwise individuals were excluded or sequenced after cloning. Sequences
resulting from PCR-mediated, artificial recombinations of alleles were sometimes observed
and excluded.
PAUP* version 4.0b10 (Swofford, 2003) has been used for all analyses using maximumparsimony with unordered and equally weighted characters. A two-step heuristic search
method was used: (1) 1,000 random sequence additions, TBR branch swapping holding 10
trees each step, and saving 10 shortest trees per replicate; (2) trees obtained from the first
analyses were taken as starting trees and heuristic searches were conducted until all or a maximum of 10,000 shortest trees were saved, but the tree search was allowed to continue to
check all input trees for possible shorter ones (swapping to completion). Gaps were treated as
missing characters, and no gap coding procedure was applied because of the many indels within accessions that were ignored within allelic consensus sequences. The matrices including all
characters were analyzed for five nuclear DNA markers (ITS nrDNA, NIA, XDH, PRK, PHYC) and
for ITS nrDNA alone in a matrix excluding the difficult region of ITS1 (Figures 2,3), since the
alignment was ambiguous for that region and many gaps had to be introduced. For compari98
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
son, the plastid matrix of Barfuss et al. (2005) was reanalyzed with the same phylogenetic
methods, but the super-outgroup Stegolepis (Rapateaceae) has been excluded to be comparable with results of the other loci. Statistics for each marker are given in Table 11.
Support was estimated with the bootstrap (Felsenstein, 1985). Bootstrap percentages
(BP) were calculated using 1,000 replicates, TBR branch swapping, and simple sequence addition, holding 10 trees each step and saving 100 shortest trees per bootstrap replicate.
Results
Direct sequencing of the eight nuclear DNA markers was possible in nearly all accessions.
Comparisons of amplified fractions of nuclear DNA markers to the assembled RefSeq genome
of Oryza sativa showed that MS, RPB2, XDH, and PHYC are single-copy in rice (Table 1). Although present in multiple copies in Oryza sativa, blast of PGIC, NIA, and PRK fragments produced in this study showed greatest similarity to the same single paralog.
Nuclear ribosomal internal transcribed spacer (ITS nrDNA)
Previously published protocols and PCR primers did not consistently produce PCR products,
indicating that either the primers did not match the target or the protocol was inappropriate.
The newly developed primers with a significantly higher annealing temperature and higher
DMSO concentrations produced PCR products in few samples if the standard denaturing temperature was used. Therefore high strong secondary structure are likely present in bromeliad
ITS nrDNA, which is insufficiently denatured at 95°C to allow successful amplification at 72°C
(Figure 2). At higher denaturing temperatures PCR bands of the expected length began to appear but yields were variable; at 99°C, all samples amplified consistently. As would be suspected from high GC content (Figure 3), cycle sequencing was also problematic. By altering the
sequencing chemistry it was possible to get full ITS nrDNA sequences for all included accessions, although a three-step loss of signal still occurred. Some taxa gave problems even with
this modified protocol and were finally excluded.
With the primers finally used in this study, a standard ITS nrDNA sequence was composed
of 123 bp of 18S nrDNA, ITS1 (of variable length), 164 bp for 5.8S nrDNA, ITS2 (of variable
length), and 139 bp of 26S nrDNA. Fragment size ranged from 884–1,017 bp (in Tillandsia biflora and Vriesea scalaris, respectively). No pseudogenes were found, although slight changes to
the published motifs of the conserved and variable domains described previously for ITS2 were
found. The final matrices included 1,291 (including all characters) and 1,057 (excluding the
problematic ITS1 region) positions, respectively. Other statistics are given in Table 11.
For ITS nrDNA strict consensus trees (Figure 4), bootstrap consensus trees (Figure 5) and
phylograms (Figure 6) are shown, resulting from analyses of the total matrix as well as of a
matrix excluding the problematic ITS1 region. Although several clades of related species received bootstrap support, backbone relationships are insufficiently resolved. Each accession is
different from the rest (i.e. long terminal branches) but synapomorphies are few (i.e. short
internal branches), as can be seen in one of the individual parsimony trees (Figure 6, Table 11).
99
BARFUSS, M.H.J.
100
NUCLEAR DNA MARKERS
Figure 4. Strict consensus trees of the ITS nrDNA: analysis of the complete DNA sequence matrix (A), analysis excluding the problematic region of ITS 1 (B).
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
101
Figure 5. Bootstrap consensus trees of the ITS nrDNA: analysis of the complete DNA sequence matrix (A), analysis excluding the problematic region of ITS 1 (B).
BARFUSS, M.H.J.
102
NUCLEAR DNA MARKERS
Figure 6. Phylograms selected randomly from 10,000 trees saved during maximum parsimony analyses of the ITS nrDNA: analysis of the complete DNA sequence
matrix (A), analysis excluding the problematic region of ITS 1 (B).
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
Malate synthase (MS) and RNA polymerase II, beta subunit (RPB2)
MS (Figure 1, B) was initially attempted using the primers “ms400f”, “ms526f”, “ms943r” and
“ms1488r” of Lewis & Doyle (2001) and the modified or new primers “ms356f” and “ms1408r”
(D. Springate, RBG Kew, London, unpublished) in all possible combinations. Since no amplifications were obtained, new primers were designed based on MS sequences obtained from GenBank. Three forward and three reverse primers were developed covering a region from partial
exon 1 to partial exon 4. The position of internal primers was designed to amplify three overlapping fragments that might be assembled as one piece. New primers were tested in all possible combinations, but only amplification of the fragment containing intron 2 using the primers “ms428f” and “ms960r” was successful and further optimized (Table 2). PCR primers
were used as sequencing primers. Details of all primers tested for MS are given in Table 4.
RPB2 (Figure 1, C) was tested using primers “P6F”, “P7R”, “P7F”, “10R”, “P10F”, and “11aR”
(Table 5) of Denton & al. (1998) in all possible combinations for a region covering partial exon
11 to partial exon 24. Only the primer combination “P10F”/”P11aR” successfully amplified a
fragment that contains intron 23. PCR conditions were further optimized (Table 2) and the
product finally sequenced using PCR primers.
Both markers were tested for two small sets from Tillandsia species complexes (T. tectorum, T. plumosa) with Mezobromelia hutchisonii as outgroup. The amplified fragment of MS
(659–707 bp) covered a region from partial exon 2 (279–282 bp) to partial exon 3 (261 bp)
(Figure 1, B), that of RPB2 (520–522 bp) a region from partial exon 23 (148 bp), to partial exon
24 (134 bp) (Figure 1, C). Both markers displayed indel variation, but only few substitutions. No
phylogenetic analysis was conducted due to the limited number of taxa and sequence variability.
Glucose-6-phosphate isomerase, cytosolic (PGIC)
Amplification of PGIC (Figure 1, D) was attempted using primers of Ford & al. (2006) in different combinations. Primer combination “AA11F”/”AA16R” for a fragment covering parts of exon 11 to exon 16, respectively, was the only successful one. PCR primers were used as sequencing primers. Optimized PCR conditions and primer details are given in Table 2 and Table
6, respectively.
PGIC was currently tested only for two species, one each from Catopsis and Alcantarea.
The amplified fragment (983 bp) covered a region from partial exon 11 (17 bp) to partial exon
16 (6 bp) (Figure 1, D). Two 1-bp insertions and 53 substitutions were observed. No phylogenetic analysis was performed, due to limited sampling.
Nitrate reductase 1, [NADH] (NIA)
NIA (Figure 1, E) was initially tested using the primers combinations “NIA2F”/”NIA2R” (targeting intron 2) and “NIA3F”/”NIA3R” (targeting intron 3) of Howarth & Baum (2002). Amplification of intron 2 failed and the primers for intron 3 only amplified a ca. 200 bp fragment. Therefore new primers were designed to cover a region from partial exon 1 to partial exon 3. Several
primer combinations have been tested, but only the combination “nia410f”/”nia1042r” covering a part of exon 1 and the complete intron 1 gave satisfactory results and was therefore optimized further (Table 2). PCR primers were used as sequencing primers. Primer details are
given in Table 7.
103
BARFUSS, M.H.J.
104
NUCLEAR DNA MARKERS
Figure 7. Strict consensus tree (A) and bootstrap consensus tree (B) of NIA.
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
105
Figure 8. Strict consensus tree (A) and bootstrap consensus tree (B) of XDH.
BARFUSS, M.H.J.
NUCLEAR DNA MARKERS
NIA was investigated for an enlarged sample set (72 accessions). The amplified fragment (729–
798 bp) covered a region from parts of exon 1 (629–638 bp), intron 1 (of variable length), to
partial exon 2 (15 bp) (Figure 1, E). Fragment length ranged from 649–800 bp (Brocchinia micrantha, Tillandsia duratii, respectively). Some accessions displayed a microsatellite at the 3'
end of intron1. A strict consensus and a bootstrap consensus tree are shown in Figure 7. Backbone relationships are not resolved, but several terminal clades receive support.
Xanthine dehydrogenase (XDH)
Amplification of XDH (Figure 1, F) was attempted using primers that were newly designed for
partial exon 4 and complete intron 5 using sequences downloaded from GenBank. Primers of
the phylogenetic studies of Górniak & al. (2010) and Morton (2011) became available only
after the current study was completed. Amplification of intron 5 failed so only the successfully
amplified part of exon 4 was further optimized (Table 2). Best amplicons were achieved using
the primer combination “xdh479f”/”xdh1611r”. These were initially also used as sequencing
primers, but in addition internal sequencing primers were developed to produce acceptable
traces for the whole fragment. All primers are listed in Table 8.
XDH was investigated for an enlarged sample set (64 accessions). PCR primers produced
fragments with part of exon 4 (1,107–1,110 bp). No indels within accessions were found, and
only a 3-bp indel for Brocchinia micrantha was observed in the entire DNA matrix. Resolution is
restricted to some terminal clades (Figure 8).
Phosphoribulokinase (PRK)
For PRK (Figure 1, G) initial gradient PCR runs were performed using degenerate primers originally designed for angiosperms, monocots, and/or palms (Lewis & Doyle 2002; D. Springate,
RGB Kew, London, unpublished). Primer combinations “prk663f”/”prk1040r” and
“prk663f”/”prk1167r” spanning a region of partial exon 2 to partial exon 5 amplified single,
weak PCR products only in samples having high-quality DNA extracts, whereas any combination using “prk488f” located in exon 1 yielded no amplification. After successful sequencing of
PCR products, a second set of bromeliad-specific PCR primers (“prk622f”, “prk1069r”) and
additional internal primers (“prk734f”, “prk889r”) sitting in exon 4 were designed, which were
initially used for Bromelioideae (Bromeliaceae) by Schulte & al. (2009). However, these primers often showed primer-dimer formation. Therefore in a last phase of optimization, three
primers were slightly modified (“prk621f”, “prk890r”, “prk1069r-2”) to allow higher annealing
temperatures and two additional nested sequencing primers (“prk630f”, “prk1057r”) were
developed to avoid sequencing problems caused by incorporation of dimers into the products.
For the earliest diverging genus of Bromeliaceae, Brocchinia, “prk734f” had to be adjusted
because of mismatches present at the 3' end (prk734f-2). The optimized PCR conditions and all
primers tested are given in Table 3 and Table 9, respectively.
Final PCR primers (“prk621f”, “prk1069r-2”) amplified fragments ranging from 831–1,692
bp in Tillandsia ionochroma and Guzmania patula, respectively (see Supplementary Data of
chapter 5). A standard PRK sequence was composed of parts of exon 2 (9 bp), intron 2 (of variable length), exon 3 (85 bp), intron 3 (of variable length) exon 4 (245 bp), intron 4 (of variable
length) and parts of exon 5 (108 bp) (Figure 1, G). Primers “prk621f” and “prk1069r-2” were
designed to be highly specific for PRK of Bromeliaceae, yielding clean, single PCR bands even
for accessions for which initial and second primer sets produced faint or no bands.
106
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
107
Figure 9. Simplified strict consensus trees of equally-most-parsimonious trees found in parsimony analyses of individual datasets supplemented with bootstrap support from parsimony bootstrap analysis either above or below branches; triangles sitting in terminal position on a branch indicate monophyletic groups, whereas triangles emerging from within
a node indicate several branches of that group on a polytomy. The tree for plastid DNA was modified according to results of Barfuss & al., (2005) and is shown for comparison.
BARFUSS, M.H.J.
NUCLEAR DNA MARKERS
Direct sequencing was successful in about ⅔ (67.2%) of the accessions with few SNPs (23.6%).
Intra-accession indels were detected in 146 (32.8%) accessions. The number of indels within a
given accession ranged from 1–5. Indel variation was only found in the less conserved introns
and never in exons. Three taxa had to be cloned, since the number of indels was too great to
be unambiguously edited: Vriesea psittacina, Guzmania graminifolia, and Tillandsia platyrhachis. Occasionally others were cloned to unambiguously verify the length of both
alleles [see Appendix]. SNPs were found in 241 (54.3%) out of 444 sampled accessions. Only
193 (43.5%) samples were found to be homozygous for the region sequenced; inter- and intraindividual variation in introns was high. The number of SNPs in a given accession ranged from
1–18 (highest number in Tillandsia fasciculate, B0076) were primarily found in introns and less
frequently in exons, which primarily had synonymous substitutions. Homopolymers were
found in several accessions in intron 2 and 4, respectively (see Supplementary Data, chapter
5).
Statistics for the PRK matrix and trees are given in Table 11. Indels are common at all hierarchical levels, yielding a final matrix of 3,291 characters. Evaluation of SNPs and indels within
accessions showed no significant impact on subsequent data analysis (signal conflict), although
homoplasy was present. Therefore coded SNPs were used except for the three cloned taxa.
Phylogenetic relationships based on PRK variation are shown in Figure 9, which is the
strict consensus tree with bootstraps indicated. Strongly supported are the sister group relationship (BP 100) of Glomeropitcairnia (BP 100) and Catopsis (BP 100) and core Tillandsioideae
(BP 100). Tribes Vrieseeae and Tillandsieae as well as core Tillandsieae are not supported. The
Cipuropsis and Vriesea groups are strongly supported (BP 91 and 100, respectively). Werauhia
is strongly supported (BP 100), whereas the Cipuropsis-Mezobromelia group has only weak BP
(58). The same picture is seen for Alcantarea (BP 100) and Vriesea (BP 61). Vriesea is monophyletic only when Andean members located in the Cipuropsis-Mezobromelia group, Tillandsia malzinei and former xerophytic gray-leaved members, the latter two now placed within
Tillandsia s. str., are excluded (data not shown). The sister group position of the Vriesea group
and a clade containing Tillandsia s. str. and the T. dodsonii group was surprising but only weakly supported (BP 63). Monophyly of Guzmania is not supported, and three clades of Guzmania
species (data not shown) occur in a polytomy with several others. The T. wagneriana (BP 64)
and T. lindenii (BP 100) groups are resolved as sisters (BP 65). Racinaea is strongly supported
(BP 96), but its relationships to other groups remain unresolved. Tillandsia s. str. and T. dodsonii group are resolved as sister groups with a moderate support (BP 81) with the T. dodsonii
group being strongly supported (BP 96), but unresolved with several other clades of Tillandsia
species.
Phytochrome C (PHYC)
For partial exon 1 of PHYC (Figure 1, H), initial PCRs were performed using the four degenerate
primers “phyc0503f-mo”, “phyc0515f-mo”, “phyc1699r-mo”, and “phyc1705r-mo”, which
were obtained from primer sequences originally published by Mathews & Donoghue (1999)
and modified according to monocot sequences downloaded from GenBank. All primer combination worked, but showed strong primer-dimer formation. Best amplifications were achieved
with “phyc0515f-mo” and “phyc1699r-mo”. However, all combinations were successfully sequenced, but showed strong signals of dimers at the start of the traces. Therefore bromeliadspecific PCR primers (“phyc0515f-br”, “phyc1699r-br”) were produced and optimized (Table 2)
based on sequence information obtained from “phyc0503f-mo” and “phyc1705r-mo” since
108
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
their annealing position is outside the annealing positions of these primers. In addition, nested
sequencing primers (“phyc0524f-br”, “phyc1690r-br”) were designed to avoid sequencing
problems as much as possible. Several internal primers creating overlapping reads towards the
end of the fragment were also developed to sequence the whole PCR product. Monocotspecific ones were also tested successfully for Polystachya (Orchidaceae) by Russell & al.
(2010). Four internal primers giving the best reads were finally selected for sequencing. Details
of all primers are given in Table 10.
Only fragments of partial exon 1 (Figure 1, H) were amplified ranging from 1,159–1,192 bp
in Werauhia insignis and Guzmania acorifolia/G. condensata, respectively (see Supplementary
Data of chapter 5), but mostly 1,177 bp. Direct sequencing was straightforward in nearly all
cases. In contrast to PRK, no length differences between the two alleles of an accession were
detected; this is likely due to the fact that only conserved parts of the exon 1 were amplified in
PHYC. Only two obvious hybrids of distantly related taxonomic groups, where one of the possible parental taxa displayed two 3 bp insertions, were observed; these were finally excluded.
Sequences of PHYC generally showed clean raw data with no or only few polymorphic sites in
the electropherograms. Most individuals are either homozygous or show only little intraspecific allelic variation, as can be expected for relatively conserved exon. SNPs were found in 220
taxa out of 444 sampled individuals. About 224 samples were found to be homozygous for the
amplified region. The number of SNPs in a given heterozygous taxon ranged from 1–15 (the
upper limit seen in T. aff. cucaensis Wittm. B0735, see Supplementary Data of chapter 5). Distribution of polymorphic sites was even across the amplified portion of PHYC with no particular
preference for a specific region, but significantly higher at the 3rd codon position (i.e. synonymous substitutions).
Information on the analyzed PHYC matrix and trees are given in Table 9. Indels are rare,
and present either in single species or few closely related species, leading to a final matrix size
of 1,228 characters. Like in PRK, all polymorphic sites have been checked for their impact on
subsequent data analysis, which did not show significant conflicts. Therefore the polymorphism-coded PHYC dataset was used for all analyses, with the exception of three taxa, which
were cloned for PRK and therefore also for PHYC, to allow the combination of corresponding
allelic sequences.
Figure 9 shows the strict consensus tree of a MP analysis with BP, which summarizes phylogenetic relationships based on PHYC sequence data. As in PRK tree, both Glomeropitcairnia
and Catopsis are strongly supported (BP 100) as sisters (BP 100), with the latter clade sister to
a strongly supported core Tillandsioideae (BP 100). Tribe Vrieseeae is paraphyletic, but the
resulting grade has only a weak support (BP 58). The Vriesea group (BP 100 ), Alcantarea (BP
97) and Vriesea (BP 89) are monophyletic, the last as found in PRK, but only when the Andean
members, Tillandsia malzinei and former xerophytic gray-leaved species are excluded (data
not shown). The Cipuropsis group splits into two lineages, Werauhia (BP 100) and the Cipuropsis-Mezobromelia group (BP 67). Mezobromelia is weakly supported (BP 57) as nested in the
Cipuropsis-Mezobromelia group (data not shown), which in addition contains taxa previously
assigned to Vriesea and Tillandsia (data not shown). Tribe Tillandsieae are strongly supported
(BP 92). The first lineage that splits from core Tillandsieae is Guzmania (BP 85), with Mezobromelia hutchisonii nested inside (data not shown). Core Tillandsieae is only weakly supported
(BP 58) and relationships of genera and groups are unresolved. The T. lindenii group (89 BP),
Racinaea (no BP) and the T. dodsonii group (BP 86) are monophyletic but all three are placed in
a polytomy with different clades of Tillandsia s. str. and the T. wagneriana group.
109
BARFUSS, M.H.J.
NUCLEAR DNA MARKERS
Discussion
Challenges of nuclear DNA markers
The fact that nuclear DNA sequences are much less used than plastid ones became evident in
initial phases of the current study. Several problems highlighted earlier were also encountered
during the optimization of nuclear markers for Tillandsioideae. The most time-consuming steps
were primer design, extraction of high-quality DNA, PCR optimization, improving sequencing
results of ITS nrDNA, and editing as well as aligning intron-containing markers and ITS nrDNA.
The only advantage of Tillandsioideae is that it presumably consists of more than 98% diploid
species, which clearly helps in data collection, evaluation and interpretation. The major challenges of nuclear DNA sequences for phylogenetic studies in Tillandsioideae are: (1) limited
availability of effective primers; (2) occurrence of strong secondary structure (ITS nrDNA, NIA)
that make PCR and sequencing difficult; (3) a short fragment length and consequentially a
small number of PICs (e.g., amplified fragments of MS, RPB2); (4) heterozygous individuals with
SNPs and indel mutations, the latter mainly in markers containing polymorphic introns (e.g.,
PRK), making sequence editing time-consuming and increased laboratory costs if cloning is
required; and (5) alignment of markers containing polymorphic introns with indel variation. A
proper pre-evaluation of nuclear DNA markers and the selection of more conserved regions
(e.g., PHYC, PGIC, XDH) would have minimized these complications. A review article on nuclear
DNA markers used in the Department of Systematic and Evolutionary Botany at the University
of Vienna for different angiosperm lineages is in progress (Barfuss & al., in prep.).
Nuclear versus plastid DNA markers
The current results support the hypothesis that several nuclear DNA markers evolve much
more rapidly than do plastid markers. The number of potential PICs per sequenced base pair in
PHYC is 2-fold, in PRK 3-fold higher than in plastid DNA markers, which means sequencing
these more variable nuclear DNA markers requires much lesser sequencing effort to have the
same level of resolution as combined plastid DNA markers. However, homoplasy in three out
of five nuclear data sets examined here was also higher (Table 11). Conserved regions like XDH
evolve at about the same rate as plastid regions. Barfuss & al. (2005) and Givnish & al. (2011)
have shown that the combination of up to eight plastid markers is insufficient to resolve phylogenetic relationships of Bromeliaceae down to the subgeneric rank (Figure 9). Inclusion of
properly chosen plastid markers will definitely improve the already published plastid analyses,
but this is costly and time-consuming. Whether sequencing more plastid or additional nuclear
markers is more efficient and useful for the scientific questions being investigated needs to be
evaluated before large datasets are sequenced.
Phylogenetic utility of nuclear markers
ITS nrDNA is not suitable for well resolved backbone relationships, although a lot of intraindividual variation was present (Figure 6). This is remarkable, since in many other angiosperm
lineages (e.g., Barnadesioidae (Asteraceae): Gruenstaeudl & al., 2009) and especially in monocots (e.g., Musaceae: Hřibová, 2011; Polystachya (Orchidaceae): Russell & al., 2010;), ITS nrDNA has been applied successfully below family level. The strong secondary structure could
have ecological significance for adaptation to hot and dry environments (Hurst & Merchant,
2001), as epi- and lithophytes are often exposed to changing microclimatic conditions. Howev110
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
er, orchid twig epiphytes in subtribe Oncidiinae that occupy the same micro-environments as
many species of Tillandsia exhibit much higher levels of ITS nrDNA variation than do those
species occupying more mesic epiphytic habitats (Chase, 2009). In this context, it is interesting
to note that mesophytic bromeliad taxa (e.g., Vriesea, Werauhia) display a shorter problematic
ITS1 region than xerophytic species (Tillandsia). Because of low resolution and great difficulties
in amplification, sequencing and alignment, ITS nrDNA cannot be recommended as marker
generally applied in phylogenetic studies of Bromeliaceae, but might be currently the only
marker to discriminate species for barcoding purpose (Michael H. J. Barfuss, unpublished data).
Amplified fragments of MS and RPB2 are relatively short and information content is limited compared to other markers studied. Therefore these genomic regions were not further
investigated for an enlarged set of samples. Although PGIC seems promising regarding ease of
sequencing and alignment at various taxonomic levels within Bromeliaceae, it was not further
tested for other samples of Tillandsioideae. Trees of NIA and XDH show limited resolution of
deeper nodes (Figures 7, 8, respectively). However, several terminal clades receive bootstrap
support, and both markers might be useful in adding more phylogenetic information in a combined analysis of nuclear DNA markers. Results of XDH show that this marker is better suited to
higher taxonomic levels (as in Orchidaceae; Gorniák et al., 2010), whereas NIA might help better discriminate closely related taxa.
Preliminary results indicated that PRK and PHYC were the two most promising nuclear
DNA markers tested in terms of amplification and sequencing ease as well as in providing high
numbers of PICs. Therefore these regions were sequenced for all available Tillandsioideae accessions. As for combined plastid data, individual nuclear DNA markers are unable to resolve
relationships completely (Figure 9). Although several clades are retrieved and highly supported
by most markers, they still provides different degrees of resolution and levels of support below
the subfamily rank. PHYC is easy to align across the analyzed taxa, but it fails to provide resolution in more terminal nodes, especially in core Tillandsieae. PRK is difficult and not suitable,
especially at higher levels, in the light of the numerous indels that have to be introduced into
the alignment, but it helps to provide more resolution at intermediate levels.
Recently diverged species
To address questions concerning relationships of recently diverged species within genera of
Tillandsioideae, our sampling was far too limited and turned out to be problematic in taxonomically complicated species complexes, especially when consensus allelic sequences are
used. However, in the complete data sets of PRK and PHYC we sometimes included two or
sometimes more accessions of the same taxon, which mostly cluster together or into clades of
closely related species. We also found some cases where accessions of the same species but of
a different subspecific rank did not cluster, although these accessions were still not far from
each other. Whether these patterns are results of missing PICs, homoplasy, allelic consensus
sequences, or questionable species concepts would need to be investigated further. It is clearly necessary to include multiple accessions of the same species/taxon, if possible over their
whole distribution, and preferably complete gene sequences including all exons and introns to
gain more linked information. In addition more cloning for divergent alleles of an individual,
especially in complicated species complexes, is required to have pure allelic data.
111
BARFUSS, M.H.J.
NUCLEAR DNA MARKERS
Hybrids, hybrid speciation and reticulate evolution
The two species suspected to be of hybrid origin, T. dorotheae and T. marconae, could be
clearly confirmed by sequencing nuclear DNA. Detection was possible since parental taxa belong to different lineages within Tillandsia with relatively high sequence divergence. The two
alleles of each sample cluster with high support with the clade of their parents. Parents of T.
dorotheae are T. albertiana and a species of the T. argentina complex, that of T. marconae, T.
landbeckii and a species of the T. purpurea complex. Most living plants in the botanical gardens
or private collections are of vegetatively propagated origin from one or a few wild collections.
Whether these species are allopolyploids, homoploid hybrids, or just F1 individuals of an occasional recent hybridization event would need to be investigated by more documented material. Except for these two cases, we currently do not have well-supported instances of hybridization, reticulate evolution or incomplete lineage sorting in other species.
Acknowledgements
The authors thank Tod F. Stuessy for providing lab facilities; many botanical gardens and
individual people for providing plant material; Walter Till for taxonomic expertise, Mark W.
Chase for a critically review of the manuscript. Financial support was provided by the University of Vienna, the Commission for Interdisciplinary Ecological Studies (KIÖS) at the Austrian
Academy of Sciences (ÖAW) to W. Till and M.H.J. Barfuss (2007-02), and the SYNTHESYS (Synthesis of Systematic Resources) program funded by European Union.
References
Álvarez, I., Costa, A. & Feliner, G.N. 2008. Selecting single-copy nuclear genes for plant phylogenetics: A preliminary analysis for the Senecioneae (Asteraceae). J. Molec. Evol. 66: 276–
291.
Baldwin, B.G., Sanderson, M.J., Porter, J.M., Wojciechowski, M.F., Campbell, C.S. & Donoghue, M.J. 1995. The ITS region of nuclear ribosomal DNA: A valuable source of evidence on angiosperm phylogeny. Ann. Missouri Bot. Gard. 82: 247–277.
Barfuss, M.H.J., Samuel, R., Till, W. & Stuessy, T.F. 2005. Phylogenetic relationships in subfamily Tillandsioideae (Bromeliaceae) based on DNA sequence data from seven plastid regions. Amer. J. Bot. 92: 337–351.
Blattner F.R. 1999. Direct amplification of the entire ITS region from poorly preserved plant
material using recombinant PCR. BioTechniques 27: 1180–1186.
Borsch T. & Quant, D. 2009. Mutational dynamics and phylogenetic utility of noncoding chloroplast DNA. Pl. Syst. Evol. 282: 169–199.
Chapman, M.A., Chang, J., Weisman, D., Kesseli, R.V. & Burke, J.M. 2007. Universal markers
for comparative mapping and phylogenetic analysis in the Asteraceae (Compositae). Theor. Appl. Gen. 115: 747–755.
Chase, M.W., Williams, N.H., Donisete de Faria, A., Neubig, K.M., do Carmo E. Amaral, M. &
Whitten, W.M. 2009. Floral convergence in Oncidiinae (Cymbidieae; Orchidaceae): an expanded concept of Gomesa and a new genus Nohawilliamsia. Ann. Bot. (Oxford) 104:
387–402.
112
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
Chew, T. ,De Luna, E. & González, D. 2010. Phylogenetic relationships of the pseudobulbous
Tillandsia species (Bromeliaceae) inferred from cladistic analyses of ITS 2, 5.8S ribosomal
RNA gene, and ETS sequences. Syst. Bot. 35: 86–95.
Clarkson, J.J., Kelly, L.J., Leitch, A.R., Knapp, S. & Chase, M.W. 2010. Nuclear glutamine synthetase evolution in Nicotiana: Phylogenetics and the origins of allotetraploid and homoploid (diploid) hybrids. Molec. Phylog. Evol. 55: 99–112.
Crayn, D.M., Winter, K. & Smith, J.A.C. 2004. Multiple origins of crassulacean acid metabolism
and the epiphytic habit in the Neotropical family Bromeliaceae. Proc. Natl. Acad. Sci.
U.S.A. 101: 3703–3708.
Cronn, R.C., Small, R.L., Haselkorn, T. & Wendel, J.F. 2002. Rapid diversification of the cotton
genus (Gossypium: Malvaceae) revealed by analysis of sixteen nuclear and chloroplast
genes. Amer. J. Bot. 89: 707–725.
Denton, A.L., McConaughy, B.L. & Hall, B.D. 1998. Usefulness of RNA polymerase II coding
sequences for estimation of green plant phylogeny. Molec. Biol. Evol. 15: 1082–1085.
Douzery, E.J.P., Pridgeon, A.M., Kores, P., Linder, H.P., Kurzweil, H. & Chase, M.W. 1999.
Molecular phylogenetics of Diseae (Orchidaceae): A contribution from nuclear ribosomal
ITS sequences. Amer. J. Bot. 86: 887–899.
Doyle, J.J. & Doyle, J.L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf
tissue. Phytochem. Bull. Bot. Soc. Amer. 19: 11–15.
Edgar, R.C. 2004a. MUSCLE: A multiple sequence alignment method with high accuracy and
high throughput. Nucl. Acids Res. 32: 1792–1797. http://www.drive5.com/muscle/
Edgar, R.C. 2004b. MUSCLE: A multiple sequence alignment method with reduced time and
space complexity. B.M.C. Bioinf. 5: 113.
Emshwiller, E. & Doyle, J.J. 1999. Chloroplast-expressed glutamine synthetase (ncpGS): Potential utility for phylogenetic studies with an example from Oxalis (Oxalidaceae). Molec. Phylog. Evol. 12: 310–319.
Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783–791.
Ford, V.S., Lee, J., Baldwin, B.G. & Gottlieb, L.D. 2006. Species divergence and relationships in
Stephanomeria (Compositae): PGIC phylogeny compared to prior biosystematic studies.
Amer. J. Bot. 93: 480–490.
Gaut, B.S., Muse, S.V., Clark, W.D. & Clegg, M.T. 1992. Relative rates of nucleotide substitution at the rbcL locus in monocotyledonous plants. J. Molec. Evol. 35: 292–303.
Givnish, T.J., Barfuss, M.H.J., Van Ee, B., Riina, R., Schulte, K., Horres, R., Gonsiska, P.A.,
Jabaily, R.S., Crayn, D.M., Smith, J.A.C., Winter, K., Brown, G.K., Evans, T.M., Holst, B.K.,
Luther, H.E., Till, W., Zizka, G., Berry, P.E. & Sytsma, K.J. 2011. Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: Insights from an eight-locus plastid
phylogeny. Amer. J. Bot. 98: 872–895.
Givnish, T.J., Millam, K.C., Berry, P.E. & Sytsma, K.J. 2007. Phylogeny, adaptive radiation, and
historical biogeography of Bromeliaceae inferred from ndhF sequence data. Pp. 3–26 In:
Columbus, J.T., Friar, E.A., Porter, J.M., Prince, L.M. & Simpson M.G. (eds.), Monocots:
Comparative Biology and Evolution – Poales. Claremont: Rancho Santa Ana Botanic Garden (RSABG).
113
BARFUSS, M.H.J.
NUCLEAR DNA MARKERS
Givnish, T.J., Milliam, T.M., Evans, T.M., Hall, J.C., Berry, P.E. & Terry, R.G. 2004. Ancient vicariance or long-distance dispersal? Inferences about phylogeny and South AmericanAfrican disjunctions in Rapateaceae and Bromeliaceae based on ndhF sequence data. Int.
J. Plant Sci. 165 (4, Suppl.), 35–54.
Górniak, M., Paun, O. & Chase, M.W. 2010. Phylogenetic relationships within Orchidaceae
based on a low-copy nuclear coding gene, XDH: Congruence with organellar and nuclear
ribosomal DNA results. Molec. Phylog. Evol. 56: 784–795.
Gruenstaeudl, M., Urtubey, E., Jansen, R.K., Samuel, R., Barfuss, M.H.J. & Stuessy, T.F. 2009.
Phylogeny of Barnadesioideae (Asteraceae) inferred from DNA sequence data and morphology. Molec. Phylog. Evol. 51: 572–587.
Hall, T.A. 1999. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41: 95–98.
Hershkovitz, M.A. & Zimmer E.A. 1996. Conservation patterns in angiosperm ITS2 sequences.
Nucleic Acids Res. 24: 2857–2867.
Hřibová, E.,Čížková, J., Christelová, P., Taudien, S., de Langhe, E. & Doležel, J. 2011. The ITS15.8S-ITS2 sequence region in the Musaceae: Structure, diversity and use in molecular phylogeny. PLoS one 6: e17863 (1–11), doi: 10.1371/journal.pone.0017863.
Horres, R., Zizka, G., Kahl, G. & Weising, K. 2000. Molecular phylogenetics of Bromeliaceae:
Evidence from trnL (UAA) intron sequences of the chloroplast genome. Pl. Biol. 2: 306–
315.
Hurst, L.D. & Merchant, A.R. 2001. High guanine-cytosine content is not an adaptation to high
temperature: a comparative analysis amongst prokaryotes. Proc. Biol. Sci. 268: 493–497.
Howarth, D.G. & Baum, D.A. 2002. Phylogenetic utility of a nuclear intron from nitrate reductase for the study of closely related plant species. Molec. Phylog. Evol. 23: 525–528.
Jabaily, R.S. & Sytsma, K.J. 2010. Phylogenetics of Puya (Bromeliaceae): Placement, major
lineages, and evolution of Chilean species. Amer. J. Bot. 97: 337–356.
Jobes, D.V. & Thien, L.B. 1997. A conserved motif in the 5.8S ribosomal RNA (rRNA) gene is a
useful diagnostic marker for plant internal transcribed spacer (ITS) sequence. Plant Mol
Biol Rep 15: 326–334.
Kelchner, S.A. 2000. The evolution of noncoding chloroplast DNA and its application in plant
systematics. Ann. Missouri Bot. Gard. 87: 482–498.
Lewis, C.E. & Doyle, J.J. 2001. Phylogenetic Utility of the Nuclear Gene Malate Synthase in the
Palm Family (Arecaceae). Molec. Phylogen. Evol. 19: 409–420.
Lewis, C.E. & Doyle, J.J. 2002. A phylogenetic analysis of tribe Areceae (Arecaceae) using two
low-copy nuclear genes. Pl. Syst. Evol. 236: 1–17.
Liu, J.-S. & Schardl, C.L. 1994. A conserved sequence in internal transcribed spacer 1 of plant
nuclear rRNA genes. Plant Mol. Biol. 26: 775–778.
Lukas, B. 2010. Molekulare Phylogenie und Phytochemie der Gattung Origanum. Dissertation,
University of Vienna.
Ma, J.X., Li, Y.N., Vogl, C., Ehrendorfer, F. & Guo, Y.P. 2010. Allopolyploid speciation and ongoing backcrossing between diploid progenitor and tetraploid progeny lineages in the
Achillea millefolium species complex: Analyses of single-copy nuclear genes and genomic
AFLP. B.M.C. Evol. Biol.10: 100 (1–11), doi:10.1186/1471-2148-10-100.
Mathews, S. & Donoghue, M.J. 1999. The root of angiosperm phylogeny inferred from duplicate phytochrome genes. Science 286: 947–950.
114
IN TILLANDSIOIDEAE (BROMELIACEAE)
PART 2, CHAPTER 3
Morton, C.M. 2011. Newly sequenced nuclear gene (XDH) for inferring angiosperm phylogeny.
Ann. Missouri Bot. Gard. 98: 63–89.
Oxelman, B., Yoshikawa, N., McConaughy, B.L., Luo, J., Denton, A.L. & Hall, B.D. 2004. RPB2
gene phylogeny in flowering plants, with particular emphasis on asterids. Molec. Phylog.
Evol. 32: 462–479.
Piñeiro, R., Costa, A., Aguilar, J.F. & Feliner, G.N. 2009. Overcoming paralogy and incomplete
lineage sorting to detect a phylogeographic signal: A GapC study of Armeria pungens.
Botany-Botanique 87: 164–177.
Russell, A., Samuel, R., Klejna, V., Barfuss, M.H.J., Rupp, B. & Chase, M.W. 2010. Reticulate
evolution in diploid and tetraploid species of Polystachya (Orchidaceae) as shown by plastid DNA sequences and low-copy nuclear genes. Ann. Bot. (Oxford) 106: 37–56.
Safer, S. (2011). Molecular and phytochemical investigations on the genus Leontopodium. Dissertation, University of Innsbruck.
Samuel, R., Kathriarachchi, H., Hoffmann, P., Barfuss, M.H.J., Wurdack, K.J., Davis, C.C. &
Chase, M.W. 2005. Molecular phylogenetics of Phyllanthaceae: Evidence from plastid
matK and nuclear PHYC sequences. Amer. J. Bot. 92: 132–141.
Sang, T. 2002. Utility of low-copy nuclear gene sequences in plant phylogenetics. Critical Reviews in Biochemistry and Molecular Biology 37: 121–147.
Sang, T., Pan, J., Zhang, D., Ferguson, D., Wang, C., Pan, K.-Y. & Hong, D.-Y. 2004. Origins of
polyploids: an example from peonies (Paeonia)and a model for angiosperms. Biological
Journal of the Linnean Society. 82: 561–571.
Sang, T. & Zhang, D. 1999. Reconstructing hybrid speciation using sequences of low-copy nuclear genes: Hybrid origins of five Paeonia species based on Adh gene phylogenies. Syst.
Bot. 24: 148–163.
Sass, C. & Specht, C.D. 2010. Phylogenetic estimation of the core bromelioids with an emphasis on the genus Aechmea (Bromeliaceae). Mol. Phyl. Evol. 55: 559–571.
Schulte, K., Barfuss, M.H.J. & Zizka, G. 2009. Phylogeny of Bromelioideae (Bromeliaceae) inferred from nuclear and plastid DNA loci reveals the evolution of the tank habit within the
subfamily. Molec. Phylogen. Evol. 51: 327–339.
Schulte, K. & Zizka, G. 2008. Multi locus plastid phylogeny of Bromelioideae (Bromeliaceae)
and the taxonomic utility of petal appendages and pollen characters. Candollea 63: 209–
225.
Shaw, J., Lickey, E.B., Beck, J.T., Farmer, S.B., Liu, W.S., Miller, J., Siripun, K.C., Winder, C.T.,
Schilling, E.E. & Small, R.L. 2005. The tortoise and the hare II: Relative utility of 21
noncoding chloroplast DNA sequences for phylogenetic analysis. Amer. J. Bot. 92: 142–
166.
Shaw, J., Lickey, E.B., Schilling, E.E. & Small, R.L. 2007. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms:
The tortoise and the hare III. Amer. J. Bot. 94: 275–288.
Steger, G. 1994. Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction. Nucleic Acids Res. 22: 2760–2768.
Strand, A.E., LeebensMack, J. & Milligan, B.G. 1997. Nuclear DNA-based markers for plant
evolutionary biology. Molecular Ecology 6: 113–118.
Su, X.-Z., Wu, Y., Sifri, C.D. & Wellems, T.E. 1996. Reduced extension temperatures required
for PCR amplification of extremely A+T-rich DNA. Nucleic Acids Research 24: 1574–1575.
115
Sun, Y., Skinner, D.Z., Liang, G.H. & Hulbert, S.H. 1994. Phylogenetic analysis of Sorghum and
related taxa using Internal Transcribed Spacers of nuclear ribosomal DNA. Theor. Appl.
Genet. 89: 26–32.
Swofford, D.L. 2003. PAUP*: Phylogenetic analysis using parsimony (*and other methods),
version 4.0b10. Sunderland: Sinauer Associates.
Tank, D.C. & Sang, T. 2001. Phylogenetic utility of the glycerol-3-phosphate acyltransferase
gene: Evolution and implications in Paeonia (Paeoniaceae). Molec. Phylog. Evol. 19: 421–
429.
Tel-Zur, N., Abbo, S., Myslabodski, D. & Mizrahi, Y. 1999. Modified CTAB procedure for DNA
isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Pl.
Molec. Biol. Reporter 17: 249–254.
Terauchi, R., Terachi, T. & Miyashita, N.T. 1997. DNA polymorphism at the Pgi locus of a Wild
Yam, Dioscorea tokoro. Genetics 147: 1899–1914.
Terry, R.G., Brown, G.K. & Olmstead, R.G. 1997a. Examination of subfamilial phylogeny in
Bromeliaceae using comparative sequencing of the plastid locus ndhF. Amer. J. Bot. 84:
664–670.
Terry, R.G., Brown, G.K. & Olmstead, R.G. 1997b. Phylogenetic relationships in subfamily Tillandsioideae (Bromeliaceae) using ndhF sequences. Syst. Bot. 22: 333–345.
Thomas, M.M., Garwood, N.C., Baker, W.J., Henderson, S.A., Russell, S.J., Hodel, D.R. &
Bateman, R.M. 2006. Molecular phylogeny of the palm genus Chamaedorea, based on the
low-copy nuclear genes PRK and RPB2. Molec. Phylogen. Evol. 38: 398–415.
Vaezi, J. & Brouillet, L. 2009. Phylogenetic relationships among diploid species of Symphyotrichum (Asteraceae: Astereae) based on two nuclear markers, ITS and GAPDH. Molec.
Phylog. Evol. 51: 540–553.
Versieux, L.M., Barbará, T., Wanderley, M.d.G.L., Calvente, A., Fay, M.F. & Lexer, C. 2012.
Molecular phylogenetics of the Brazilian giant bromeliads (Alcantarea, Bromeliaceae): implications for morphological evolution and biogeography. Molec. Phylog. Evol. 64: 177–
189.
Wendel, J.F. 2000. Genome evolution in polyploids. Plant Molecular Biology 42: 225–249.
Werle, E., Schneider, C., Renner, M., Volker, M. & Fiehn, W. 1994. Convenient single-step,
one tube purification of PCR products for direct sequencing. Nucleic Acids Research 22:
4354–4355.
White, T.J., Bruns, T., Lee, S. & Taylor, J. 1990. Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics, Pp. 315–322 in Innis, M.A., Gelfand, D.H., Sninsky,
J.J. & White, T.J. (eds.), PCR protocols: A guide to methods and applications. San Diego:
Academic Press.
Willyard, A., Cronn. R. & Liston, A. 2009. Reticulate evolution and incomplete lineage sorting
among the ponderosa pines. Molec. Phylog. Evol. 52: 498–511.
Yockteng, R. & Nadot, S. 2004. Phylogenetic relationships among Passiflora species based on
the glutamine synthetase nuclear gene expressed in chloroplast (ncpGS). Molec. Phylog.
Evol. 31: 379–396.
Zuker, M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic
Acids Res 31: 3406–3415.
116
Part 3
Classification of
Bromeliaceae subfamily Tillandsioideae
Chapter 4
Progress towards a new classification
of Tillandsioideae
Walter Till & Michael H.J. Barfuss
Status: published, Journal of the Bromeliad Society 56 (6): 253–259. 2006.
Contribution: data collection, data analysis, manuscript writing/editing
121
BARFUSS, M.H.J.
122
PROGRESS TOWARDS A NEW
CLASSIFICATION OF TILLANDSIOIDEAE
PART 3, CHAPTER 4
123
BARFUSS, M.H.J.
124
PROGRESS TOWARDS A NEW
CLASSIFICATION OF TILLANDSIOIDEAE
PART 3, CHAPTER 4
125
BARFUSS, M.H.J.
126
PROGRESS TOWARDS A NEW
CLASSIFICATION OF TILLANDSIOIDEAE
PART 3, CHAPTER 4
127
Chapter 5
A new classification of Bromeliaceae subfamily
Tillandsioideae inferred from DNA sequences data
of two genomes and morphology
Michael H.J. Barfuss, Walter Till & Rosabelle Samuel
University of Vienna, Faculty of Life Sciences, Department of Systematic and Evolutionary Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria
Author for correspondence: Michael H.J. Barfuss, michael.h.j.barfuss@univie.ac.at
Keywords: Alcantarea; Catopsis; Cipuropsis; plastid DNA; Glomeropitcairnia; Guzmania; Josemania; Lectotypifications; Lemeltonia; Mezobromelia; PHYC; PRK; Racinaea; R. subg. Pseudophytarrhiza; Rothowia; low-copy nuclear DNA; Tillandsia; T. subg. Pseudovriesea; T. subg. Viridantha; Vriesea, Werauhia
Running Title: New classification of Tillandsioideae (Bromeliaceae)
Status: in preparation, intended to be submitted to Taxon
Contribution: data collection, data analyses, manuscript writing
Abstract
In order to establish a natural and stable classification system for Tillandsioideae we conducted phylogenetic analyses of newly generated low-copy nuclear DNA sequence data (PHYC,
PRK) and previously published plastid DNA markers (atpB-rbcL, matK, rbcL, partial rbcL-accD,
rps16 intron, partial trnK intron, trnL intron, trnL-trnF), together with a re-evaluation of morphological characters. Parsimony analysis revealed the following, mostly well-supported general branching pattern for Tillandsioideae: ((Glomeropitcairnia, Catopsis) (((Alcantarea,
Vriesea) (Werauhia, Cipuropsis-Mezobromelia clade)) (Guzmania, ((Josemania, Racinaea)
(Rothowia, (Lemeltonia, Tillandsia)))))). Core Tillandsioideae are well supported by all analyses,
but Bayesian analysis yielded a slightly different branching pattern for core Tillandsieae: (Guzmania, ((Josemania, Rothowia) (Racinaea (Lemeltonia, Tillandsia))). Based on the results a new
classification of Tillansioideae is presented. Two new subtribes (Cipuropsidinae, Vrieseinae),
three new genera (Josemania, Lemeltonia, Rothowia), and three new subgenera (Racinaea
subg. Pseudophytarrhiza, Tillandsia subg. Viridantha, T. subg. Pseudovriesea) are described and
several species are reclassified. A key to the genera of Tillandsioideae is also provided. Lectotypes are selected for Catopsis subg. Tridynandra, Tillandsia sect. Conostachys, and Tillandsia
sect. Eriophyllum. Classification of some unresolved phylogenetic units remains informal and
needs further attention, especially within the Cipuropsis-Mezobromelia clade and the genus
Tillandsia.
131
Introduction
Tillandsioideae Burnett is the largest and morphologically most diverse subfamily of Bromeliaceae Juss. with more than 1300 species in nine generally accepted genera (Smith & Downs,
1977; Smith & Till, 1998; Till, 2000a; Grant & Zijlstra, 1998; Luther & Rabinowitz, 2010; species
numbers according to Luther, 2010): Alcantarea (E. Morren ex Mez) Harms (28 spp.), Catopsis
Griseb. (18 spp.), Glomeropitcairnia (Mez) Mez (2 spp.), Guzmania Ruiz & Pav. (210 spp.), Mezobromelia L.B. Sm. (9 spp.), Racinaea M.A. Spencer & L.B. Sm. (65 spp.), Tillandsia L. (626 spp.,
in 6 subgenera), Vriesea Lindl. (266 spp., in two sections), and Werauhia J.R. Grant (87 spp., in
2 sections). The genus Viridantha Espejo (8 spp.; Espejo-Serna, 2002; Espejo-Serna & al., 2007;
López-Ferrari & Espejo-Serna, 2009) is included in Tillandsia, following most recent authors,
and is subdivided into two sections, sect. Viridantha (5 spp.) and sect. Caulescens Espejo (3
spp.).
A well-established taxonomic concept of the whole subfamily is of much interest, because
tillandsioids are an important component of neotropical ecosystems accounting for a high
portion of recorded (epiphytic) biodiversity, are of special importance for conservationists
since many species are endemic to certain regions, have the greatest number of horticulturally
important Bromeliaceae species, and are popular amongst bromeliad collectors and amateur
botanists. Despite its importance, a well-supported and broadly accepted classification scheme
has not been achieved to date. The main reasons are the lack of definite morphological characters to delimit genera, subgenera and sections, and the low variability of DNA markers studied
up to now, weakening the possibility to reconstruct a solid phylogenetic framework with wellsupported, monophyletic entities (see Hörandl & Stuessy (2010) for the definitions of evolutionary terms used in this study).
Traditional morphological classifications
A large number of species in the subfamily exhibit considerable interspecific morphological
variation especially in reproductive organs (e.g., inflorescences and flowers) and in general
habit, with life forms ranging from entirely mesophytic and water-impounding to strongly xerophytic, rarely terrestrial to commonly epiphytic or lithophytic (e.g., Gilmartin, 1983; Gilmartin & Brown, 1986; Benzing, 2000; Stefano & al., 2008). Early bromeliad taxonomists considered floral morphological characters to be most important for classification, since there is
sometimes no diagnostic variation in the vegetative structures of distantly related species
(Baker, 1889; Mez, 1896, 1934–35; Smith, 1951; Smith & Downs, 1977). Smith & Downs, authors of the last monograph of Bromeliaceae (Pitcairnioideae Harms: 1974; Tillandsioideae:
1977; Bromelioideae: 1979) maintained the use of these floral characteristics in their taxonomic treatments: the four main characters used to differentiate genera of Tillandsioideae are the
position of the ovary, the structure of seeds, the presence vs. absence of petal appendages,
and free vs. conglutinate petals. However, in their "Preface" Smith & Downs (1977: 663) state
about the difficulty of identifying Tillandsioideae genera "once the crucial petals and stamens
are lost" and the sometimes inconsistent placement of species by Mez (1934–35; cf. the different treatment of several xerophytic, grey-leaved Vriesea species by both authors). Therefore,
many species are misplaced in both monographs according to these generic definitions, since
their classification was often based on herbarium specimens without perfect flowers (e.g.,
Utley, 1978; Weber & Smith, 1983; Utley & Luther, 1991; Grant, 1993a).
133
BARFUSS, M.H.J.
NEW CLASSIFICATION
Revisions of floral morphological characters used and neglected in Smith & Down's monographs pointed out the potential and limits of these features to classify Bromeliaceae species
(petal appendages: Brown & Terry, 1992; stigmas and papillae: Brown & Gilmartin, 1984,
1989b; Schill & al., 1988; Gortan, 1991; septal nectaries: Böhme, 1988; pollen: Halbritter, 1988,
1992; and seeds: Gross, 1988). No taxonomic change was proposed in any of these studies,
because species sampling was too limited to generally apply recognized characteristics to
higher taxonomic units. Resurrections and segregations of subgenera and closely related species complexes at generic status were done later based on the consideration of these additional and reevaluated morphological characters (Alcantarea: Grant, 1995a; Racinaea: Spencer &
Smith, 1993; Werauhia: Grant, 1995a; and Viridantha: Espejo-Serna, 2002). Some of these new
genera are not always accepted, because remaining species of Tillandsia and Vriesea are left in
their old generic circumscriptions without alteration of their morphological definitions. Even
more surprising and hardly acceptable for some bromeliad specialists are rearrangements of
former xerophytic, grey-leaved Vriesea species done by Grant (1993b, 1994b, 1995b, 2005),
since the new placement clearly contradicts the definition of the genus Tillandsia established
by earlier botanists, although for example Mez (1934–35) did not always follow strictly this
characterization when new morphological evidence arose.
The controversies about the correct generic placement of certain species have led to the
situation that modern bromeliad taxonomists follow different opinions regarding a generally
accepted classification scheme (Grant & Zijlstra, 1998; Smith & Till, 1998; Till, 2000a, b; Barfuss
& al., 2005; Luther & Sieff, 1994, 1997; Luther, 2001, 2008, 2010; Luther & Rabinowitz, 2010),
since some of these modifications were based on morphological dissimilarity and not on a
shared character or character combinations (see Tables 4 and 5 for a comparison of traditional
and most recent classification systems). The most important morphological character at the
generic level within Tillandsioideae (and Bromeliaceae in general) is the presence vs. absence
of petal appendages (Smith & Downs, 1974, 1977, 1979; Brown & Terry, 1992), which delimits
the genus pairs Tillandsia/Vriesea and Guzmania/Mezobromelia. The taxonomic value of this
character has been questioned (e.g., Read, 1968; Utley, 1978; Gardner, 1982; Gilmartin, 1983;
Beaman, 1989) although all bromeliad monographers used it in their taxonomic treatments
(Wittmack, 1888; Baker, 1889; Mez, 1896; Harms, 1930; Mez, 1934–35; Smith & Downs, 1977).
Field botanists discovered that within populations of a single species, individuals with and
without petal appendages can coexist (Read, 1968; W. Till, pers. com.), which would mean,
following the strict generic concept of Smith and Downs (1977), these individuals should be
classified in different genera. Despite these problems with using petal appendages as a strict
and definite character, Benzing & al. (2000) countered that "...the frequently disparaged petal
appendage would regain some lost currency as one of the more useful among the traditional
characters... [if] occasional exceptions" are accepted.
Defining infrageneric units morphologically (e.g., within Tillandsia: Till, 2000b) is an analogous difficulty. Only a few classical taxonomic studies have treated whole subgenera or species
complexes of Tillandsia: T. subg. Anoplophytum (Tardivo, 2002), T. subg. Diaphoranthema (Till,
1984, 1992), T. subg. Phytarrhiza (Gilmartin, 1983; Gilmartin & Brown, 1986), T. subg. Pseudalcantarea (Beaman, 1989; Beaman & Judd, 1996), T. subg. Tillandsia (Gardner, 1982, 1986a, b;
including some taxa from T. subg. Allardtia sensu Smith & Downs, 1977), the Tillandsia gardneri complex (Ehlers, 1997), Tillandsia macdougallii complex (Granados Mendoza, 2008), the
Tillandsia plumosa complex (= Viridantha) (Espejo-Serna, 2002; Ehlers, 2009), and the Tillandsia tectorum complex (Hromadnik, 2005). Tillandsia subg. Allardtia was lastly only treated
134
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
within the "Tillandsia and Racinaea" chapter by Till (2000b). The separation of T. subg. Allardtia and T. subg. Anoplophytum is based on the character "straight vs. plicate filaments" and
T. subg. Tillandsia is distinguished from both by its "exserted stamens and style". Till (2000b)
stated that "the distinction between subgenera Allardtia and Anoplophytum is weak, and their
separation may not be justified, at least not as proposed by Smith & Downs" (1977). The existence of closely related species (Till, 2000b) showing either of these subgeneric characteristics
(straight vs. plicate filaments, respectively) and previous ontogenetic studies on filament plication (Evans & Brown, 1989) demonstrate that this distinction is inappropriate. This is reflected
by the opinion of Till (2000b), where he moved the Andean species with plicate filaments from
T. subg. Anoplophytum sensu Smith & Downs (1977) into T. subg. Allardtia. Several species of
other subgenera of Tillandsia, especially of T. subg. Pseudalcantarea and T. subg. Phytarrhiza
sensu Smith & Downs (1977), show similar patterns of morphological convergence or parallelism.
Since traditional generic, subgeneric and sectional treatments are exclusively based on
flower morphology, the most plausible explanation for their evolution is that pollination syndromes can adapt relatively fast to new environmental situations, leading to floral characteristics which could have evolved in parallel or convergently in different phylogenetic lineages.
This might be caused by a high capability of bromeliads in general to adapt easily to different
ecological niches and modified environmental conditions (e.g., Gardner, 1986a; Benzing, 2000;
Kessler, 2002; Schmidt-Lebuhn & al., 2007; Krömer & al., 2008). Despite known problematic
issues of previously-used floral morphological characters, their usage still persists, since no
other diagnostic characters or character combinations have yet been identified or are sufficiently known to unambiguously circumscribe natural taxonomic units.
Previous molecular phylogenetic studies
Molecular phylogenetic studies which included members of Tillandsioideae revealed a holophyletic (= monophyletic s.str.; see Hörandl & Stuessy, 2010) subfam. Tillandsioideae (e.g.,
Terry & Brown, 1996, Terry & al., 1997a, b; Horres & al. 2000; Crayn & al., 2004; Givnish & al.,
2004; Givnish & al., 2007; Givnish & al., 2011). Also, the early diverging clades comprising
Catopsis and Glomeropitcairnia were well supported in most studies. However, resolution in
core Tillandsioideae genera (Alcantarea, Guzmania, Mezobromelia, Racinaea, Tillandsia, Viridantha, Vriesea, and Werauhia; = Tillandsioideae s.str., Terry & al., 1997b) was greatly lacking,
because phylogenetic studies only relied on a single or few plastid DNA regions. First reliable
insights into relationships of core Tillandsioideae were brought by the seven marker analyses
of Barfuss & al. (2005). The main results of this study were the split of core Tillandsioideae into
two main lineages and the para- and/or polyphyly of the largest tillandsioid genera Tillandsia
and Vriesea based on the information content of these plastid DNA markers. All other lastly
segregated genera were found to be holophyletic, but they were clearly nested either within
Vriesea (Alcantarea, Werauhia) or within Tillandsia (Racinaea, Viridantha) according to their
traditional generic circumscriptions. Taxonomic changes proposed since the monograph of
Smith & Downs's (1977) were only partly supported and clearly showed, that morphology
based taxonomy is in conflict with molecular results. However, the taxon sampling was too
limited for making a conclusive decision regarding holophyly/paraphyly of these genera. The
study also showed that none of the previously applied infrageneric concepts of Tillandsia,
Vriesea, and Werauhia using traditional morphological characters were fully supported by
plastid DNA sequence data indicating convergent or parallel evolution of certain features. A
135
BARFUSS, M.H.J.
NEW CLASSIFICATION
preliminary report of Till & Barfuss (2006) including results of one single-copy nuclear gene
(Phosphoribulokinase = PRK; not explicitly stated in the text but used to create Figures 1 and 2
of Till & Barfuss, 2006; 254–255) further corroborates these findings. However, the nested
position within Tillandsia of meso- and semi-mesophytic taxa of T. subg. Phytarrhiza and Racinaea is no longer supported whereas Viridantha still remains nested within Tillandsia. Nevertheless, the shortcomings of all previous DNA studies were that they suffered from an extremely low number of parsimony-informative nucleotide characters (PICs), a serious undersampling of taxa in relation to the actually accepted species and the dependence on few analyzed plastid DNA regions.
Despite the split of Tillandsioideae into 4 main lineages, which were considered as separate tribes (Pogospermeae Brongn. (1864) = Catopsideae Harms (1930), Glomeropitcairnieae
Harms, Tillandsieae Rchb., Vrieseeae W. Till & Barfuss) in Barfuss & al. (2005) and the transfer
of W. insignis (Mez) W. Till, Barfuss & Samuel in Barfuss & al. (2004) no further taxonomic conclusions were drawn in any previous phylogenetic study, leaving the most interesting questions unanswered: what are the generic and infrageneric units within Tillandsioideae and how
can these be defined genetically and morphologically?
Aiming at answering these question we conducted phylogenetic analyses of previouslyused plastid and newly-generated nuclear DNA sequences, i.e., (1) plastid atpB-rbcL, matK,
rbcL, partial rbcL-accD, rps16 intron, partial trnK intron, trnL intron, and trnL-trnF (taken from
Barfuss & al., 2005) and (2) nuclear PHYC (phytochrome C) and PRK (phosphoribulokinase)
together with a re-evaluation of potentially useful morphological characters of the habit and
various floral elements.
Materials and Methods
Taxon selection
Plant material was primarily selected based on results of Barfuss & al. (2005) and the taxonomic treatment of Smith & Downs (1977). Ninety-seven accessions out of 122 from the first study
together with 347 new accessions were included in the present investigation, making a total of
444 individuals of tillandsioid species. A complete list of all plant material examined including
nomenclatoric authors can be found in the supplementary data (Appendix). Sampling within
recognized genera, subgenera and sections was significantly increased: Alcantarea (23 acc.),
Catopsis (10 acc.); Glomeropitcairnia (3 acc.), Guzmania (52 acc.), Mezobromelia (7 acc.), Racinaea (23 acc.), Vriesea sect. Vriesea (29 acc.), V. sect. Xiphion p.p., typo excluso (23 acc.),
Werauhia sect. W. (9 acc.), and W. sect. Jutleya (7 acc.); the main focus has been within the
species-richest genus Tillandsia (258 acc.): T. subg. Allardtia (100 acc.), T. subg. Anoplophytum
(44 acc.), T. subg. Phytarrhiza (meso- and semi-mesophytic: 31 acc., xerophytic: 10 acc.),
T. subg. Pseudalcantarea (7 acc.), T. subg. Diaphoranthema (12 acc.), and T. subg. Tillandsia
(54 acc.). Members of T. subg. Pseudalcantarea and meso- and semi-mesophytic taxa of
T. subg. Phytarrhiza were nearly completely sampled (missing only T. narthecioides Presl), to
clarify issues of high levels of polyphyly within these subgenera (Gilmartin & Brown, 1986;
Beaman & Judd, 1996; Till, 2000b), and to test for either an early split of species from the rest
of Tillandsia (T. subg. Pseudalcantarea) or affinities to the genus Racinaea (meso- and semimesophytic T. subg. Phytarrhiza) based on earlier plastid DNA results (Barfuss & al., 2005).
Additional tillandsioid taxa were chosen to include species with a distinct morphology, which
136
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
indicates an inadequate generic, subgeneric or sectional placement by Smith & Downs (1977),
an ancestral position within recognized taxa or probable separate phylogenetic units within
Tillandsioideae, e.g., the large, broad-leaved and water-impounding or three-pinnate species
of T. subg. Tillandsia occurring in north-western South America (e.g., Till, 2000b; León &
Sagástegui, 2008), the former xerophytic, grey-leaved Vriesea species (e.g., Grant, 1993b), or
the Tillandsia tectorum complex (e.g., Hromadnik, 2005) which is associated with the Tillandsia
plumosa complex (= Viridantha) in an earlier study (Barfuss & al., 2005). As an outgroup accessions of Glomeropitcairnia and Catopsis were used, since in all previous investigations these
genera came out as two early diverging lineages, both being sister to each other and together
being sister to the core Tillandsioideae. Relevant type species for genera and infrageneric units
were investigated for nearly all taxa except Racinaea and Cipuropsis, i.e., R. cuspidata
(L.B. Sm.) M.A. Spencer & L.B. Sm. and Ci. subandina Ule (syn. V. subandina (Ule) L.B. Sm. &
Pittendr.), respectively.
Most material came from the botanical garden of the University of Vienna and the private
collection of H. & L. Hromadnik. Additional samples were donated by the botanical gardens of
Berlin-Dahlem, Göttingen, Heidelberg, Kew, München, Linz, and Sarasota and the private collections of W. Adlassnig, R. Ehlers, P. Lechner, E.M.C. Leme, J.M. Manzanares, J.P. Pinzon,
M. Speckmaier and M. Winkler. Some accessions were also collected by the first and/or second
author during scientific fieldtrips and/or student expeditions to Venezuela (2000), the Dominican Republic (2001, 2002), and Ecuador (2003, 2004, 2006). Species nomenclature and infrageneric classification basically follows Smith & Downs (1977) with alterations by Luther & Sieff
(1994, 1997), Luther (2001, 2008, 2010), Luther & Rabinowitz (2010) and Grant (1993b, 1994b,
1995b, 2005). Additional taxa accepted, but classified differently or missing in the taxonomic
literature followed are Guzmania fusispica Mez & Sodiro [≠ G. osyana (E. Morren) Mez], Tillandsia buseri var. nubicola Gilmartin [≠ T. buseri Mez], T. fosteri Gilmartin [≠ T. demissa
L.B. Sm.], T. macropetala Wawra [≠ T. viridiflora (Beer) Baker], T. malzinei (E. Morren) Baker [=
Vriesea malzinei E. Morren], T. recurvifolia var. subsecundifolia (W. Weber & Ehlers) W. Till [≠
T. leonamiana E. Pereira], and the recently described new species Racinaea tillii Manzan. &
Gouda (Manzanares & Gouda, 2010). However, from the results chapter onwards the new
nomenclature is used, which is presented in the chapter "Classification of Tillandsioideae" and
can be visualized in Figure 2 (see Appendix) and Tables 4 and 5.
Gene selection
Data from seven plastid DNA regions were taken from a preceding study (Barfuss & al., 2005).
No additional plastid sequences were generated, because the original sampling already covered most of the accepted taxonomic lineages and preference was given to new nuclear DNA
sequences. Eight nuclear gene regions were initially screened for a subset of Tillandsioideae
accessions used in Barfuss & al. (2005) to select appropriate genomic regions for sequencing
the more exhaustive sample set. DNA regions surveyed included the multi-copy internal transcribed spacer of the nuclear ribosomal DNA (ITS nrDNA; e.g., Chew & al., 2010), and the lowcopy nuclear genes glucose-6-phosphate isomerase, cytosolic (PGIC; e.g., Ford & al., 2006),
malate synthase (MS; e.g., Lewis & Doyle, 2001, 2002), nitrate reductase (NIA: e.g., Howarth &
Baum, 2002), phosphoribulokinase (PRK; e.g., Lewis & Doyle, 2002; Thomas & al., 2006), phytochrome C (PHYC; e.g., Mathews & Donoghue, 1999; Samuel & al., 2005), RPB2 (RNA polymerase II, beta subunit; e.g., Denton & al., 1998; Oxelman & al., 2004), and xanthine dehydrogenase (XDH; e.g., Górniak & al., 2010). Parameters for the selection of effective DNA regions
137
BARFUSS, M.H.J.
NEW CLASSIFICATION
were (1) the number of loci obtained, (2) the performance of primers and PCR conditions for
both amplification and sequencing, (3) the occurrence of homopolymers and microsatellites,
which are often hard to sequence, (4) the length of the PCR products and (5) the number of
potentially phylogenetically informative characters. The primary target was to obtain one or
more genomic regions which are either well homogenized (ITS nrDNA) or effectively singlecopy (other nuclear regions) to be mostly sequenced directly. PHYC and PRK were finally selected for phylogenetic analyses (see "Results" for the reasons and chapter 3). All nuclear sequences where generated newly for this study and have been archived in NCBI's GenBank
(http://www.ncbi.nlm.nih.gov/genbank).
Molecular data and analyses
Total DNA extractions were done from fresh or silicagel-dried material mainly following a sorbitol/CTAB-based method for difficult plant tissue (Tel-Zur & al., 1999) with modifications according to Russell & al. (2010a). In some cases the 2x CTAB procedure described by Doyle &
Doyle (1987) adopted for mini columns, the DNeasy® Plant Mini Kit (QIAGEN®) following the
manufacturers protocol, or a combination of both were used; details for the latter can be requested from the first author. Initial primer sequences and PCR conditions for PRK and PHYC
were described by Schulte & al. (2009) and Russell & al. (2010b), respectively. Details for general PCR and sequencing reactions, occasional cloning and other laboratory materials and
methods followed chapter 3 of this thesis. Both the PHYC and PRK genes were amplified and
sequenced using sometimes slightly modified or additional primers. Details of all primers used
are given in Tables 1 (PHYC) and 2 (PRK) (see also chapter 3 for more primer details).
Raw sequences were initially analysed and edited using the Sequencing Analysis Software
v5.3 (Applied Biosystems®, Life Technologies™ Corporation). A contig of forward and reverse
DNA strands from both PCR and/or internal sequencing primers was generated with the SeqMan Pro module of the Lasergene® v8.1 software package (DNASTAR, Inc.). Sequences were
edited and a consensus was exported as text file in fasta format. Usually fasta files were
pooled together and aligned initially with MUSCLE v3.8 (Edgar, 2004a, b) and then adjusted by
eye with the BioEdit v7.0.5 (Hall, 1999) following the guidelines of Kelchner (2000) and Borsch
& Quandt (2009). In very polymorphic DNA regions containing numerous indels related taxa
were aligned first. Relationships were pre-estimated from previous results or preliminary phylogenetic analyses. Only then were individual alignments aligned together using the profile
alignment function of MUSCLE and adjusted by eye using BioEdit. Aligned matrices can be obtained from the first author upon request.
When allelic length variation between priming sites was observed, editing of sequences
coming from both directions was not straightforward and done in such a way that the missing
DNA sequence parts of one allele (largely small inserted repeats or deletions) were ignored
and allelic consensus sequences were used further. This was accomplished by identifying the
type of indel and extracting the longer allele from shifted electropherograms, which are a result of non-homologous parts of the two different alleles to be overlaid. If the number of indels
was too high with two or more indels between priming sites in both alleles, editing of sequences became difficult and doubtful, so that in such cases PCR products were reinvestigated
with a cloning step included. Edited indel containing consensus sequences were only included
for analyses, when prior evaluation steps on polymorphic sites (see below) indicated that allelic indels are also most likely due to variation within a single species or within a closely related
species complex.
138
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Table 1. Primers used for PHYC with their location given according to base positions in exon 1 of the PHYC reference
sequence of Oryza sativa (AF141942).
name
sequence (5'–3')
position (direction)
comments
reference
phyc515f-br
AAGCCCTTYTACGCTATCCTGCACCG
490–515 (up)
Bromeliad-specific PCR primer
This study
phyc524f-br
GCTATCCTGCACCGGATCGAYGT
502–524 (up)
Bromeliad-specific internal sequencing primer
This study
phyc974f-br
GCTCCTCACGGCTGCCACGCTCA
952–974 (up)
Bromeliad-specific internal sequencing primer
This study
phyc1145r2-mo
CAACAGGAACTCACAAGCATATC
1,167–1,145 (down)
monocot-specific internal sequencing primer
This study
phyc1690r-br
TCAACATCTTCCCAYGGGAGGCT
1,712–1,690 (down)
Bromeliad-specific internal sequencing primer
This study
phyc1699r-br
ATWGCATCCATTTCAACATCTTCCCA
1,724–1,699 (down)
Bromeliad-specific PCR primer
This study
Table 2. Primers used for PRK with their location given according to base positions in exon regions of the PRK reference sequence of Oryza sativa (NM_001054360); exon 1 (1–545), exon 2 (546–630), exon 3 (631–715), exon 4 (716–
960), exon 5 (961–1,212).Bold italicized numbers denote positions where primers are located partly within intron
parts of PRK.
name
sequence (5’–3’)
position (direction)
comment
reference
prk621f
TCAGCAATGAGGTTAAATTTGCATGG
exon 2, 596–621 (up)
Bromeliad-specific PCR primer
This study
prk630f
AAATTTGCATGGAAAATTCAGGTC
exon 2, intron 2, 610–633 (up)
Bromeliad-specific internal sequencing primer
This study
prk734f
CTGCAGATCCGCAGAAGAAATATGC
intron 3, exon 4, 710–735 (up)
Bromeliad-specific internal sequencing primer
Schulte & al., 2009
prk890r
GGGTATGAGCATGTCAATTTCCTCC
exon 4, 914–890 (down)
Bromeliad-specific internal sequencing primer
This study
prk1057r
CTTCAGCATTTGTTGTGTCACCTC
exon 5, 1,080–1,057 (down)
Bromeliad-specific internal sequencing primer
This study
prk1069r2
GGAAAATCTGCRTGCTTCAGCATTTG
exon 5, 1,094–1,069 (down)
Bromeliad-specific PCR primer
This study
Table 3. Attributes of analyzed matrices of the five datasets D1–5 and parsimony scores of equally most parsimonious trees after analysis using PAUP*. The sequence length is given for individual markers only. bp = base pairs, var.
char. = variable characters, PIC = parsimony informative characters, CI = consistency index, RI = retention index.
matrix
lenght [bp]
plastid DNA (D1)
no. of char.
no. of var. char.
no. of PICs
no. of trees
tree length
CI
RI
6,115
744 (12%)
378 (6%)
>100,000
1,210
0.685
0.831
PHYC (D2)
1,159–1,192
1,228
485 (39%)
369 (30%)
>100,000
1,398
0.473
0.867
PRK (D3)
831–1,692
3,291
737 (22%)
534 (16%)
>100,000
2,232
0.478
0.870
PHYC + PRK (D4)
4,519
1,222 (27%)
903 (20%)
>100,000
3,775
0.458
0.859
PHYC + PRK + plastid DNA (D5)
10 634
1,966 (18%)
1,297 (12%)
>100,000
5,043
0.507
0.853
139
BARFUSS, M.H.J.
NEW CLASSIFICATION
Table 4. Accepted tribal, subtribal and generic concepts and informal clades of Tillandsioideae (Bromeliaceae) in
comparison to earlier classification systems (based on the placement of the nomenclatoric types). i.s. = incertae
sedis.
Smith & Till (1998)
Barfuss & al. (2005)
genus
genus
genus
tribe
genus/subtribe/clade
tribe/clade
Glomeropitcairnia
Glomeropitcairnia
Glomeropitcairnia
Glomeropitcairnieae
Glomeropitcairnia
Glomeropitcairnieae
Catopsis
Catopsis
Catopsis
Catopsideae
Catopsis
Pogospermeae
Alcantarea
Alcantarea
Alcantarea
Vriesea
Vriesea
Vriesea
Werauhia
Werauhia
Werauhia
Vriesea
Chrysostachys clade
Vriesea tuerckheimii (i.s.)
Mezobromelia
Mezobromelia
Mezobromelia
Mezobromelia
Vrieseeae
Core Tillandsioideae
Splendens clade
Vriesea
Cipuropsidinae
Cipuropsis
Cipuropsis-Mezobromelia clade
Vrieseeae
Vriesea
Barfuss, Till & Samuel
Vrieseinae
Smith & Downs (1977)
Singularis clade
Josemania
Tillandsia
Tillandsieae
Tillandsia
Rothowia
Tillandsia
Viridantha
Guzmania
140
Racinaea
Racinaea
Racinaea
Guzmania
Guzmania
Guzmania
Core Tillandsieae
Lemeltonia
Tillandsia
Tillandsieae
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Table 5. Accepted subgeneric concept, informal clades, and unclassified species of Tillandsia (Bromeliaceae) and
recent generic segregates in comparison to earlier classification systems (based on the placement of the nomenclatoric type). A dashed line indicates further informal subdivision of a given taxon. subg. = subgenus, p.p. = pro parte,
i.s. = incertae sedis.
Smith & Downs (1977)
Till (2000)
Barfuss & al. (2005)
Barfuss, Till & Samuel
Tillandsia subg. Pseudalcantarea
Tillandsia subg. Pseudalcantarea
Tillandsia subg. Pseudalcantarea
Tillandsia subg. Pseudalcantarea
Vriesea p.p., typo excluso
(xerophytic, grey-leaved)
Tillandsia subg. Pseudovriesea
Tillandsia subg. Tillandsia
Rauhii clade
Tillandsia subg. Tillandsia
Tillandsia subg. Tillandsia
Tillandsia subg. Tillandsia
(syn. Tillandsia subg. Allardtia)
Viridantha
Tillandsia subg. Viridantha
Biflora clade
Tillandsia australis (i.s.)
Tillandsia subg. Allardtia
Tillandsia edithae (i.s.)
Tillandsia subg. Allardtia
Tillandsia subg. Allardtia p.p. majore, typo excluso
Tillandsia disticha (i.s.)
Tillandsia pseudomicans (i.s.)
Tillandsia sphaerocephala (i.s.)
Xiphioides clade
Tillandsia esseriana (i.s.)
Tillandsia subg. Anoplophytum
Tillandsia subg. Anoplophytum
Tillandsia subg. Anoplophytum
Gardneri clade
Tillandsia subg. Anoplophytum
Tillandsia albertiana (i.s.)
Tillandsia nana (i.s.)
Tillandsia subg. Diaphoranthema
Tillandsia subg. Diaphoranthema
Tillandsia subg. Diaphoranthema
Tillandsia subg. Phytarrhiza
(xerophytic)
Tillandsia subg. Phytarrhiza
(xerophytic)
Tillandsia subg. Diaphoranthema
Tillandsia subg. Phytarrhiza
Purpurea clade
Josemania
Tillandsia subg. Phytarrhiza
Tillandsia subg. Phytarrhiza
(meso-/semi-mesophytic)
Tillandsia subg. Phytarrhiza
(meso-/semi-mesophytic)
Lemeltonia
Rothowia
Racinaea subg. Pseudophytarrhiza
Tillandsia subg. Pseudo-Catopsis
Racinaea
Racinaea
Racinaea subg. Racinaea
141
BARFUSS, M.H.J.
142
NEW CLASSIFICATION
Figure 1. Simplified strict consensus trees of equally most-parsimonious trees found in parsimony analyses of individual datasets (D1–D5) supplemented with BP from parsimony
bootstrap analysis either above or below branches; triangles sitting in terminal position on a branch indicate holophyletic units, whereas triangles emerging from within a node
indicate several branches of that unit on a polytomy. Only relationships of all except two recognized genera and one informal clade are displayed. Cipuropsis and Mezobromelia are
included in the Cipuropsis-Mezobromelia clade including informal clades. D1 = plastid DNA, D2 = PHYC, D3 = PRK, D4 = PHYC + PRK, D5 = plastid DNA ( + PHYC + PRK.
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Nuclear alignments were evaluated prior to phylogenetic analyses for the impact of polymorphic sites (Single Nucleotide Polymorphism = SNP). The need for these additional steps before
phylogenetic analyses has been pointed out by Jabaily & Sytsma (2010) and Russell & al.
(2010b) in detail. The occurrence of SNPs in resulting electropherograms of nuclear allelic consensus sequences was inferred as allelic variation within the given sample. In the final alignments these positions were carefully evaluated to see if their patterns suggest the presence of
similar alleles in more distantly related taxa indicating polyploidization, hybridization, reticulation, introgression events or the occurrence of ancient alleles (incomplete lineage sorting).
The aligned plastid DNA matrix with some taxa deleted (because of missing nuclear DNA
sequence information) was taken from Barfuss & al. (2005). A 25 bp region from atpB-rbcL (a
partly inverted composed poly-A/poly-T region) where homology could not be unambiguously
assessed, was excluded here from the analyses (positions 4,321–4,345 of the plastid DNA dataset and positions 8,840–8,864 of the combined dataset, respectively). Five datasets were
analyzed, i.e., (D1) plastid DNA, (D2) PHYC, (D3) PRK, (D4) PHYC + PRK, and (D5) PHYC + PRK +
plastid DNA, and compared with results from Barfuss & al. (2005). In four cases sequences of
the plastid DNA dataset had to be combined with nuclear sequences from a different source of
the same species (G. variegata L.B. Sm., T. barclayana Baker, T. lindenii Regel, T. stricta Sol. ex
Sims), since the original sample of Barfuss & al. (2005) was not available anymore. These species could not be excluded because they represent genetic entities that would otherwise be
either underrepresented or non-existent in the combined dataset. Alleles resulting from
cloned nuclear sequences were combined with duplicated plastid DNA sequences only when
their position in individual nuclear trees did not significantly contradict the plastid DNA tree
topologies.
Analyses of the aligned and evaluated individual and combined datasets included maximum parsimony (MP) and Bayesian metropolis-coupled MCMC inference (BI). MP analyses
were implemented in the program PAUP* 4.0b10 (Swofford, 2003) using a two-step heuristic
search strategy due to the complexity of DNA sequence data. The first step of heuristic searches was done with 1,000 random sequence additions, TBR branch swapping and holding 10
trees each step to reduce time spent swapping on non-optimal trees, and saving always 100
shortest trees per replicate, even if the trees were not the shortest over all replicates (to check
for multiple island in the resulting tree space). In the second round trees obtained from the
first analyses were taken as starting trees and heuristic searches were conducted until all or a
maximum of 100,000 shortest trees were saved, but the tree search was allowed to continue
to swap on all input trees for shorter ones (swapping to completion). Gaps were always treated as missing characters and no gap coding procedure was applied, mainly because gaps resulting from allelic length variation in the very indel-rich PRK dataset (D3) were ignored.
BI analyses were done with the program MrBayes 3.1 (Huelsenbeck & Ronquist, 2001;
Ronquist & Huelsenbeck, 2003) using the MPI version (Altekar & al., 2004) at the University of
Oslo Bioportal (http://www.bioportal.uio.no/). Best-fitting nucleotide substitution models
were determined in advance using MrModeltest 2.3 (Nylander, 2004) following the Akaike
information criterion. Although the best model for each dataset was determined as GTR + I +
G, analyses of the two combined datasets D4 and D5 were still partitioned (PHYC, PRK, plastid
DNA) to allow different parameters for each individual region to be estimated separately. Four
independent runs of four MCMC chains were done for ten million generations, sampling every
1,000 generations and with a burn-in of 8 to 25 %, which was evaluated for each run individually. Other parameters and priors were left as default.
143
Genus/clade
Habit
Petals
Petal
appendages
Filaments
Petalfilament
Anthers
Aperture type
Glomeropitcairnia
m
f
p
f
f
f
diffuse
Catopsis
m
f
a
f
f
f
Catopsis type
Guzmania
m
c
a / (p)
f
c / (f)
f / (c)
diffuse / inaperturate
Josemania
m / sm
f
a
f
f
f
insulae
Lemeltonia
sm / (m)
f
a
c
f
f
insulae
Ovary
position
½–⅔
inferior
superior–
⅛ inferior
max. ⅓
inferior
max. ⅓
inferior
max. ⅓
inferior
Ovule
appendage
(chalazal)
Setal
nectary
ducts
Stigma
type
Pseudopappus
Endostome
type
Embryo
type
p: ap > ov
?
cb
micropylar
?
?
pp
ascending
se
chalazal
d
g
a
horizontal
se / cb
micropylar
a / b / (e)
a / (b)
a
?
pi
micropylar
?
?
a
horizontal
cf
micropylar
d
b
micropylar
c / d / f (?)
b
m
f
a
f
f
f
insulae to diffuse
max. ⅓
inferior
a
horizontal
Rothowia
m
f
a
f
f
f
insulae to diffuse
max. ⅓
inferior
a
?
cb
micropylar
e
b / (–a)
max. ⅓
inferior
p: ap ≤ ov or
(ap > ov) / a
horizontal
se/ cs / (cb)
/ (cp)
micropylar
e/f/g/h/i
/k/l/m/n
b/c/d/e/
f / (–g)
a
?
se
micropylar
c
a
a
?
se
micropylar
?
?
a
descending
cup
micropylar
d
a / (b)
a
?
se
micropylar
?
?
a
?
se
micropylar
?
?
a
?
cs
micropylar
c/d
a
p: ap ≤ ov
?
cupp
micropylar
?
?
p: ap > ov
?
cp / (ce)
micropylar
?
?
p: ap ≤ ov /
(a)
descending
cb / tl
micropylar
e / (–c)
b / (a)
Tillandsia
m / (sm) /
(sx) / x
f
a / (p)
f
f
f
operculum /
diffuse, insulae,
Alcantarea type
Cipuropsis
m
c (ca. ¼)
p
f
c (ca. ¼)
f
diffuse to insulae
Mezobromelia
m
c (ca. ½)
p
f
c (ca. ½)
c
diffuse / inaperturate
Werauhia
m
f
p / (a)
f
f
f
insulae
Chrysostachys
clade
m
c (ca. ¼)
p
f
c (ca. ¼)
?
?
Singularis clade
m
c (ca. ¼)
a
f
c (ca. ¼)
f
fine insulae
Splendens clade
m
f
p
f
f
f
?
V. tuerckheimii
m
f
p
f
c (ca. ¼)
f
?
Alcantarea
m / (sm)
f
Vriesea
m / (sm) /
(sx)
f / (c)
short
p
p
f
f
f
f / (c) short
f
f
Alcantarea type
insulae
max. ⅓
inferior
max. ⅓
inferior
max. ⅓
inferior
max. ⅓
inferior
max. ⅓
inferior
max. ⅓
inferior
max. ⅓
inferior
max. ⅓
inferior
max. ⅓
inferior
NEW CLASSIFICATION
Racinaea
se /
(slightly cs)
/ (cf)
BARFUSS, M.H.J.
144
Table 6. Morphological characters used to differentiate among genera and informal clades of Tillandsioideae. Taxa are arranged according to the "Classification of Tillandsioideae" chapter. Values for the septal nectary ducts, endostome type, and embryo type were exclusively taken from literature. m = mesophytic, sm = semi-mesophytic,
sx = semi-xerophytic, x = xerophytic; f = free, c = connate/conglutinate; p = present, a = absent; ap = appendage, ov = ovule, pp = pseudopappus; cb = convolute-blade,
ce = conduplicate-erect, cf = coralliform, cp = conduplicate-patent, cs = conduplicate-spiral, cup = cupulate, cupp = cupulate-papillate, se = simple-erect, tl = tubolaciniate,
pi = pinnatisect; ? = missing data; (?) = information questionable in original source; data in () = rarely.
Habit
Petal shape
Petal appendages
Ovule appendage (chalazal)
Stigma type
Endostom type
Embryo type
Tillandsia subg. Anoplophytum
(sx) / x
lingulate
a
a / p: ap ≤ ov
se / cs
m
(e–f) / f
Tillandsia subg. Diaphoranthema
x
lingulate / unguiculate
a
p: ap ≤ ov
se
m/n
e
Tillandsia subg. Phytarrhiza
x
unguiculate
a
p: ap ≤ ov
se
m
e/f
Tillandsia subg. Pseudalcantarea
m
lingulate
a
a
(cs) / cp
f
b
Tillandsia subg. Pseudovriesea
sx / (x)
lingulate
p / (a)
p: ap ≤ ov
cs / ce
f
(b–) / ± f
Tillandsia subg. Tillandsia
m / sm / sx / x
lingulate
a / (p)
p: ap ≤ ov / ap > ov
cs / (se)
e/g/i/k
b / c / f (–g)
Tillandsia subg. Viridantha
x
lingulate
a
a / p: ap ≤ ov
se / cb
h/l
c/f
Biflora clade
m / sm / (sx)
lingulate
a
a / p: ap ≤ ov
cs / (se)
e/g/h
b/f
Gardneri clade
x
lingulate
a
a / (p: ap ≤ ov)
se
m
(e–f) / f
Purpurea clade
x
unguiculate
a
a / (p: ap ≤ ov)
cs
h/l
c–f
Rauhii clade
m
lingulate
a
p: a ≤ o/( ap > ov)
cs / (cp)
h
d
Xiphioides clade
x
unguiculate
a
p: ap ≤ ov
se / (cs)
m
f
145
PART 3, CHAPTER 5
Subgenus/clade
OF TILLANDSIOIDEAE (BROMELIACEAE)
Table 7. Morphological characters used to differentiate among subgenera and informal clades of Tillandsia. Taxa are arranged according to the “Classification of Tillandsioideae” chapter. m = mesophytic, sm = semi-mesophytic, sx = semi-xerophytic, x = xerophytic; p = present, a = absent; ap = appendage, ov = ovule, cb = convolute-blade,
ce = conduplicate-erect, cp = conduplicate-patent, cs = conduplicate-spiral, se = simple-erect; data in () = rarely.
BARFUSS, M.H.J.
NEW CLASSIFICATION
Support was obtained from 50 % Bayesian majority rule consensus trees (Posterior Probabilities, PP) and from parsimony Bootstrap analyses done in PAUP* 4.0b10. Bootstrap percentages
(BPs) were calculated using 1,000 replicates, TBR branch swapping, simple sequence addition,
holding 10 trees each step and saving 100 shortest trees per bootstrap replicate.
Combinability of plastid DNA, PHYC and PRK datasets was determined by visual inspection
of individual bootstrap consensus trees (Whitten & al., 2000). Measures of incongruence like
the incongruence length difference test (ILD; Farris & al, 1994, 1995; Lee, 2001) have been
demonstrated not always to be useful indicators of data combinability, specifically in the light
of relatively recently divergent plant groups with a lower sequence divergence in plastid DNA
compared to nuclear DNA (Yoder & al., 2001; Reeves & al., 2001). We use the following descriptions for categories of BP: unsupported, <50%; weak, 50–79%; moderate, 80–89%; strong,
90–100%. In the case of only weakly- to moderately-supported incongruent topologies between the individual trees, direct combination was regarded as appropriate. The artifact of
long-branch attraction (LBA) can sometimes yield high moderate or strong BP for a phylogenetically wrong clade, therefore the number of autapomorphies for and the taxon sampling
within the specific clades are also considered (Bergsten, 2005).
Morphological data and analyses
Morphological characters were collected and documented over several years from herbarium
sheets, liquid flower preservations, fresh material cultivated in the botanical garden of Vienna
or from several donors (see "Taxon selection") using a stereo microscope (Olympus SZ60 Stereo Zoom Microscope) or SEM technology (either with a JEOL T-300 or JEOL JSM-6390 Scanning Electron Microscope). Characters of some taxa were extracted and carefully evaluated
from the original description of the respective taxon, the last monograph (Smith & Downs,
1977) or selected literature (e.g., Brown & Gilmartin 1984, 1989b; Böhme, 1988; Groß, 1988;
Brown & Terry, 1992; Halbritter, 1992). Except for the general habit, the gathered morphological information mainly comprised characters of diverse floral elements like sepals, petals (incl.
petal appendages), stamens (incl. filaments, anthers and pollen), carpels (incl. stigma and ovules) as well as ovary position and seed morphology (Tables 6, 7). Characters were manually
mapped on resulting phylogenetic trees and empirically useful morphological traits taken for
the circumscription of obtained clades. No phylogenetic analysis of a morphological data matrix was performed, because of missing data in many of accessions studied. Although several
species could not be investigated for all used morphological traits, it was assumed that characteristics can be applied to higher taxonomic ranks.
Results
Table 3 provides the attributes of the analyzed matrices, together with the details of the equally-most-parsimonious trees found in parsimony analyses of each of the five datasets. Tables 6
and 7 summarize morphological traits found useful so far and used to differentiate between
genera, subgenera and informal clades. Simplified strict consensus trees of shortest trees from
parsimony analyses supplemented with BP are shown in Figure 1, D1–5 for comparing main
differences in tree topologies between each dataset. One most parsimonious phylogram (Figure 2, see Appendix) from total combined analysis that reflects our understanding on phylogenetic relationships best was chosen and supplemented with BP and PP. Alternative topologies
of terminal branches and backbone relationships from Bayesian analysis are displayed in boxes
146
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
mostly in front of the corresponding clades (terminals) or on the upper-left side (backbones) of
Figure 2. Their positions within the parsimony tree are highlighted by capital letters (Figure 2,
A–P: terminals, Q–S: backbones). Other trees from different analyses (parsimony and Bayesian) of each individual dataset are not shown and can be requested from the first author. Only
relationships of genera accepted in the present investigation (except Cipuropsis and Mezobromelia) and one informal infrasubtribal clade (Cipuropsis-Mezobromelia clade) are shown and
their results presented for each individual dataset (Figure 1, D1–5). Results including all tribal,
subtribal, generic, and infrageneric units and informal infrageneric clades are displayed for the
total combined dataset only (Figure 2). The nomenclature, taxonomy and morphological characterization of clades, and the taxonomic position of each species studied can be obtained
from the chapter "Classification of Tillandsioideae", Figure 2 or Tables 4 and 5, respectively.
Main geographic distributions are given in the key to the genera.
Gene selection
From eight genomic regions screened initially, only PRK and PHYC were selected because (1)
they were easy to amplify and sequence in most cases, (2) had the most variation in relation to
their average length, and (3), most importantly, amplifications resulted in single, clear bands.
This indicates an effectively single-copy nature of both nuclear regions and therefore allows
PCR products to be sequenced directly without cloning, at least in individuals without allelic
indel variation or hybrid origin. This is in agreement with previous studies of these genes in
other monocot genera like Puya Molina (Bromeliaceae: Jabaily & Sytsma, 2010), Oryza sativa L.
(Poaceae: Chen & al., 2004), or Chamaedorea Willd. (Arecaceae: Thomas & al., 2006). Reports
of possible multiple loci (paralogs) of PRK in subfamily Bromelioideae (Schulte & al., 2009)
cannot be confirmed for Tillandsioideae and are possibly due to a gene duplication event within Bromelioideae (D. Silvestro & K. Schulte, unpubl. data). Within PRK introns few polynucleotide runs (SSRs) were observed and could not be avoided (see supplementary data): (1) in intron four a more frequent SSR was detected in most tillandsioid accessions (except for taxa
were the SSR containing region has been lost), (2) in intron two few Guzmania species exhibit a
large insertion containing a second SSR, and (3) in intron three of G. multiflora (André) André
ex Mez, which also exhibits a larger insertion, a third SSR was observed. The other regions
were discarded either because they made problems in amplifications and sequencing (ITS
nrDNA), primer combinations only amplified a comparatively short DNA fragment (RPB2, MS),
or sequence variation and taxon sampling was to low (NIA, PGIC, XDH).
Nuclear data evaluation
Details of allelic length variation (heterozygous indels) and polymorphic sites (SNPs) are given
separately for each nuclear marker (see below). Evaluation of both PHYC and PRK data revealed that none of the investigated samples showed evidence for polyploidization, hybridization, reticulation, introgression or incomplete lineage sorting involving more distantly related
species. Although polyploidy (either auto- or allopolyploidy) can have an major impact on the
allele distribution and frequency within an/all affected species, it can be rejected for most
species analyzed here since in most cases allelic variation can be traced back to two alleles (see
supplementary data). This is also in agreement with previous findings that most Tillandsioideae
species (and Bromeliaceae species in general) show a diploid chromosome number of 2n = 2x =
50 (e.g., Brown & Gilmartin, 1989a; Palma-Silva & al., 2004). In two species within T. subg.
Diaphoranthema (T. capillaris Ruiz & Pav., T. virescens Ruiz & Pav.), allelic variation of SNPs
147
BARFUSS, M.H.J.
NEW CLASSIFICATION
supports already documented cases of polyploidy with 2n = 4x = 100 (Till, 1992), either autopolyploidization or allopolyploidization involving closely related species. However, these were
ignored since they have no impact on the current results (incl. branching patterns and statistical support) and are insignificant for the current study. A possible case of a hybrid origin involving sister taxa was found in the sample determined as Ro. platyrhachis. One allele of this
accession is strongly associated with Ro. wagneriana. Sequence variation between or within
other closely related species in terminal branches was too low to either reject or confirm any
of the above mentioned hypotheses. Therefore, these were ignored, since the scope of the
present study is to resolve deeper nodes primarily and not individual relationships of recently
diverged species. Otherwise it would also be necessary to question and investigate the species
concept used involving a very broad sampling and other molecular markers. However, polytomies and less resolved or supported clades at any hierarchical level in resulting trees were
primarily caused by a lack of phylogenetic signal rather than conflicting polymorphic sites.
Plastid DNA data and analyses (dataset D1)
Barfuss & al. (2005) already explored attributes and detailed results from the plastid DNA data
analyses. A summary of generic relationships based on a strict consensus tree of a maximum
parsimony analysis supplied with BP is given in Figure 1, D1. Differences in the attributes between the first plastid DNA study and the present investigation (Table 3) are due to the deletion of several taxa from the plastid DNA matrix, where no nuclear DNA data was available,
and the exclusion of a 25 bp region from atpB-rbcL. Deleting these taxa and base pairs had no
significant effect on the topology of obtained trees, identical to those of Barfuss & al. (2005).
PHYC data and analyses (dataset D2)
Characteristics of the analyzed PHYC matrix are given in Table 3. PCR primers amplified fragments of partial exon one ranging from 1,159 (Werauhia insignis) to 1,192 bp (Guzmania acorifolia (Griseb.) Mez, G. condensata Mez & Wercklé), but mostly a fragment of 1,177
bp. According to the tree topology, the latter size can be inferred as the ancestral state. Indel
events are rare and derived, and present either in single species or few closely related species
in terminal branches. Indication of allelic length variation within individuals caused by heterozygous indels was not found in any of the sampled taxa, since in PHYC only conserved parts of
the exon 1 were amplified. Allelic nucleotide variation as indicated by obvious double peaks at
a few positions was found in 220 taxa out of 444 sampled individuals. 224 samples were found
to be homozygous for the region sequenced. The number of SNPs in a given heterozygous taxon ranged from 1–15 (the latter seen in T. aff. cucaensis Wittm. B0735). Distribution of polymorphic sites was evenly across the whole range of PHYC with no particular preference for a
specific gene region, but significantly higher at the 3rd codon position caused mostly by synonymous substitutions. Evaluation of the effects of SNPs in PHYC data indicate that there is not a
strong phylogenetic bias in not having allelic data and allelic consensus sequences can be used.
We therefore only used the polymorphism-coded PHYC dataset for all analyses, with the exception of three taxa, which were cloned for PRK and therefore also for PHYC, to be able to
combine corresponding allelic sequences.
Figure 1, D2 is the strict consensus tree of a MP analysis with BP, which summarizes the
generic relationships based on PHYC sequence data. As in plastid DNA tree, both Glomeropitcairnia (tribe Glomeropitcairnieae) and Catopsis (tribe Pogospermeae) are strongly supported
(Bootstrap percentage = BP 100) and sisters (BP 100), with the latter clade sister to a strongly148
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
supported core Tillandsioideae (BP 100). Tribe Vrieseeae is paraphyletic in PHYC results, but
the resulting grade of subtribes Vrieseinae and Cipuropsidinae has only a weak BP (58). Vriesea
is holophyletic (BP 89) only when Andean members located in the Cipuropsis-Mezobromelia
clade, T. malzinei and former xerophytic gray-leaved members, the latter two now placed
within Tillandsia, are excluded. The sister group relationship of Vriesea and Alcantarea (= subtribe Vrieseinae) is strongly supported (BP 100), as is the holophyletic origin of Alcantarea (BP
97). Subtribe Cipuropsidinae is holophyletic (BP 90) and splits into two lineages, Werauhia (BP
100) and the Cipuropsis-Mezobromelia clade (BP 67). Mezobromelia is a weakly-supported
holophyletic lineage (BP 57) nested within the Cipuropsis-Mezobromelia clade (not shown),
which in addition contains taxa previously assigned to Vriesea and Tillandsia. Tribe Tillandsieae
is strongly supported (BP 92). The first lineage which splits from core Tillandsieae is Guzmania
(BP 85), with G. hutchisonii (syn. M. hutchisonii) nested inside (not shown). Core Tillandsieae is
only weakly supported (BP 58) and relationships of genera are unresolved. Holophyletic taxa
are Josemania (BP 89), Racinaea (no BP) and Lemeltonia (BP 86), but all three are placed on a
polytomy with different clades of Tillandsia and Rothowia.
PRK data and analyses (dataset D3)
Attributes of the analyzed PRK matrix are given in Table 3. PCR primers amplified fragments
ranging from 831 (G. patula Mez & Wercklé) to 1,692 bp (T. ionochroma André ex Mez), with a
mean of 1,086 bp. Indel events are common at all hierarchical levels of obtained trees, both
between individuals of a single species and different higher clades. Unlike PHYC, evidence of
allelic length variation within individuals was detected in 146 of sampled taxa, since the amplified region covered three less conserved introns and four exons (exon 2 to exon 5), with the
second and the fifth exon being sequenced only partially. The number of heterozygous indels
ranged from 1 to 5 in a given taxon. Indels were only found in the less conserved intron regions
and never in the PRK exons. Three taxa had to be cloned, since indel variation between alleles
was too high to perform unambiguous editing, i.e., V. psittacina (Hook.) Lindl., G. graminifolia
(André ex Baker) L.B. Sm., and Ro. platyrhachis. Some others were occasionally cloned to verify
allele length (see supplementary data). Allelic nucleotide variation was found in 241 taxa out
of 444 sampled individuals. Only 193 samples were found to be homozygous for the whole
region sequenced concerning indels and SNPs, which seems to be due to the relatively high
rate of evolution in intron sequences. The number of SNPs in a given heterozygous taxon
ranged from 1 to 18 (the latter seen in T. fasciculata Sw. B0076). Polymorphic sites were primarily found in the less conserved intron parts of the gene and less frequent in exons with
mostly synonymous substitutions. Like in PHYC, evaluation of the effects of SNPs in PRK data
indicate that there is not a strong phylogenetic bias in not having allelic data and allelic consensus sequences can be used. We therefore also only use the polymorphism-coded PRK dataset for all analyses, with the exception of the three cloned taxa.
Generic relationships based on PRK sequence data are shown in Figure 1, D3, which is the
strict consensus tree of a MP analysis supplemented with BP. Backbone relationships are much
less resolved than in the PHYC tree. Topologically identical are the sister group relationship (BP
100) of Glomeropitcairnia (BP 100) and Catopsis (BP 100) and core Tillandsioideae (BP 100).
Subtribes Cipuropsidinae and Vrieseinae are strongly supported (BP 91 and 100, respectively),
whereas tribes Vrieseeae and Tillandsieae as well as core Tillandsieae are not supported.
Werauhia is strongly supported by PRK sequence data (BP 100), whereas the CipuropsisMezobromelia clade has only weak BP (BP 58). The same picture is seen for Alcantarea (BP
149
BARFUSS, M.H.J.
NEW CLASSIFICATION
100) and Vriesea (BP 61), respectively. The relationship of Vriesea and Alcantarea to a clade
containing Tillandsia and Lemeltonia is surprising, but this is only weakly supported (BP 63).
Guzmania is not supported and three different clades of Guzmania species (not shown) occur
in a polytomy with several other clades. Rothowia and Josemania are resolved as sisters (BP
65), both also being holophyletic (BP 64 and 100, respectively). Racinaea is strongly supported
(BP 96), but relationships to other genera remain unresolved. Tillandsia and Lemeltonia are
resolved as sister taxa with a moderate BP (BP 81) with Lemeltonia being strongly supported
(BP 96), but in a polytomy with several other clades of Tillandsia species.
Combined nuclear data (dataset D4)
Analyses of concatenated nuclear datasets D2 and D3 (Figure 1, D4) combine tree topologies
obtained from individual analyses of PHYC and PRK (Figure 1, D2–3). Most accepted infrasubfamilial clades and genera are holophyletic with mostly moderate or high BP, i.e., Glomeropitcairnia (BP 100), Catopsis (BP 100), core Tillandsioideae (BP 100), Vrieseinae (BP 100), Vriesea
(BP 89), Alcantarea (BP 100), Cipuropsidinae (BP 99), Werauhia (BP 100), CipuropsisMezobromelia clade (BP 81), Tillandsieae (BP 81), core Tillandsieae (BP 77), a sister relationship
of Josemania and Rothowia (BP 79), Josemania (BP 100), Racinaea (BP 99), Lemeltonia (BP
100), and the sister relationship of Lemeltonia and Tillandsia (BP 95). The grade of paraphyletic
Vrieseeae taxa (BP 60), Guzmania (BP 61), and Rothowia (BP 70) are weakly supported. No BP
is given for the holophyletic Tillandsia and the sister group position of Racinaea to the clade
containing Josemania and Rothowia.
Combined plastid and nuclear data (dataset D5)
Results of combined analyses of all markers used are shown in two figures. Figure 1, D5 presents a strict consensus tree summarizing mostly generic relationships. Figure 2 is more inclusive and displays relationships of all accessions investigated including subgeneric units and
informal infrasubfamilial and -generic clades. These are either displayed as a phylogram from
parsimony analysis with BP and PP (Figure 2, complete tree) or parts of a phylogram from
Bayesian analysis showing alternative topologies (Figure 2, A–S). As in the combined nuclear
dataset (Figure 1, D4), all generic entities and accepted infrasubfamilial units are holophyletic,
but are here supported by mostly high or moderate values: Glomeropitcairnia (tribe Glomeropitcairnieae; BP 100/PP 100), Catopsis (tribe Pogospermeae; BP 100/PP 100), Werauhia (BP
100/PP 100), Splendens clade (BP 100/PP 100), Chrysostachys clade (BP 85/PP 100), Cipuropsis
(BP 82/PP 98), Alcantarea (BP 100/PP 100), Vriesea (BP 89/PP 100), Josemania (BP 100/PP
100), Racinaea (BP 100/PP 100), Rothowia (BP 81/PP 100), and Lemeltonia (BP 100/PP 100);
weak BP is currently given only for Mezobromelia (BP 66/PP 100), Guzmania (BP 68/PP 100),
and Tillandsia (BP 62/PP 96). Tribes and subtribes are also strongly supported: Vrieseeae (BP
93/PP 100), Cipuropsidinae (BP 100/PP 100), Vrieseinae (BP 100/PP 100), and Tillandsieae (BP
100/PP 100). Unclassified units that were also considered in this study, but not treated taxonomically are: core Tillandsioideae (BP 100/PP 100), and core Tillandsieae (BP 93/PP 100). Relationships of the latter including genera Josemania, Lemeltonia, Racinaea, Rothowia and Tillandsia are resolved differently in parsimony and Bayesian analysis (Fig 2) and show currently
either no BP or only PP. Infrageneric units that are well characterized and strongly to moderately supported (referring to BP only) are (1) within Racinaea: R. subg. Pseudophytarrhiza (BP
100/PP 100) and R. subg. Racinaea (BP 100/PP 100); (2) within Tillandsia: T. subg. Pseudalcantarea (BP 99/PP 100), T. subg. Viridantha (BP 95/PP 100), the Purpurea clade (BP 100/PP 100),
150
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
the Gardneri clade (BP 100/PP 100), T. subg. Anoplophytum (BP 99/PP 100), T. subg. Phytarrhiza (BP 100/PP 100), and the Xiphioides clade (BP 91/PP 100). Weakly supported are
T. subg. Pseudovriesea (BP 68/PP 99), T. subg. Tillandsia, the Rauhii clade (BP 57/no PP), and
the Biflora clade. Tillandsia subg. Diaphoranthema is paraphyletic, although internal clades
receive strong statistical support (Figure 2, Q). Additional clades which are neither used formally nor named, but present in most markers studied are (1) a clade containing the Gardneri
clade, T. subg. Anoplophytum, T. subg. Diaphoranthema, T. subg. Phytarrhiza, the Xiphioides
clade and the unclassified species T. albertiana, T. edithae, and T. esseriana (BP 84/PP 100),
and (2) the same but without the earlier branching Gardneri clade (BP 63/PP 100); and a terminal clade comprising T. subg. Diaphoranthema, T. subg. Phytarrhiza, and the Xiphioides clade
(BP 91/PP 100).
Morphological data and analysis
Mapping morphological characters onto phylogenetic trees (data not shown) resulted in the
following diagnostic traits to be used for delimiting generic clades in the present study (Table
6): ovary position, seed morphology, stigma structure, corolla structure, stamen structure, the
presence vs. absence of petal appendages, and the mesophytic vs. xerophytic habit. Other
characters considered to be important, but currently not used as diagnostic characters here
are ovule and pollen morphology (Table 6 and 7). The main reason for not using these characters for defining genera is the high amount of missing data for many accessions, since pollen
and ovules have been rarely available for detailed study. However, these characters are used
to characterize new generic and subgeneric units, where data was available and most taxa
could be examined. Despite of the limited sampling, the congruence between morphological
characters and the molecular phylogeny is convincing. The key characters for delimiting genera
in the core Tillandsioideae (all genera except Catopsis and Glomeropitcairnia) are the stigma
morphology, followed by the corolla and stamen structure. Three new stigma types are recognized (conduplicate-erect, conduplicate-patent, and pinnatisect) in addition to the already
described ones (Brown & Gilmartin, 1989b).
An initial trial to completely avoid the use of petal appendages as diagnostic characters
failed, since some genetic entities (Guzmania, Tillandsia, internal clades of the CipuropsisMezobromelia clade) share similar character states in their morphological traits currently used
for differentiation. Therefore petal appendages are also used in the present taxonomic concept, but with a reduced taxonomic value, since no other morphological characters have yet
been identified or are satisfactorily known. Petal appendages still have to be utilized for the
differentiation of Mezobromelia from Guzmania and Cipuropsis from Tillandsia, with the following exceptions: (1) T. subg. Pseudovriesea and T. malzinei within Tillandsia (petal appendages present in these taxa, whereas absent from the rest of Tillandsia species), (2) G.
hutchisonii within Guzmania (petal appendages present in this taxon, whereas absent from the
rest of Guzmania species), (3) W. insignis within Werauhia (petal appendages absent from this
taxon, whereas present in the rest of Werauhia species), and (4) T. asplundii and T. singularis
within the Cipuropsis-Mezobromelia clade (petal appendages absent from these taxa, whereas
present in all other Cipuropsis-Mezobromelia clade species). The species Ci. amicorum (syn.
T. amicorum) and M. schimperiana (syn. T. schimperiana) are clearly misplaced in the genus
Tillandsia, not only from the genetic side: all investigated material of these species exhibits
petal appendages. The placement of Ci. amicorum into Tillandsia was most probably based on
herbarium-preserved flowers, where petal appendages can be easily overlooked, and the
151
BARFUSS, M.H.J.
NEW CLASSIFICATION
placement of M. schimperiana was based on a post-anthetic herbarium specimen, assuming
that according to the overall morphology it would best fit into Tillandsia.
DISCUSSION
In contrast to "Classification system of Tillandsioideae", arrangement of discussed taxonomic
units and clades is not strictly hierarchical and alphabetical and more orientated towards the
tree topology (Figure 2, see Appendix). This is necessary, since additional clades that were not
formally treated (e.g., core Tillandsioideae and core Tillandsieae and the CipuropsisMezobromelia clade), and not accepted units (e.g., T. subg. Allardtia) are included as well.
However, within higher units taxa and clades are arranged alphabetically to maintain clarity.
DNA data and analysis
Individual datasets D1–D3 exhibit big differences in their attributes (Table 3) and in their usefulness in resolving phylogenetic relationships, also evident from individual bootstrap consensus trees (Figure 1, D1–D3). No marker alone, neither plastid DNA (Figure 1, D1) nor individual
nuclear DNA (Figure 1, D2–D3), is able to completely resolve branching patterns, but nuclear
sequences in combination (on an average of about 2,300 bp) are yielding even better resolution within Tillandsioideae than combined plastid sequences (on an average of about 5,000
bp). Although the information content of nuclear datasets D2 and D3 is much higher, the
amount of homoplasy also increased compared to plastid DNA data.
The easiest markers to work with so far are the matK gene within the plastid DNA and
PHYC within nuclear DNA markers. PRK is difficult and not very suitable, especially at higher
levels, in the light of the numerous indels that have to be introduced into the alignment.
Several species that were genetically investigated by multiple accessions are apparently of
non-holophyletic origin (Figure 2); for some this can be explained as a result of missing DNA
sequence information, evident from very short branches in respective terminal clades: e.g.,
L. scaligera, M. schimperiana, and M. bicolor; some taxa appear paraphyletic: e.g., A. nevaresii
Leme and R. fraseri (Baker) M.A. Spencer & L.B. Sm.; and for others the results support a polyphyletic origin: e.g., varieties of R. tetrantha (Ruiz & Pav.) M.A. Spencer & L.B. Sm., T. fendleri
Griseb., T. guatemalensis, some varieties and forms of T. tectorum E. Morren, and subspecies
of T. xiphioides. Although this suggests the need of a critical revision of species boundaries
within Tillandsioideae, sampling needs to be increased to really address this question. Therefore species circumscription is not treated within our study as it has no relevance for the outcome.
Morphological data and analysis
As already shown by previous phylogenetic and morphological studies, the four diagnostic
characters used by Smith& Downs (1977) to differentiate genera within Tillandsioideae are
insufficient for achieving an evolutionary based, phylogenetically accurate classification system; these morphological characters are the ovary position, apical and/or basal seed appendages, free vs. conglutinate/connate petals, and present vs. absent petal appendages. The first
two characters are applied in this study in the same way for separating Glomeropitcairnia and
Catopsis from core Tillandsioideae. The other two characters cannot be applied as proposed by
Smith & Downs (1977), since they involve either convergent/parallel evolution or symplesiomorphic character states, evident in the evolution of a fused corolla tube in both tribes Til152
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
landsieae (Guzmania) and Vrieseeae (Mezobromelia) or the occurrence of petal appendages in
different phylogenetic lineages. Petal appendages are primarily found in tribe Vrieseeae with
only a few exceptions (see "Results"); in contrast, petal appendages are mostly absent in Tillandsieae, but also here a few exceptions are present.
Most morphological characters used in this study need a future, much detailed investigation of an increased sample of Tillandsioideae, e.g., stigma or septal nectary morphology.
Glomeropitcairnia
Glomeropitcairnia Harms. is well supported, isolated, genetically and morphologically well
differentiated and comprises only two species, i.e., Gl. erectiflora Mez and the type species
Gl. penduliflora (Griseb.) Mez. Till & al. (1997) illustrated and portrayed both species side by
side for the first time showing important, previously undocumented morphological characteristics, i.e., a convolute-blade like stigma with unique papillae and a diffuse aperture of the pollen. The genus was originally described by Mez (1896) as a subgenus of Pitcairnia L'Hér. based
on the character state of the position of the ½–⅔ inferior ovary. Initial controversies over the
correct subfamilial placement have mostly been solved since Harms (1930) established the
separate, monotypic tribe Glomeropitcairnieae under subfam. Tillandsioideae based on the
common character of plumose seed appendages. Gilmartin & al. (1989) confirmed the placement within Tillandsioideae based on cladistic analyses of morphological characters and concluded the genus to be the sister taxon to Guzmania and Mezobromelia. Ranker & al. (1990) on
the other hand analyzed restriction site variation of the plastid genome and suggested to treat
Glomeropitcairnia as a monotypic subfamily within Bromeliaceae because of its genetic divergence from the other tillandsioid genera. Whereas the placement within Tillandsioideae is well
supported by all published DNA sequence analyses (originally using the ndhF gene by Terry &
Brown, 1996) a close relationship to any of the other genera and the exclusion from Tillandsioideae is not supported.
Catopsis
Other than Glomeropitcairnia, Catopsis (type species C. nitida (Hook.) Griseb.) was always
placed within Tillandsioideae. Harms (1930) emphasized the recognition of a monotypic tribe
for the genus based on its unique seed characteristics. Catopsis is very isolated within Tillandsioideae showing apparent pollen, septal nectary, and seed morphological characters that
differentiate it from the rest of the genera within the subfamily (e.g., Böhme, 1988; Halbritter,
1992; Palací, 1997). Because of its unique position, which is also supported by a larger number
of autapomorphies for the genus in the phylogenetic trees, a separate, monotypic tribe is also
justified by DNA sequence data. Not Harms (1930), who established Catopsideae for Catopsis
within Tillandsioideae as originally indicated in Barfuss & al (2005), but Brongniart (1864) was
the first who proposed Pogospermeae as a monotypic tribe for Pogospermum, which later
became a synonym of Catopsis. Therefore Pogospermeae is the oldest available and correct
name at tribal level which has to be used.
Core Tillandsioideae = Tillandsioideae s.str.
This term for a clade found in phylogenetic investigations of Bromeliaceae was first introduced
by Terry & al. (1997b) and comprised the genera Guzmania, Mezobromelia, Tillandsia (incl.
Racinaea), and Vriesea (incl. Werauhia). They did not investigate any Alcantarea species, but
Horres & al. (2000) showed that Alcantarea is also part of the core Tillandsioideae. Crayn & al.
153
BARFUSS, M.H.J.
NEW CLASSIFICATION
(2004) had a different view in circumscribing this group and excluded Werauhia and Mezobromelia, maybe because they did not include any Alcantarea or true Vriesea species. Their
only "Vriesea" species was T. malzinei, which is actually not part of the true Vriesea alliance
(see below) but belongs to Tillandsia and is in sister position to T. funckiana Baker (Terry & al.,
1997b). We are following the original idea of core Tillandsioideae and restrict the term to all
currently accepted genera and informal clades except Catopsis and Glomeropitcairnia. In all
phylogenetic trees obtained from different datasets (Figure 1, D1–5), regardless which genome
(plastid, nuclear) was studied, core Tillandsioideae always resolve as a holophyletic unit with
maximal BP.
Vrieseeae
Tribe Vrieseeae corresponds roughly to Vriesea s.l. (= Vriesea sensu Smith & Downs, 1977),
with the exception of some taxa, which are now either excluded (T. malzinei and former xerophytic, gray-leaved Vriesea species) or included (Mezobromelia sensu typo and elements of
former T. subg. Allardtia, i.e., Ci. amicorum, M. schimperiana, T. asplundii, and T. singularis).
Mezobromelia sensu typo was only excluded in Barfuss & al. (2005), because conclusions were
based on a misidentified Guzmania species. In the current study the tribe is subdivided into
two main lineages, i.e., subtribe Cipuropsidinae with Cipuropsis, Mezobromelia, Werauhia and
three informal clades (see below), and subtribe Vrieseinae with Alcantarea and Vriesea. Both
subtribes can be distinguished by morphological characters of stigmas and ovules (see below).
Vrieseeae is holophyletic in the plastid DNA tree (Figure 1, D1, BP 100), but not in the case of
individual and combined nuclear markers (Figure 1, D2–D4). Although topologies of these are
conflicting, supports for these alternative branching patterns involving taxa of Vrieseeae are
generally weak. The reason seems to be the lack of characters to resolve backbone relationships in this part of the tree with better confidence. We still trust in the resolution of the plastid DNA tree and are fairly confident that more nuclear DNA sequences will provide the same
result as plastid DNA data, since BP of the alternative topology from combined nuclear data
(Figure 1, D4) still remains weak (BP 60).
Cipuropsidinae
Subtribe Cipuropsidinae is mainly composed of the Andean, Caribbean, and Mesoamerican
species of Mezobromelia and Vriesea sensu Smith & Downs (1977), which do not show
Vrieseinae-type stigmas. In contrast to Vrieseinae, which usually have very long appendages on
the ovules, taxa of this clade generally display no or only very short appendages, except for
Vriesea tuerckheimii. The subtribal name is based on the oldest generic name available for a
species falling within this clade (Ci. subandina). It is divided into two main lineages, one corresponding to taxa assigned to the Cipuropsis-Mezobromelia clade, the other one to Werauhia.
154
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Cipuropsis-Mezobromelia clade
Presently, the Cipuropsis-Mezobromelia clade is an assemblage of previously recognized species of Mezobromelia sensu typo, Tillandsia, and Vriesea (incl. Cipuropsis). Besides Tillandsia
s.str, this is the second clade where a fully resolved classification system cannot yet be applied.
Species and clades found in this phylogenetic unit are (1) V. glutinosa and V. splendens (Splendens clade), (2) V. chrysostachys and V. ospinae (Chrysostachys clade), (3) V. tuerckheimii, (4)
T. asplundii and T. singularis (Singularis clade), (5) Ci. amicorum and Ci. zamorensis (syn. V.
zamorensis) (Cipuropsis), (6) M. bicolor, M. pleiosticha, and M. schimperiana (Mezobromelia).
Vriesea species found here differ from Vriesea s.str. in not having the convolute-blade or the
tubolaciniate stigma morphology but either simple-erect or conduplicate-spiral, respectively.
Cipuropsis amicorum and M. schimperiana have been misplaced in Tillandsia (petal appendages present in both species), and the Singularis clade lacks petal appendages and was therefore
previously treated within T. subg. Allardtia, but differs clearly from that subgenus in other
morphological features, e.g., pollen with fine insulae on the aperture, leaf sheaths drying silvery-grey, and tubular yellow corolla. V. tuerckheimii remains unclassified and without assignment to a group. We are presently proposing generic rearrangements only for three species
within the Cipuropsis and Mezobromelia clades (Ci. amicorum, Ci. zamorensis, and
M. schimperiana) mainly because of limited access to plant material for additional species that
are supposed to group within the Cipuropsis-Mezobromelia clade. Although investigated species of this clade form distinct, moderately or strongly-supported phylogenetic units, the application of morphological characters is currently too vague to propose new genera.
Cipuropsis
Cipuropsis was proposed as a monotypic genus for Ci. subandina (syn. V. subandina). Its generic status was later accepted by Harms (1930), but not by Mez (1934–35) and Smith & Downs
(1977), who treated the genus as a synonym of Vriesea. The only diagnostic character believed
to be natural and discriminative for species of Tillandsia and Vriesea by the latter two have
been petal appendages while other morphological differences between species complexes
were considered to be within the morphological range of these two genera. Unfortunately, it
has not been possible for us to collect or receive fresh or well-preserved plant material for
genetic analyses of Ci. subandina, but because of its strong morphological similarity with and
obvious relationships to Ci. zamorensis based on herbarium specimens (incl. type material) of
both species (W. Till & M.H.J. Barfuss, unpubl. data), it is most likely that Ci. subandina species
groups within this clade. As Cipuropsis is the oldest generic name available for a group of species within the Cipuropsis-Mezobromelia clade, it is restored from the synonymy of Vriesea and
treated as a separate genus within subtribe Cipuropsidinae. Two species previously treated
either under Tillandsia or Vriesea are transferred here, i.e., Ci. amicorum and Ci. zamorensis,
respectively. Further taxa not investigated but supposed to belong to Cipuropsis are V. dubia
(L.B Sm.) L.B. Sm., V. duidae (L.B Sm.) Gouda, V. elata (Baker) L.B. Sm., V. lutheriana J.R. Grant,
and V. rubra (Ruiz & Pav.) Beer. This taxonomic change is justified, since Ci. subandina will always be part of the Cipuropsis-Mezobromelia clade. Even if the taxonomic circumscription of
Cipuropsis will change, other species of the Cipuropsis-Mezobromelia clade would have to be
assigned to Cipuropsis for reasons of priority.
155
BARFUSS, M.H.J.
NEW CLASSIFICATION
Mezobromelia
Species previously classified within Mezobromelia s.l. belong to two phylogenetically unrelated
units. The first lineage, of which we have only investigated G. hutchisonii, is placed within
Guzmania; the second one, in which the type species M. bicolor is located (= Mezobromelia
s.str.), is nested within the Cipuropsis-Mezobromelia clade as sister to Cipuropsis. Mezobromelia schimperiana is genetically definitely not a Tillandsia species, as evident also from flower
morphology, because petals are connate and appendaged in all investigated specimens. It
shares great morphological similarity with species assigned to Mezobromelia s.str.
(J.M. Manzanares, unpubl. data), which is also evident genetically; the species is therefore
assigned to this genus.
Chrysostachys clade
This clade is composed of two Vriesea species (the name-giving V. chrysostachys, and V. ospinae) which were already considered to be a distinct species complex under Vriesea (W. Till,
unpubl. data). Both species show simple-erect stigma morphology.
Singularis clade
Tillandsia asplundii and the name-giving T. singularis are always nested within the CipuropsisMezobromelia clade, despite having no petal appendages. Both species are sister taxa and
together they are in sister position to the clade containing both genera Cipuropsis and Mezobromelia. Some similar Tillandsia species like T. delicatula L.B. Sm., T. pinnata Mez & Sodiro,
and T. truncata L.B. Sm. might also cluster with these two species, but due to the lack of material, these species were not investigated genetically. This species complex was previously
treated under T. subg. Allardtia and forms a distinct clade within Cipuropsis-Mezobromelia.
Generic status might be justified, when a broader sampling has been studied.
Splendens clade
Presently it is composed of two species in northern South America. While V. glutinosa had
been placed in V. sect. Xiphion by Smith & Downs (1977), V. splendens (the name-giving taxon)
was classified as a member of V. sect Vriesea. The recognition of a formal status of this group,
to which also V. soderstromii L.B. Sm. may belong, remains to be studied further.
Werauhia
Werauhia (type species W. gladioliflora (H. Wendl.) J.R. Grant) is holophyletic and well supported by all DNA regions studied. It is also supported by morphological characters (e.g., cupulate stigma type) that were already recognized by J.R. Grant before molecular data was available. He therefore correctly excluded these taxa from Vriesea and erected the genus Werauhia
with two sections. Werauhia sect. Jutleya (type species W. pedicellata (Mez & Wercklé) J.R.
Grant) is holophyletic, but in association with taxa of W. sect. Werauhia in two grades, making
the latter paraphyletic. To retain the current classification, W. sect. Werauhia would have to
be split into two groups based on the current taxon sampling. However, such actions awaits
significantly increased species numbers in future studies.
156
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Vrieseinae
Subtribe Vrieseinae is composed of the two well supported genera Alcantarea and Vriesea as
they are defined in this study. Characteristics for this subtribe are ovules with usually long appendages and geographic distributions with centers of diversity in eastern Brazil. Stigma types
found in this clade are convolute-blade (Brown & Gilmartin, 1989b), tubolaciniate (Leme &
Brown, 2004), conduplicate-patent (Versieux & Wanderley, 2007), conduplicate-erect.
Alcantarea
Contrary to V. subg. Vriesea, V. subg. Alcantarea (Mez, 1891–94) sensu Smith & Downs (1977)
(type species A. regina (Vell.) Harms, lectotypified by Grant & Zijlstra, 1998) is a holophyletic
group if a few misplaced species are excluded (Grant, 1995a). J.R. Grant reestablished the generic status proposed by Harms (1929) based on additional morphological evidence (Grant,
1995a). Morphological characters of this genus have been recently revised by Leme (2007,
2009) and the whole Alcantarea by Versieux (2009) and Versieux & al. (2010, 2012). Brown &
Gilmartin (1989b) and Grant (1995a) originally indicated the stigma type of Alcantarea to be
the same as Vriesea (convolute-blade). Figure 23 of Brown & Gilmartin (1989b) is wrongly assigned to V. geniculata (Wawra) Wawra (= A. geniculata (Wawra) J.R. Grant) and displays a
true Vriesea species; indications of a convolute-blade stigma morphology for other Alcantarea
species (as Vriesea) in their appendix are also incorrect. Leme (2007) demonstrated that the
stigma of Alcantarea is of a different type than that of Vriesea. In his opinion, the stigma is
conduplicate-spiral, but this view is largely not in concordance with the original idea of this
stigma type by Brown & Gilmartin (1989b). Therefore two new stigma terms are used (conduplicate-patent and conduplicate-erect, respectively) as additional morphological characters to
define Alcantarea. These new stigma types are illustrated either in Versieux & Wanderley
(2007) and Leme (2007), or Leme (2009).
Vriesea
Vriesea subg. Vriesea sensu Smith & Downs (1977; type species V. psittacina) is an assemblage
of different phylogenetic units including Werauhia, species now assigned to the CipuropsisMezobromelia clade, the former xerophytic, gray-leaved Vriesea species and T. malzinei, the
latter two in fact belonging to Tillandsia (Terry & al., 1997b). Utley (1978, 1983) and Grant
(1995) already recognized that V. subg. Vriesea was an assemblage of different evolutionary
units, and Grant (1995) finally separated Werauhia (including the type species) from the rest of
V. subg. Vriesea sect. Xiphion. Former xerophytic, gray-leaved Vriesea species were excluded in
a series of four publications (Grant, 1993b, 1994b, 1995b, 2005). First evidence that T. malzinei
has to be assigned to Tillandsia s.str. was provided by Terry & al. (1997b) and this is confirmed
in the present study. Former Vriesea species of the Cipuropsis-Mezobromelia clade have never
been excluded from V. subg. Vriesea, but differ clearly in not having the convolute-blade or
tubolaciniate stigma type. These two stigma types are therefore the main characteristics of the
predominantly Brazilian Vriesea species and are used to differentiate Vriesea s.str. in conjunction with habit or petal appendages from the remaining tillandsioid genera. Sections Vriesea
and Xiphion p.p., typo excluso of Vriesea s.str. are para- and/or polyphyletic, but placement of
species assigned to different sections is not by random in the phylogenetic context. Xiphion
characters (included stamens) seem to be plesiomorphic within Vriesea, evident from clades in
a grade at the base of Vriesea. Vriesea sect. Vriesea is derived (exserted stamens), but pa157
BARFUSS, M.H.J.
NEW CLASSIFICATION
raphyletic, because one lineage within this clade shows Xiphion characters, a possible reversal
to the ancestral state. This hypothesis is supported by both the parsimony and Bayesian trees,
but has no statistical support, which demonstrates the need of further molecular investigations of Vriesea for achieving a proper infrageneric classification.
Tillandsieae
Contrary to Vrieseeae, which is currently resolved as a holophyletic unit only by plastid DNA
data, Tillandsieae shows a holophyletic origin with moderate or high BP in nearly all DNA
markers studied except in the PRK tree, where core Tillandsioideae clades display a polytomy
(Figure 1, D3). This tribe contains Guzmania, as well as the core Tillandsieae genera Josemania,
Lemeltonia, Racinaea, Rothowia, and Tillandsia. Since inclusion of Mezobromelia in Barfuss &
al. (2005) was based on a misidentified Guzmania species and the actual type of Mezobromelia
is placed in Vrieseeae, it is excluded here from Tillandsieae. The clade corresponds to what
Crayn & al. (2004) considered as the core Tillandsioideae in their study (see above).
Guzmania
Circumscription of Guzmania (type species G. monostachia) sensu Smith & Downs (1977) has
not changed over more than three decades. Some taxa were transferred from other genera to
Guzmania after publishing of the monograph only because complete floral characteristics were
available for some species and clearly indicated that they were misplaced according to the
generic definition (e.g., Utley, 1978; Luther, 1998). Current DNA results mostly confirm the old
circumscription with the exception of the inclusion of one morphologically distinct group of
former Mezobromelia with laxly-branched inflorescences and exposed stamens. Out of this
group only G. hutchisonii was investigated. This taxon was initially treated as Tillandsia species
by Smith & Downs (1977), but was transferred to Mezobromelia based on the floral character
of conglutinate/connate petals bearing appendages (Weber & Smith, 1983). It differs from
Mezobromelia sensu typo (M. bicolor) in the inflorescence architecture (candelabra-like with
compact lateral branches in Mezobromelia s.str. vs. laxly-branched in G. hutchisonii) and in the
stamen structure (conglutinate anthers and included stamens in Mezobromelia s.str. vs. free
anthers and exserted stamens in G. hutchisonii). Therefore, the exclusion from Mezobromelia
s.str. and the transfer of G. hutchisonii to Guzmania is justified genetically and morphologically. In addition, evidence from plastid DNA data (M.H.J. Barfuss, unpubl. data) suggests close
relationships to G. diffusa L.B. Sm. and G. bakeri (Wittmack) Mez. A number of Guzmania species which have not been analyzed as well (e.g., G. candelabrum (André) André ex Mez, G.
dalstroemii H. Luther, G. hirtzii H. Luther, and G. lemeana Manzan.) are showing similar inflorescences architecture and therefore might also be related to G. hutchisonii (Smith & Downs,
1977). Mezobromelia fulgens L.B. Sm. is also closely related genetically (M.H.J. Barfuss, unpubl.
data). Further Mezobromelia species with free and exposed anthers, which are supposed to be
closely related to G. hutchisonii and therefore believed to belong to the same clade but have
not been investigated, are M. brownii H. Luther and M. lyman-smithii Rauh & Barthlott. Apart
from these hypotheses concerning relationships, however, a distinction between Mezobromelia and Guzmania based just on the character of petal appendages is clearly inappropriate and
does not reflect true phylogenetic relationships. Whether the way how the corolla tube is developed (either early or late sympetalous; Leins & Erbar, 2010) could serve as a discriminative
character (most probably truly connate in Mezobromelia vs. only conglutinate in Guzmania)
needs a further, detailed investigation.
158
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
A holophyletic origin of Guzmania is supported by three datasets (PHYC D2, nuclear combined
D4, and total combined D5), whereas in dataset D3 (PRK) Guzmania clades display a polytomy
with other core Tillandsioideae clades (Figure 1). Present in all phylogenetic trees are three
main clades of Guzmania taxa (shown only in Figure 2), i.e., (1) G. hutchisonii, (2) Guzmania
s.str., and (3) the remaining Guzmania species (incl. Sodiroa). Guzmania appears paraphyletic
in the plastid DNA study of Barfuss & al. (2005), but the sample investigated as M. pleiosticha
(Griseb.) Utley & H. Luther (acc. no. MB-15 = B0015) turned out to be a misidentified G. variegata. Guzmania hutchisonii appeared as the earliest diverging lineage within Tillandsieae in
Barfuss & al. (2005). This is most probably due to numerous autapomorphies for the species
responsible for LBA, which is further enforced by the low number of parsimony-informative
plastid DNA characters and missing samples. The genetic divergence of clades 1 and 3 from
Guzmania s.str. (clade 2) in all markers investigated is also reflected by morphology and indicates derived status within the genus. The hypothesis that in clade 3 a recent radiation has
taken place is supported by short branches relative to taxa of Guzmania s.str. and G.
hutchisonii. Nevertheless, PHYC and combined nuclear DNA sequences data consistently place
all three clades into one linage (BP 85 vs. 61, respectively) and total-combined data give good
resolution, and again increasing statistical support (BP 68/PP 100) indicating the whole group
to be a natural entity. Support for the separation of a genus Sodiroa (e.g., Baker, 1889; Harms,
1930; Mez, 1934–35; not accepted by but mentioned in the key of Smith & Downs, 1977:
1284) that is defined by connate sepals is currently not provided by DNA data, and this character probably evolved convergently or in parallel, or reversals to the ancestral state (free sepals)
have occurred. This is in contrast to the acceptance by Betancur & Miranda-Esquivel (1999),
who reevaluated morphological characters phylogenetically without the application of molecular tools. Further generic segregations or infrageneric divisions of clades (e.g., the three main
clades) need a careful evaluation of morphological characters, more relatives of G. hutchisonii,
and more genetic evidence to obtain well resolved relationships within Guzmania.
Core Tillandsieae = Tillandsieae s.str.
This clade contains all segregates of Tillandsia since the treatment of Smith & Downs (1977)
and can be referred to Tillandsia s.l. (= Tillandsia sensu Smith & Downs, 1977) if wrongly assigned taxa are excluded or included (see below). The nested placement of the here segregated genera Josemania, Lemeltonia, Racinaea, and Rothowia within Tillandsia in the earlier study
of Barfuss & al. (2005) obviously was an artifact caused by the low number of parsimonyinformative characters. The current study clearly shows that all four segregates constitute holophyletic lineages not nested within Tillandsia s.str. Since Tillandsia sensu s.l. is morphologically very heterogeneous and the already earlier segregated genus Racinaea is widely accepted,
recognition of Josemania, Lemeltonia, and Rothowia is necessary for these morphologically
divergent holophyletic lineages.
Josemania
This new genus (type species J. lindenii) contains taxa previously ascribed to T. subg. Phytarrhiza based on the character of broad and conspicuous petal blades. In 1869 Regel proposed
T. sect. Wallisia for the single species T. lindenii. E. Morren (1870) changed the status and
erected the genus Wallisia which was not followed by bromeliad taxonomists. According to
Art. 52.1 of the international code of botanical nomenclature (McNeill & al., 2006), this name
is illegitimate at generic level and therefore the new name Josemania is proposed. All species
159
BARFUSS, M.H.J.
NEW CLASSIFICATION
classified here, i.e., J. anceps, J. cyanea, J. lindenii, J. pretiosa and J. umbellata, are easily recognized by their inflorescence structure, characteristic flowers and the pinnatisect stigma
morphology (already illustrated in Morren, 1871).
Lemeltonia
Species of this new genus (type species L. dodsonii) have also been part of former, polyphyletic
T. subg. Phytarrhiza sensu Smith & Downs (1977) and appear to be the sister genus to Tillandsia s.str. according to the current generic circumscriptions. The genus seems to be somehow
intermediate between Racinaea, Rothowia, Josemania on the one hand and Tillandsia s.str. on
the other. Species assigned to this genus are L. acosta-solisii, L. cornuta, L. dodsonii,
L. monadelpha, L. narthecioides, L. scaligera, and L. triglochinoides. Lemeltonia narthecioides
has not been included into the recent phylogenetic analyses, but high support for the inclusion
into this genus comes from previous (Barfuss & al.,2005) and unpublished DNA data of the first
author as well as from morphology.
Racinaea
Racinaea (Grant, 1994a; type species R. cuspidata) constitutes a separate entity within tribe
Tillandsieae, which remained questionable after investigation of plastid DNA markers (Barfuss
& al., 2005). Racinaea subg. Racinaea consists of all species formerly assigned to T. subg. Pseudo-Catopsis plus R. dyeriana (syn. T. dyeriana), a species previously classified within T. subg.
Phytarrhiza but showing similar morphological features except those of strongly asymmetrical
sepals and inconspicuous petal blades. However, subsymmetric petals and conspicuous petal
blades are also found in other Racinaea species (e.g., R. elegans (L.B. Sm.) M.A. Spencer &
L.B. Sm., R. riocreuxii (André) M.A. Spencer & L.B. Sm.), which are without doubt part of this
genus. Racinaea subg. Pseudophytarrhiza has been established for the two species R. venusta
(type species) and R. hamaleana, which have been transferred here from T. subg. Phytarrhiza
too. These two species are genetically and morphologically distinct from the rest of Racinaea,
but phylogenetically always in sister position to R. subg. Racinaea with strong statistical support.
Rothowia
This is a very isolated linage composed of three described species, i.e., Ro. laxissima,
Ro. platyrhachis, and the type species Ro. wagneriana, which previously have been treated
under T. subg. Phytarrhiza. The genus is entirely mesophytic and displays well-established phytotelmata (water-impounding rosettes). The elate inflorescence rachises and the obconic stigma are distinctive.
Tillandsia
Tillandsia s.l. (type species T. utriculata, incl. T. subg. Pseudo-Catopsis = Racinaea) is a morphologically very heterogeneous, but holophyletic group with respect to few exceptions, which are
now either found in Vrieseeae or have been transferred from there (see below). Its circumscription corresponds roughly to the clade indicated as core Tillandsieae (Figure 2). Subsequent to the monograph, parts of the genus were studied to enable better recognition of individual species groups. The segregation of Racinaea and Viridantha has caused dispute among
bromeliad taxonomists, among whom the first genus is broadly accepted and the latter not.
160
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Only on the base of our molecular data we can propose a reliable classification for several segregates (see above). However, Tillandsia s.str. remains in a provisional state.
Currently Tillandsia s.str. is circumscribed as the terminal supported backbone clade of
our phylogenetic reconstruction (Figure 2) which includes either T. subg. Pseudalcantarea
(parsimony analysis) or T. subg. Pseudovriesea (Bayesian analysis) as the most ancient lineage.
Excluded are morphologically and genetically clearly distinguishable taxa (Josemania, Lemeltonia, Racinaea, Rothowia), which were previously treated as or integrated into separate
subgenera (T. subg. Pseudo-Catopsis and meso-/semi-mesophytic T. subg. Phytarrhiza, respectively). The genus is divided into several subgenera and informal infrageneric clades, with their
formal status depending mainly on phylogenetic support values (BP and PP), the number of
investigated species, morphological characters to define them, and already well-established
taxa in literature. As indicated under individual subgenera or infrageneric clades, their species
circumscriptions have sometimes significantly changed since the monograph of Smith & Downs
(1977).
Tillandsia subg. Allardtia
Tillandsia subg. Allardtia is not accepted under Tillandsia. The subgenus in the sense of Smith
& Downs (1977) is an artificial assemblage of at least 14 phylogenetic units according to the
current species sampling: (1) the Singularis clade is placed within Cipuropsidinae, (2)
T. guatemalensis (type species of T. subg. Allardtia), T. selleana Harms, and T. leiboldiana
Schltdl. are forming one group and are nested within T. subg. Tillandsia together with (3)
T. adpressiflora Mez, (4) T. secunda Knuth, and (5) T. remota Wittm. in different positions; (6)
T. barthlottii and T. myriantha are grouping with T. subg. Pseudovriesea; (7) the Tillandsia tectorum complex and the Tillandsia plumosa complex (= Viridantha) are closely related and form
a distinct unit (T. subg. Viridantha); (8) xerophytic Andean members of T. subg. Allardtia are
located in the Xiphioides clade together with Andean species of former T. subg. Anoplophytum;
(9) T. disticha (i.s. = incertae sedis) is a taxon genetically very isolated from all other Tillandsia
species; (10) T. edithae (i.s.) and (11) T. australis (i.s.) are currently unclassified, but show affinities to T. subg. Anoplophytum s.str; (12) T. pseudomicans (i.s.) and (13) T. sphaerocephala (i.s.)
are also unclassified because of unstable positions in different types of analysis; (14) and the
rest of T. subg. Allardtia sensu Smith & Downs (1977) containing most species seems to be a
distinct holophyletic group, but presently not well supported because of low variability in both
plastid and nuclear DNA markers.
Therefore the Singularis clade (phylogenetic unit 1) is shifted to Cipuropsidinae; T. subg.
Allardtia sensu typo has to be considered as a synonym of T. subg. Tillandsia with the six taxa
mentioned earlier transferred there (2–5); T. barthlottii and T. myriantha are classified within
T. subg. Pseudovriesea (6); the Tillandsia tectorum complex is transferred to T. subg. Viridantha (7); xerophytic Andean members are transferred to the Xiphioides clade (8); T. australis,
T. disticha, T. edithae, T. pseudomicans, and T. sphaerocephala are unclassified (9–13); and the
rest of T. subg. Allardtia (14) is provisionally treated as a distinct clade without formal nomenclatoric adjustments (Biflora clade).
Tillandsia subg. Anoplophytum
Current and previous results show that T. subg. Anoplophytum sensu Smith & Downs (1977;
type species T. stricta) is (1) paraphyletic (regarding the clade that is most inclusive for species
previously classified under T. subg. Anoplophytum; see Figure 2, S (BP 84/PP 100), including
161
BARFUSS, M.H.J.
NEW CLASSIFICATION
Gardneri clade, excluding T. australis), since (a) both T. subg. Diaphoranthema and T. subg.
Phytarrhiza s.str. are nested within this clade, (b) the Xiphioides clade is in terminal position
containing also species formerly assigned to T. subg. Allardtia, and (c) the currently unclassified T. edithae (T. subg. Allardtia sensu Smith & Downs, 1977) is also included; and (2) polyphyletic because T. macbrideana and T. pseudomacbrideana, elements of former T. subg. Anoplophytum, are now part of the Biflora clade. A postulated holophyletic origin of T. subg. Anoplophytum by Tardivo (2002) based on a phylogenetic analysis of selected morphological characters is not supported, mainly because subgeneric circumscription was according to Smith &
Downs' vague morphological definition. The distinction between T. subg. Anoplophytum and
T. subg. Allardtia based on the character of "plicate vs. straight" filaments is obviously inappropriate and does not reflect true relationships. Support for this view comes not only from
the phylogenetic results but also from ontogenetic studies on species from other subgenera
(Evans & Brown, 1989), where plicate filaments are also reported for T. subg. Allardtia and
T. subg. Tillandsia. In its current circumscription T. subg. Anoplophytum is restricted to
T. stricta and its close relatives, which have their distribution center in south-eastern Brazil.
The taxon is characterized by simple inflorescences with polystichously arranged flowers.
Tillandsia subg. Diaphoranthema
In the current analyses T. subg. Diaphoranthema (type species T. recurvata (L.) L.) is a paraphyletic lineage within Tillandsia, but this is not well supported. The investigated species are forming a clade in the parsimony analysis in sister position to the Xiphioides clade, but with T. subg.
Phytarrhiza s.str. nested inside. In the Bayesian analysis the three clades composed of species
of T. subg. Diaphoranthema (Figure 2, S), i.e., Capillaris clade (T. capillaris, T. funebris A. Cast.,
T. kuehasii W. Till, and T. virescens) Usneoides clade (T. landbeckii ssp. andina W. Till, T. mollis
H. Hrom. & W. Till, T. usneoides (L.) L., and T. cf. usneoides), and T. recurvata, are associated in
different degrees with both T. subg. Phytarrhiza and the Xiphioides clade. To test for a holophyletic origin of T. subg. Diaphoranthema in the current circumscription additional DNA
markers and taxa are required. The stigma type of all species in this subgenus is simple-erect
according to Brown & Gilmartin (1989b), lobes are short rendering a capitate stigma resembling that of T. subg. Phytarrhiza s.str. This morphological similarity points towards a close
relationship of both subgenera in their actual circumscription (Till, 1992). Both taxa share an
orange pollen color which is absent in remaining Tillandsia.
Tillandsia subg. Phytarrhiza
Tillandsia subg. Phytarrhiza sensu Smith & Downs (1977; type species T. duratii) is clearly artificial and polyphyletic. The division into mesophytic, semi-mesophytic and xerophytic members was a first attempt of grouping these species into natural units (Gilmartin, 1983; Gilmartin
& Brown, 1986; Till, 2000b). This view is only partly supported by DNA sequence data. Mesophytic and semi-mesophytic members belong to four different evolutionary units, three of
them representing separate phylogenetic entities, which deserve generic status (Josemania,
Rothowia, Lemeltonia). The fourth, containing R. dyeriana, R. hamaleana and R. venusta, is
strongly associated with Racinaea and therefore these species are transferred to this genus.
Xerophytic members likewise are a polyphyletic group within Tillandsia. They also display at
least two phylogenetically distinct units, (1) species associated with T. purpurea Ruiz & Pav.
(Purpurea clade), and (2) species around T. duratii, showing strong affinities to T. subg. Diaphoranthema. In the current treatment T. subg. Phytarrhiza is restricted to T. duratii and its close
162
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
relatives and is accepted as a separate subgenus, despite of its nested position (see "Classification of Tillandsioideae" for reasons). Other taxa examined so far are T. kirschnekii Rauh & W.
Till, T. paleacea C. Presl, and T. aff. streptocarpa Baker. Whether its subgeneric status can be
kept or T. subg. Diaphoranthema must be merged as suggested by Till (1992) needs further
investigation of more species and additional DNA markers to obtain well-resolved relationships.
Polyphyletic Tillandsia subg. Phytarrhiza in the sense of Smith & Downs (1977) has been
studied by Gilmartin (1983) and Gilmartin & Brown (1986) in more detail, and by Till (1992) for
a morphological comparison to T. subg. Diaphoranthema (see above). The first two studies
used cladistic analyses of morphological characters (mainly obtained from Smith & Downs,
1977) primarily to test hypotheses concerning evolution of xerophytes and mesophytes within
the subgenus. The main problem of both studies is the assumption that T. subg. Phytarrhiza is
holophyletic, which does not match the molecular results. Their postulation for monophyly is
based on the character of broad and conspicuous petal blades, which is obviously a character
that evolved convergently or in parallel in different lineages. Identification of species groups
within T. subg. Phytarrhiza (and T. subg. Diaphoranthema as sister or outgroup) of Gilmartin &
Brown (1986) prior to the computer analyses for phylogenetic reconstruction is very critical
and resulting trees should be taken with caution. Initial analyses of Gilmartin (1983) are more
interesting, since analyses are based on single species and not on species groups. Three important clades from her phenetic analysis correspond to the newly erected genera Josemania,
Lemeltonia, and Rothowia. The morphological study of T. subg. Diaphoranthema and T. subg.
Phytarrhiza by Till (1992) support close relationships of both subgenera in their current circumscriptions.
Tillandsia subg. Pseudalcantarea
Tillandsia subg. Pseudalcantarea currently consist of three species: T. grandis Schltdl.,
T. macropetala , and T. viridiflora (type species). Further species that were previously assigned
to this subgenus belong to different phylogenetic units. T. baliophylla lies within the Biflora
clade and T. paniculata within T. subg. Tillandsia; therefore these two species are excluded
from T. subg. Pseudalcantarea. Tillandsia heterophylla E. Morren, a simulator of morphological
characters fitting the description of T. subg. Pseudalcantarea (Till, 2000b), also appears not
closely related to T. subg. Pseudalcantarea. Instead, this species is part of group III species of
Gardner's classification of T. subg. Tillandsia (Gardner, 1982, 1986b), which are, however, located in the Biflora clade and not part of T. subg. Tillandsia. The view of Beaman & Judd (1996)
that T. subg. Pseudalcantarea should be restricted only to the species T. viridiflora and
T. baliophylla, and the transfer of T. grandis to T. subg. Tillandsia is not supported by neither
plastid DNA nor nuclear DNA sequence data. Tillandsia subg. Pseudalcantarea with its three
here accepted species forms a distinct group being sister to the remaining clades of Tillandsia
in the parsimony analysis (Figure 2), but this position is only weakly supported and not present
in Bayesian analysis (Figure 2, R, here T. subg. Pseudovriesea is the earliest branch within Tillandsia s.str.). Future studies including more DNA markers will help clarifying the correct phylogenetic position of this subgenus which might merit generic status.
Tillandsia subg. Pseudovriesea
The new T. subg. Pseudovriesea (type species T. tequendamae) is composed of all xerophytic,
grey-leaved species previously assigned to Vriesea and some Tillandsia species which were
163
BARFUSS, M.H.J.
NEW CLASSIFICATION
either never formally transferred to Vriesea, despite having petal appendages (i.e.,
T. myriantha, formerly part of T. subg. Allardtia), or because the investigated material did not
show appendages on the petals so far (T. barthlottii, T. spathacea; the former being previously
part of T. subg. Allardtia, the latter of T. subg. Tillandsia). J.R. Grant already recognized the
close relationship to Tillandsia based on overall morphological similarity (habit) and differences in stigma morphology to Vriesea s.str. (convolute-blade or tubolaciniate vs. conduplicate-spiral). He transferred all xerophytic, grey-leaved Vriesea species to Tillandsia, at first
those with exserted stamens and styles (Grant, 1993b, 1994b, 1995b), later also species with
included stamens and styles (Grant 2005). The main difference to T. subg. Tillandsia is that
most of these species have retained (or regained) their petal appendages, a character which is
rarely found in other species of Tillandsia. Unfortunately, petal appendages are not synapomorphic for all taxa within T. subg. Pseudovriesea, since few members do not show petal appendages so far in the investigated flowers. To really address this issue populational, investigations on petal appendages would be necessary.
The long standing debate on whether xerophytic, grey-leaved Vriesea species are members of Vriesea (Smith & Downs, 1977), of T. subg. Tillandsia (Grant, 1993b, 1994b, 1995b), or
of a separate phylogenetic unit within Tillandsia (Barfuss & al., 2005), is now resolved. They fall
neither into Vriesea s.str. nor into T. subg. Tillandsia (as Grant originally thought when he did
the first rearrangements of taxa with exserted stamens and styles), but they are clearly members of Tillandsia deserving a subgenus of their own. Evidence from all plastid and nuclear DNA
markers are supporting this view and earlier speculations of a possible ancient hybrid origin of
this group between any Vriesea and Tillandsia species can be rejected.
Tillandsia subg. Tillandsia
Species of T. subg. Tillandsia were consistently separated from the other subgenera since Mez
(1896) based on the floral characters of exserted stamens and styles, which was followed by
Smith & Downs (1977). Gardner (1982, 1986b) established 5 groups within T. subg. Tillandsia
sensu Smith & Downs (1977), but also included some possibly wrongly classified Allardtia species. Based on results of the current phylogeny, this subgenus should be restricted (1) to group
I and II species of her classification, (2) to T. filifolia Schltdl. & Cham. of group IV, which in her
investigations shows also affinities to group II, as well as (3) to some taxa previously assigned
to T. subg. Allardtia (T. adpressiflora, T. guatemalensis, T. leiboldiana, T. remota, T. secunda,
T. selleana). As the type species of T. subg. Allardtia, T. guatemalensis, is nested within T. subg.
Tillandsia, Allardtia necessarily becomes a synonym of Tillandsia subg. Tillandsia. Group V species are actually species of T. subg. Viridantha, group III species are mainly nested within the
Biflora clade and the second species of her group IV, T. disticha, shows currently no supported
affinity to any infrageneric unit within Tillandsia and is therefore unclassified. Wrongly assigned is T. rauhii (group III, now Rauhii clade). Some species with undetermined affiliation in
Gardner's treatment can now be assigned to clades within Tillandsia: T. cryptopoda L.B. Sm.
and T. plagiotropica Rohweder clearly belong to T. subg. Tillandsia, group I; T. extensa Mez is
part of group II, T. ecarinata, T. ferreyrae and T. teres are members of the Rauhii clade and not
supported to belong to T. subg. Tillandsia. Support for subgroups of Gardner's group I (1986b)
is currently also not given (see also Granados Mendoza, 2008).
Although the subgenus has currently no statistical support, validation for the inclusion of
the investigated taxa comes from previous studies and unpublished data (Terry & al., 1997b;
Barfuss & al., 2005; M.H.J. Barfuss, unpubl. data). Four main clades can be recognized: (1) a
164
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
clade containing T. secunda (T. subg. Allardtia sensu Smith & Downs, 1977), T. malzinei (V.
sect. Vriesea sensu Smith & Downs, 1977), and T. paniculata (T. subg. Pseudalcantarea sensu
Smith & Downs, 1977); all these taxa have not been included into T. subg. Tillandsia before; (2)
a clade that W. Rauh considered as the Tillandsia mima complex (Rauh, 1973; group II species
of Gardner, 1986b) including T. adpressiflora (T. subg. Allardtia sensu Smith & Downs, 1977)
and T. hildae Rauh (group II, Gardner, 1986b); (3) a clade which contains T. utriculata and
therefore could be recognized as T. subg. Tillandsia s.str. (also group II species plus T. filifolia of
group IV, Gardner, 1986b); and (4) a clade containing the majority of species (group I species,
Gardner, 1986b, plus T. subg. Allardtia s.str.). Interesting are the geographical distributions of
these clades. Whereas the early diverging clades 1 and 2 are outside the diversity center of
T. subg. Tillandsia and mainly found in northern South America and the Caribbean, the two
core clades have their main distribution in Mexico and Central America. This pattern gives important hints for future biogeographical studies in Tillandsioideae.
Pseudobulbous taxa of T. subg. Tillandsia were studied by Chew & al. (2010). Results
based on ITS 2 and ETS data support the polyphyletic origin of this habit within T. subg. Tillandsia and even within the whole genus Tillandsia, although statistical support unfortunately is
mostly weak. An enriched taxon sampling and additional molecular markers would help clarifying the interesting question, how often the pseudobulbous habit arose within Tillandsia.
Tillandsia malzinei is a special case within T. subg. Tillandsia. Genetically, from both nuclear and plastid DNA data, this species is doubtlessly nested in the Tillandsia clade, despite
bearing appendages at the base of the petals. It is genetically very distinct from other Tillandsia species, but shows some genetic affinities to T. funckiana (Terry & al., 1997b) and other
members of T. subg. Tillandsia, but not to T. subg. Pseudovriesea. Therefore we are currently
treating this species as a strongly isolated taxon within T. subg. Tillandsia, due to its high number of molecular autapomorphies and the distinct morphology.
Tillandsia subg. Viridantha
Viridantha (Espejo-Serna, 2002) with T. plumosa as type species does not deserve generic rank
under subfam. Tillandsioideae based on the current generic circumscriptions. Taxa of Viridantha clearly are member of Tillandsia s.str., but not related to species of T. subg. Tillandsia
(group V: Gardner, 1982, 1986b) or associated with the Biflora clade (former T. subg. Allardtia
sensu Smith & Downs, 1977). This is also supported by flower morphology (Brown & Gilmartin,
1989b) since some Viridantha species resemble the convolute-blade stigma type (W. Till &
M.H.J. Barfuss, unpubl. data; but also illustrated in Gardner (1982) and Espejo-Serna (2002)), a
character not found in any other subgroup of Tillandsia. Instead, these taxa are closely related
to the Tillandsia tectorum complex (Hromadnik, 2005), which was previously included in
T. subg. Allardtia and T. subg. Anoplophytum sensu Smith & Downs (1977). We therefore consider species of Viridantha and the Tillandsia tectorum complex as members of a separate subgenus. An internal classification of this subgenus needs a deeper and more comprehensive
study of all species assigned to T. subg. Viridantha. The sectional treatment of Espejo-Serna
(2002: Vi. sect. Viridantha and Vi. sect. Caulescens) is currently not supported. Descriptions of
new taxa during the last few years and monographic treatments of both species complexes
(e.g., Espejo-Serna, 2002; Hromadnik, 2005; Ehlers, 2009) using traditional, morphology based
taxonomy show the need of a taxonomic revision also on a (phylo-)genetic basis.
The placement of T. sphaerocephala as sister to T. subg. Viridantha in the parsimony analysis seems to be an artifact with no statistical support caused by low sequence variability and
165
BARFUSS, M.H.J.
NEW CLASSIFICATION
the lack of other species related to this taxon. Bayesian analysis places T. sphaerocephala in
sister position to the Purpurea clade (Figure 2, L), which in the parsimony analysis groups with
unclassified T. nana and T. pseudomicans, a possible artifact as well (PP 60).
Biflora clade
This is one of the most critical species complexes within Tillandsia (name-giving taxon
T. biflora) and comprises mostly taxa previously assigned to T. subg. Allardtia. Some members
of this clade have also been classified in other subgenera, but clearly belong into this phylogenetic unit: (1) most group III species of Gardner's treatment of T. subg. Tillandsia (Gardner
1982, 1986b), i.e., T. deppeana; T. heterophylla, T. imperialis, and T. multicaulis, (2)
T. baliophylla of T. subg. Pseudalcantarea sensu Smith & Downs (1977), and (3) elements of
T. subg. Anoplophytum sensu Smith & Downs (1977), i.e., T. macbrideana and
T. pseudomacbrideana. Because the type species of T. subg. Allardtia, T. guatemalensis, is now
part of T. subg. Tillandsia, a new subgenus would have to be created for these taxa. DNA data
weakly links the species to a holophyletic group with no statistical support and definite morphological characters are currently missing. Therefore, we keep this complex as an informal
clade under Tillandsia. Although the huge majority of species was previously classified within
T. subg. Allardtia, included style and stamens with no filament plication can obviously not
serve as an autapomorphic character for this complex.
Gardneri clade
Earlier, this group of species (name-giving taxon T. gardneri) was classified within T. subg.
Anoplophytum. It is composed only of rose-flowered species distributed in eastern Brazil. The
DNA results suggest that this species complex has had a relatively long time of independent
evolution. Ehlers (1997) monographed them as "red-flowered" species and included 14 Brazilian members plus T. paraensis Mez. The latter belongs to T. subg. Tillandsia but was not investigated. We investigated only 3 species (T. brachyphylla, T. gardneri, T. globosa) and are not
confident enough to propose a separate formal unit for these taxa. Therefore, this complex is
recognized as an informal clade within Tillandsia.
Purpurea clade
As a part of former T. subg. Phytarrhiza, this clade contains xerophytic members (name-giving
taxon T. purpurea) and is in an isolated position within Tillandsia and not closely related to any
other subgroup. It comprises the following taxa investigated so far: T. aurea, T. cacticola,
T. purpurea, and T. straminea. Species of the Purpurea clade are distinguished from T. subg.
Phytarrhiza s.str. (simple-erect stigma type) by its conduplicate-spiral stigmas (Brown & Gilmartin, 1989b) and the often bicolored petals (crème-colored with violet tips), but for establishing a separate subgenus more taxa and accessions have to be investigated.
Tillandsia nana and T. pseudomicans are sister to this clade in the parsimony analysis, but
with weak BP (57). In Bayesian analysis T. sphaerocephala is sister to the Purpurea clade, but
also only weakly supported. Both scenarios are probably an artifact caused by missing Tillandsia taxa and sequence information.
Rauhii clade
Species of the Rauhii clade (T. ecarinata, T. ferreyrae, the name-giving T. rauhii, and T. teres)
have been previously classified within T. subg. Tillandsia (Smith & Downs, 1977). Gardner
166
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
(1982, 1986b) treated T. rauhii within group III of T. subg. Tillandsia, but this taxon is neither
related to this group of species, nor currently placed within T. subg. Tillandsia. Tillandsia ecarinata, T. ferreyrae and T. teres were not classified into any subunit of T. subg. Tillandsia and
treated as "incertae sedis" by Gardner (1982, 1986b), since floral characteristics could not be
studied at that time. Despite of missing plastid DNA sequence data for all taxa except T. rauhii,
placement of all four species within the Rauhii clade is still supported in parsimony analyses
with a weak BP (57). The Rauhii clade shows an isolated position within Tillandsia, but backbone relationships between this and other phylogenetic units are unsupported. Placement is
very likely to change if more DNA sequence data is gathered. Thus no formal unit is proposed
for species of the Rauhii clade until placement of these taxa is clarified.
Xiphioides clade
Taxa falling into this phylogenetic unit (name-giving taxon T. xiphioides) have been mainly
placed within T. subg. Anoplophytum based on the character of plicate filaments. According to
the tree topologies, this group constantly is closely related to subgenera Anoplophytum s.str.,
Diaphoranthema, Phytarrhiza s.str., the Gardneri clade and some unclassified taxa, i.e.,
T. albertiana, T. edithae and T. esseriana (Figure 2). Till (2000b) recognized close relationships
of taxa under both T. subg. Allardtia and T. subg. Anoplophytum sensu Smith & Downs (1977),
but shifted the xerophytic Andean members of T. subg. Anoplophytum with plicate filaments
to Allardtia. Genetically they are neither related to T. subg. Allardtia s.str. (which is now a synonym of T. subg. Tillandsia) nor to the rest of former Allardtia (= Biflora clade). From an evolutionary point of view it would have been better to shift xerophytic Andean members of
T. subg. Allardtia with straight filaments into T. subg. Anoplophytum. As T. subg. Anoplophytum
is currently circumscribed, the Xiphioides clade would need a separate infrageneric unit. However, due to the unresolved relationships of T. subg. Phytarrhiza and T. subg. Diaphoranthema
and the unclassified taxa mentioned earlier, as well as currently missing morphological characteristics, the Xiphioides clade is treated here as an informal unit within Tillandsia.
Incertae sedis—Unclassified Tillandsia species
Taxonomists always try to put taxa into a hierarchical scheme. Unfortunately there are a number of Tillandsia species that show either no or only unsupported affinities to certain groups,
but appear as separate, maybe relict ancient lineages, where their ancestors either did not
diversify or related species became extinct or have yet not been discovered. Unclassified investigated taxa so far are T. albertiana, T. australis, T. disticha, T. edithae, T. esseriana, T. nana,
T. pseudomicans and T. sphaerocephala. Morphological characteristics of these species seem
to be either plesiomorphic or derived involving convergence or parallelism. Currently we cannot assign these species with good phylogenetic support values to any infrageneric unit. Additional taxa and new molecular markers may help to place these taxa with better confidence in
the phylogenetic framework.
167
BARFUSS, M.H.J.
NEW CLASSIFICATION
Classification of Tillandsioideae
In classifying Tillandsioideae we considered aspects of biological (evolutionary) classification of
Hörandl & Stuessy (2010) and agree in principle with statements of Nickrent & al. (2010), who
gave a good overview of their philosophy in reclassifying Santalales into different families.
Although their classification was done on a higher level, their principal points apply in our view
to any taxonomic level. Classifications are most useful when they simultaneously serve many
functions as stressed by both studies mentioned above. Nickrent & al. (2010) list several criteria used to decide how to circumscribe taxonomic units. For the taxonomy of Tillandsioideae
we are currently emphasizing the following (Nickrent & al., 2010, slightly modified), arranged
according to their importance : (1) rejection of polyphyly, support for holophyly (= monophyly
s.str.) and paraphyly, if well supported, (2) phylogenetic information (= minimizing redundancy), (3) stability (= minimizing nomenclatural changes), (4) ease of identification (= recognizability), (5) recognition of groups that are well-established in the literature, but where supporting information is currently partly missing. Hörandl & Stuessy (2010: 1650) list also four main
criteria (theoretical foundation based on natural processes, predictivity, information content,
and practicability), which are similar to those mentioned above, and they recommend to adhere to these principles of biological classification by following a protocol of five procedural
steps. They emphasized the recognition of paraphyletic groups as natural units of biological
classification. Contrary to Nickrent & al. (2010) we agree with their opinion that paraphyly is a
natural stage in the evolution of taxa, and that it occurs regularly along with holophyly. However, in our view it should only be accepted for a final classification if all possible biological
sources are considered and well-supported conclusions can be drawn. For an acceptance of
conclusive, well-supported paraphyletic groups within Tillandsioideae, information from biological sources (in this study from DNA sequence data and morphology) is currently insufficient. Statistically unsupported paraphyly is preliminarily accepted for one already wellestablished taxon (T. subg. Diaphoranthema), where additional information could also lead to
holophyly; unsupported paraphyly may also be tentatively accepted in a not yet fully resolved
classification for a group, in which additional information is currently missing; it may be most
relevant for most-recently divergent plant groups and at the species level. Therefore, until
conclusive and well-supported clades in the complete phylogeny of all Tillandsioideae are presented, we are not describing or accepting new taxonomic units based on paraphyly, but it
could be of major importance in a future, more detailed classification than presented here.
Our main attempts are to adhere to the above emphasized five principles. Units that are well
characterized are formally recognized, whereas others, for which significant supporting information is missing, are treated as informal clades or unclassified taxa (= incertae sedis) in our
classification system.
The subfamilial classification including accepted tribal, subtribal, generic and subgeneric
ranks and informal clades is listed below in hierarchical, followed by alphabetical order. Two
new subtribes (Cipuropsidinae, Vrieseinae), three new genera (Josemania, Lemeltonia,
Rothowia), and three new subgenera (Racinaea subg. Pseudophytarrhiza, T. subg. Viridantha,
and T. subg. Pseudovriesea) are described. Several species are therefore reclassified. Lectotypes are selected for Catopsis subg. Tridynandra Mez, Tillandsia sect. Conostachys Griseb.,
Tillandsia sect. Eriophyllum K. Koch. A comparison of accepted genera of Tillandsioideae and
subgenera of Tillandsia to previous classification systems of either Smith & Downs (1977),
Smith & Till (1998), Till (2000a, b), or Barfuss & al. (2005) can be found in Tables 4 and 5, re168
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
spectively. Phylogenetic relationships of all investigated species and their current classification
are summarized in Figure 2 (see Appendix). A complete synonymy (homotypic and heterotypic)
is presented for all accepted taxa above the species level and for all reclassified species, taxonomic synonyms with the monograph of Smith & Downs (1977) being the primary reference
are given with investigated species mentioned to allow a comparison of the new vs. old taxonomic circumscriptions in the most critical groups. Morphological characteristics are listed for
all taxa above the subgeneric level and for newly described infrageneric units and the geographical distributions of well-characterized clades can be obtained from the key to the genera.
Bromeliaceae Juss. subfamily Tillandsioideae Burnett, Outl. Bot.: 442 (Jun 1835), as "Tillandsidae".—Type: Tillandsia L.
Fruit a septicidal capsule, seeds with hairs or hairlike appendages, wind dispersed.
Tribe Glomeropitcairnieae Harms, in Engl. & Prantl, Nat. Pflanzenfam. ed. 2, 15a: 100, 115
(1930).—Type: Glomeropitcairnia (Mez) Mez.
Ovary about ½–⅔ inferior, capsule only partly septicidal; petal appendages present; stigma of
the convolute-blade type; seeds with a long filiform, undivided chalazal appendage and a plumose micropylar flight apparatus; pollen with a diffuse aperture, exine reticulate.
1. Glomeropitcairnia (Mez) Mez, Bull. Herb. Boissier, sér. 2, 5: 232 (1905) ≡ Pitcairnia subg.
Glomeropitcairnia Mez, in C.DC., Monogr. phan 9: 463 (1896).—Type: Tillandsia penduliflora Griseb.
Tribe Pogospermeae Brongn., Ann. Sci. Nat. Bot., sér. 5, 1: 327 (1864).—Type: Pogospermum
Brongn. (= Catopsis Griseb.).
= Bromeliaceae tribe Catopsideae Harms, in Engl. & Prantl, Nat. Pflanzenfam. ed. 2, 15a:
100, 130 (1930).—Type: Catopsis Griseb.
Ovary max. ⅛ inferior to completely superior, capsule septicidal; petal appendages lacking;
stigma of the simple-erect type; flight apparatus of the seeds of multicellular chalazal hairs;
pollen with sharply cut aperture margins, exine reticulate (Catopsis type).
2. Catopsis Griseb., Nachr. Königl. Ges. Wiss. Georg-August-Univ. [1]: 10, 12 (13 Jan 1864) ≡
Catopsis subg. Eucatopsis Mez, in C.DC., Monogr. phan. 9: 619 (1896).—Type: Tillandsia
nitida Hook.
= Tussacia Willd. ex Beer, Fam. Bromel.: 99 (1856), non Rchb., Iconogr. bot. exot. 1: x, 28
(1827: Gesneriaceae) nec Willd. ex Schult. & Schult. f., in Roem. & Schult., Syst. veg. 7(1):
x, 57 (1829: Incertae sedis), nom illeg. (Art. 53.1) ≡ Tussaria Griseb., Fl. Brit. W. I.: 599
(1864), orth. var.—Type: Tillandsia vitellina Klotzsch = Catopsis nutans (Sw.) Griseb.
= Pogospermum Brongn., Ann. Sci. Nat. Bot. Sér. 5, 1: 327 (Jun 1864).—Type: Pogospermum flavum Brongn. = Catopsis nutans (Sw.) Griseb.
= Catopsis subg. Tridynandra Mez, in C.DC., Monogr. phan. 9: 620 (1896).—Lectotype
(proposed): Catopsis morreniana Mez.
169
BARFUSS, M.H.J.
NEW CLASSIFICATION
Tribe Tillandsieae Rchb., Consp. regn. veg.: 62 (Dec 1828–Apr 1829) ≡ Bromeliaceae tribe Tillandsieae Dumort., Anal. Fam. Pl.: 55 (Oct 1829) ≡ Bromeliaceae tribe Tillandzieae Rchb.,
in Uphof, Pflanzengattungen: 205 (1910), orth. var.—Type: Tillandsia L.
Ovary for max. ⅓ inferior, capsule septicidal; petal appendages usually lacking; stigma mainly
of the conduplicate-spiral or simple-erect type, rarely of the convolute-blade type (Guzmania p.p., Rothowia, Tillandsia subg. Viridantha p.p.), occasionally of the coralliform type
(Lemeltonia, Racinaea p.p.) or with pinnatisect margins (Josemania); seeds with a flight apparatus of pseudohairs at the micropylar end, endostome of type d (Groß, 1988) and embryo of
type g (Groß, 1988); pollen mainly with diffuse apertures, occasionally of the insulae type,
rarely of the Alcantarea type (former Vriesea imperialis type; Halbritter, 1988, 1992), or inaperturate.
3. Guzmania Ruiz & Pav., Fl. peruv. 3: 37, pl. 261 (1802).—Type: Guzmania tricolor Ruiz & Pav.
= Guzmania monostachia (L.) Rusby ex Mez.
= Caraguata Plum. ex Lindl., Bot. Reg. 13: sub pl. 1068 (1827), nom. illeg. (Art. 6.4), non
Plum. ex Adans. (1763).—Type: Tillandsia lingulata L.
= Devillea Bertero ex Schult. & Schult. f., in Roem. & Schult., Syst. veg. 7(2): 1229 (1830).—
Type: Devillea speciosa, nom. nud.
= Tillandsia sect. Conostachys Griseb., Nachr. Königl. Ges. Wiss. Georg-August-Univ. [1]: 19
(13 Jan 1864) ≡ Tillandsia subg. Conostachys (Griseb.) Baker, J. Bot. 26: 167 (1888) ≡
Vriesea subg. Conostachys (Griseb.) Mez, in Mart., Eichler & Urban, Fl. bras. 3(3): 516
(1894).—Lectotype (proposed): Tillandsia acorifolia Griseb.
= Massangea E. Morren, Belgique Hort. 27: 59, 199, pl. 8, 9 (1877) ≡ Caraguata [Plum. ex
Lindl.] subg. Massangea (E. Morren) Baker, Handb. Bromel.: 149 (1889).—Type: Tillandsia
musaica Linden & André.
= Sodiroa André, Bull. Soc. Bot. France 24: 167 "1877" (1878).—Lectotype (Smith &
Downs, 1977: 1275): Sodiroa graminifolia André ex Baker.
= Schlumbergeria E. Morren, Belgique Hort. 28: 311 (1878) ≡ Schlumbergera E. Morren,
Belgique Hort. 33: 46 (1883), orth. var., non Lem. (1858: Cactaceae) ≡ Caraguata [Plum. ex
Lindl.] subg. Schlumbergeria (E. Morren) Baker, Handb. Bromel.: 149 (1889).—Type:
Schlumbergeria roezlii E. Morren.
= Thecophyllum André, Bromel. Andr.: 107 (1889).—Type: Thecophyllum wittmackii André.
= Chirripoa Suess., Bot. Jahrb. Syst. 72: 293, pl. 4, Fig. 11 (1942).—Type: Chirripoa solitaria
Suess. = Guzmania polycephala Mez & Wercklé ex Mez
– Mezobromelia L.B. Sm. p.p., typo excluso.
Mesophytic, usually acaulescent; flowers mostly polystichously arranged; petals connate/conglutinate and mostly unappendaged; filaments free, anthers free or connate; pollen
inaperturate or with diffuse aperture; stigma of the simple-erect or convolute-blade type;
seeds with an endostome of type a and b, rarely e (Groß, 1988) and embryo of type a, rarely b
(Groß, 1988).
170
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Guzmania hutchisonii (L.B. Sm.) Barfuss & W. Till, nom. prov. ≡ Tillandsia hutchisonii L.B. Sm.,
Phytologia 13: 145, pl. 7, figs. 23, 24 (1966) ≡ Mezobromelia hutchisonii (L.B. Sm.)
W. Weber & L.B. Sm., J. Bromeliad Soc. 33(3): 121 (1983).—Type: Hutchison & Wright
6801: US (holo), UC, USC (iso).
= Mezobromelia trollii Rauh, Trop. Subtrop. Pflanzenwelt 21: 5, figs. 1–3 (1977).—Type:
Rauh 40104: HEID (holo), US (iso).
4. Josemania Barfuss & W. Till, nom. prov. pro Wallisia (Regel) E. Morren ≡ Tillandsia sect.
Wallisia Regel, Index Seminum Hort. Bot. Petrop. "1868": 92 (Mar 1869) ≡ Wallisia (Regel)
E. Morren, Belgique Hort. 20: 97 (1870), nom. illeg. (Art. 52.1) ≡ Vallesia C. Wright ex Sauvalle, Anales Acad. Ci. Méd. Fís. Nat. Habana 8: 53 (1871), orth. var., non Vallesia Ruiz &
Pav. (1794: Apocynaceae) ≡ Tillandsia subg. Wallisia (Regel) Baker, J. Bot. 26: 46 (1888).—
Type: Tillandsia lindenii Regel.
– Tillandsia subg. Phytarrhiza (Vis.) Baker p.p., typo excluso.
Leaves narrowly triangular, usually longitudinally reddish striped near the base, inflorescence
distichous, usually ovate-laceolate in outline (except Josemania umbellata), petals blue-violet
with a strongly widened blade and constricted basally into a claw, stamens and style deeply
included within the corolla, anthers subbasifixed, stigma lobes with pinnatisect and papillate
margins, ovules slenderly cylindric, obtuse.
Named after José Manuel Manzanares (1957–) from Quito, Ecuador, leading authority of Ecuadorian Bromeliaceae.
Josemania anceps (Lodd.) Barfuss & W. Till, nom. prov. ≡ Tillandsia anceps Lodd., Bot. Cab. 8:
pl. 771 (1823) ≡ Platystachys anceps (Lodd.) Beer, Fam. Bromel.: 80 (1856) ≡ Vriesea anceps (Lodd.) Lem., Ill. Hort. 6 (Misc.): 15 (1859) ≡ Phytarrhiza anceps (Lodd.) E. Morren,
Belgique Hort. 29: 368, pls. 20, 21 (1879).—Type: Adam in Loddiges Hort. s.n.: CGE?, K?
(holo), in absence: Original illustration.
= Tillandsia lineatifolia Mez, in C.DC., Monogr. phan. 9: 686 (1896).—Type: Fendler 2447:
GOET (holo).
Josemania cyanea (Linden ex K. Koch) Barfuss & W. Till, nom. prov. ≡ Tillandsia cyanea Linden
ex K. Koch, Wochenschr. Vereines Beförd. Gartenbaues Königl. Preuss. Staaten 10: 140
(1867).—Type: Linden Hort. s.n.: B? (holo).
= Tillandsia lindenii E. Morren, Belgique Hort. 19: 321, pl. 18 (Nov 1869), as „lindeni", nom.
illeg., non Regel (Mar 1869) ≡ Vriesea lindenii (E. Morren) Lem., Ill. Hort. 16: pl. 610 (1869)
, nom. illeg. ≡ Tillandsia morreniana Regel, Gartenflora 19: 41 (1870) ≡ Wallisia lindenii
(E. Morren) E. Morren, Belgique Hort. 20: 102 (1870), nom. illeg. ≡ Phytarrhiza lindenii
(E. Morren) E. Morren, Belgique Hort. 29: 297 (1879), nom. illeg. ≡ Tillandsia lindenii
[E. Morren] var. genuina E. Morren, Gard. Chron., ser. 2, 12: 460 (1879) ≡ Phytarrhiza lindenii var. genuina E. Morren, Belgique Hort. 29: 297 (1879).—Type: Wallis in Linden Hort.
s.n.: LG? (holo).
= Tillandsia coerulea Linden ex K. Koch, Wochenschr. Vereines Beförd. Gartenbaues Königl. Preuss. Staaten 13: 197 (1870), nom. nud.
= Tillandsia lindenii [E. Morren] vera Dombrain, Floral Mag. 11: pl. 44 (1872).—Type: Original illustration.
?= Tillandsia lindenii [E. Morren] var. violacea hort. ex André, Rev. Hort. 58: 61 (1886).—
Type: not indicated.
171
BARFUSS, M.H.J.
NEW CLASSIFICATION
= Tillandsia lindenii [E. Morren] superba rosea Dauthenay, Rev. Hort. 70: 539 (1898), nom.
illeg.—Type: not indicated.
= Tillandsia lindenii [E. Morren] vera superba Duval, Gartenwelt 5: 164, Fig. (1901), nom.
illeg.—Type: not indicated.
Josemania lindenii (Regel) Barfuss & W. Till, nom. prov. ≡ Tillandsia lindenii Regel, Index Seminum Hort. Bot. Petrop. "1868": 92 (Mar 1869) ≡ Wallisia lindenii (Regel) E. Morren, Belgique Hort. 20: 97 (1870), nom. illeg.—Type: Wallis in St. Petersburg Hort. s.n.: LE (holo).
?= Tillandsia lindeniana Regel, Gartenflora 18: 193, pl. 619 (Jul 1869).—Type: Linden Hort.
s.n.: LE? (holo).
= Tillandsia lindenii [E. Morren] var. luxurians E. Morren, Belgique Hort. 21: 289, pls. 20,
21 (1871) ≡ Phytarrhiza lindenii var. luxurians E. Morren (E. Morren), Belgique Hort. 29:
299 (1879) ≡ Tillandsia lindenii [E. Morren] var. luxurians (E. Morren) L.B. Sm., Contr. U. S.
Natl. Herb. 29: 494 (1951), comb. illeg. superfl. ≡ Tillandsia lindenii [E. Morren] var. abundans L.B. Sm., Phytologia 20: 166 (1970), nom. nov. superfl.—Type: Belgique Hort. 21: pls.
20, 21 (1871).
= Phytarrhiza lindenii var. koutsinskyana E. Morren, Belgique Hort. 30: 80 (1880) ≡ Tillandsia lindenii [E. Morren] var. koutsinskyana (E. Morren) L.B. Sm., Contr. U. S. Natl. Herb. 29:
494 (1951).—Type: Warsaw Hort. s.n.: LG?, WA? (holo).
?= Tillandsia lindenii [E. Morren var.] latispatha Van Houtte ex André, Rev. Hort. 60: 201
(1888).—Type: Van Houtte s.n.: K? (holo).
= Tillandsia lindenii [E. Morren] var. duvalii Duval ex André, Rev. Hort. 71: 516 (1899) ≡ Tillandsia lindenii [Regel] var. × duvalii (Duval ex André) L.B. Sm., Contr. U. S. Natl. Herb. 29:
493 (1951) ≡ Tillandsia lindenii [Regel] var. duvaliana L.B. Sm., Phytologia 20: 166 (1970),
nom. nov. superfl.—Type: Duval Hort. s.n.: ?
= Tillandsia lindenii Hasack, Möllers Deutsche Garten-Z. 15: 93, Fig. (1900) non Regel
(1869), nom. illeg.—Type: Original illustration.
= Tillandsia × duvali Duval, Gartenwelt 5: 164, Fig. (1901).—Type: Duval Hort. s.n.: ? (holo), in absence: Original illustration.
= Tillandsia lindenii [Regel] var. caeca D. Barry, Bromeliad Soc. Bull. 12: 5 (1962) ≡ Tillandsia lindenii [Regel] var. × caeca D. Barry, in Smith & Downs, Fl. Neotrop. 14(2): 846
(1977).—Type: Barry Hort. s.n.: US (holo).
Josemania pretiosa (Mez) Barfuss & W. Till, nom. prov. ≡ Tillandsia pretiosa Mez, Repert.
Spec. Nov. Regni Veg. 16: 78 (1919).—Type: Sodiro 171/39: B (holo).
= Tillandsia cyanea var. elatior L.B. Sm., Phytologia 5: 181 (1955).—Type: Fagerlind & Wibom 1947: S (holo).
Josemania umbellata (André) Barfuss & W. Till, nom. prov. ≡ Tillandsia umbellata André, Rev.
Hort. 58: 60, pl. (1886).—Type: André K-317: K (holo).
= Tillandsia lindenii [E. Morren] var. regeliana E. Morren, Belgique Hort. 20: 225, pl. 12
(1870) ≡ Phytarrhiza lindenii var. regeliana (E. Morren) E. Morren, Belgique Hort. 29: 298
(1879).—Type: Wallis s.n.: LG? (holo).
= Tillandsia lindenii [E. Morren] var. major Dombrain, Floral Mag. 10: pl. 529 (1871) ≡ Tillandsia lindenii [E. Morren] var. intermedia E. Morren, Rev. Hort. 50: 390 (1878), nom.
nov. superfl. ≡ Phytarrhiza lindenii var. intermedia E. Morren, Belgique Hort. 29: 298
(1879), nom. illeg.—Type: Veitch Hort. s.n.: ? (holo), in absence: Original illustration.
172
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
?= Vriesea violacea hort. ex Houllet, Rev. Hort. 44: 230 (1872), nom. nud.
= Tillandsia lindenii [E. Morren] var. rutilans Linden ex Houllet, Rev. Hort. 44: 230 (1872),
nom. nud.
= Tillandsia lindenii [E. Morren] var. tricolor André, Ill. Hort. 24: 190 (1877) ≡ Tillandsia cyanea var. tricolor (André) L.B. Sm., Contr. U. S. Natl. Herb. 29: 491 (1951).—Type: André
4040: K (holo).
= Tillandsia lindenii [E. Morren var.] splendida Carrière, Rev. Hort. 54: 12, pl. (1882).—
Type: Thibaut & Keteleer Hort s.n.: ? (holo).
?= Tillandsia lindenii [E. Morren] var. violacea hort. ex André, Rev. Hort. 58: 61 (1886),
nom. nud.
= Tillandsia lindenii [E. Morren] vera major Duval, Gartenwelt 5: 164, Fig. (1901), nom. illeg.—Type: Original illustration.
5. Lemeltonia Barfuss & W. Till, nom. prov.—Type: Tillandsia dodsonii L.B. Sm.
A genere Tillandsia L. s.str. filamentis basi connatis, stigmate coralliforme et ovulis obtusis
claviformibusque, seminibus cum endostomio breviter cylindrico et embryone cum radicula
plusminusve distincta differt. Petala alba vel rariter flavescentia.
– Tillandsia subg. Phytarrhiza (Vis.) Baker p.p., typo excluso.
Plants acaulescent or rarely caulescent; leaves very narrowly triangular, not forming a tank
rosette; inflorescence green or brown, lax with the often fragrant flowers mostly spreading;
petals white or rarely yellowish; stamens and style deeply included within the corolla, filaments connate among themselves at least at the base; stigma coralliform (Brown & Gilmartin
1989b); ovules clavate, obtuse; seeds with a short-cylindric endostome (type d, Groß 1988)
and embryo with the radicule ± separated (type b, Groß 1988).
Named after Elton Martinez Carvalho Leme (1960–) from Rio de Janeiro, Brazil, leading authority of Brazilian Bromeliaceae.
Lemeltonia acosta-solisii (Gilmartin) Barfuss & W. Till, nom. prov. ≡ Tillandsia acosta-solisii
Gilmartin, Phytologia 16: 160 (1968).—Type: Teuscher 2275-56: US (holo).
Lemeltonia cornuta (Mez & Sodiro) Barfuss & W. Till, nom. prov. ≡ Tillandsia cornuta Mez &
Sodiro, Bull. Herb. Boissier, sér. 2, 5: 106 (1905).—Type: Sodiro 171/42: B (holo).
Lemeltonia dodsonii (L.B. Sm.) Barfuss & W. Till, nom. prov. ≡ Tillandsia dodsonii L.B. Sm., Phytologia 28: 32, pl. 2, figs. f, g (1974).—Type: Dodson 5225: US (holo).
Lemeltonia monadelpha (E. Morren) Barfuss & W. Till, nom. prov. ≡ Phytarrhiza monadelpha
E. Morren, Belgique Hort. 32: 168, pl. 7 (1882) ≡ Tillandsia monadelpha (E. Morren) Baker,
J. Bot. 25: 281 (1887).—Type: Linden Hort. s.n.: LG (holo).
= Tillandsia graminifolia Baker, J. Bot. 25: 281 (1887).—Type: Martin s.n.: BM (lecto); Poiteau s.n.: P? (syn); Sagot 859: P (syn); Fendler 828: K (syn); Parker s.n.: K (syn).
= Catopsis (Andrea) alba E. Morren ex Baker, Handb. Bromel.: 192 (1889), nom. nud.
= Tillandsia monobotrya Mez, Repert. Spec. Nov. Regni Veg. 16: 77 (1919).—Type:
Wercklé 150 = Inst. Costar. 17444: B (holo).
Lemeltonia narthecioides (C. Presl) Barfuss & W. Till, nom. prov. ≡ Tillandsia narthecioides C.
Presl, Reliq. haenk. 1: 125 (1827).—Type: Haenke s.n.: PR (holo).
173
BARFUSS, M.H.J.
NEW CLASSIFICATION
Lemeltonia scaligera (Mez & Sodiro) Barfuss & W. Till, nom. prov. ≡ Tillandsia scaligera Mez &
Sodiro, Bull. Herb. Boissier, sér. 2, 5: 107 (1905).—Type: Sodiro 171/4: B (holo).
Lemeltonia triglochinoides (C. Presl) Barfuss & W. Till, nom. prov. ≡ Tillandsia triglochinoides
C. Presl, Reliq. haenk. 1: 125 (1827).—Type: Haenke s.n.: PR (holo).
= Tillandsia hartwegiana Brongn. ex Baker, Handb. Bromel.: 171 (1889), nom. nud.
6. Racinaea M.A. Spencer & L.B. Sm., Phytologia 74: 152 (1993).—Type: Tillandsia cuspidata
L.B. Sm.
6.1. Racinaea subg. Pseudophytarrhiza Barfuss & W. Till, nom. prov.—Type: Tillandsia venusta
Mez & Wercklé.
A subgenere typica petalis latioribus unguiculatibusque et stigmate coralliforme differt.
Petals with broader blade and distinct claw; filaments free; stigma coralliform.
– Tillandsia subg. Phytarrhiza (Vis.) Baker p.p., typo excluso.
Racinaea hamaleana (E. Morren) Barfuss & W. Till, nom. prov. ≡ Tillandsia hamaleana
E. Morren, Gard. Chron. "1869"(2): 460 (1869) ≡ Wallisia hamaleana (E. Morren)
E. Morren, Belgique Hort. 20: 97, pl. 5 (1870), nom. illeg. ≡ Phytarrhiza hamaleana
(E. Morren) E. Morren, Belgique Hort. 29: 297 (1879).—Type: Wallis s.n. in Hort.
E. Morren: LG? (holo).
= Tillandsia commelyna E. Morren, Belgique Hort. 20: 97 (1870), nom. nud.
= Tillandsia platypetala Baker, J. Bot. 26: 46 (1888).—Type: Hartweg s.n.: K (holo).
= Tillandsia nubis Gilmartin, Phytologia 16: 161 (1968).—Type: Naundorff s.n.: US (holo).
Racinaea venusta (Mez & Wercklé) Barfuss & W. Till, nom. prov. ≡ Tillandsia venusta Mez &
Wercklé, Bull. Herb. Boissier, sér. 2, 5: 108 (1905).—Type: Wercklé Bromel. Costar. 95: B
(holo).
6.2. Racinaea subg. Racinaea.
= Tillandsia subg. Pseudo-Catopsis Baker, Handb. Bromel.: 157, 192 (Aug–Oct 1889).—
Type: Tillandsia parviflora Ruiz & Pav.! (Baker 1889: 192).
= Tillandsia sect. Pseudo-Catopsis André, Bromel. Andr.: 62, 66 (Sept–Dec 1889), nom. illeg. (Art. 53.4).—Lectotype (Smith & Downs, 1977: 670): Tillandsia ropalocarpa André.
– Tillandsia subg. Phytarrhiza (Vis.) Baker p.p., typo excluso.
Mesophytic, usually acaulescent; petals free and unappendaged; filaments and anthers free;
pollen aperture diffuse or with insulae; stigma simple-erect or slightly conduplicate-spiral,
rarely coralliform.
Racinaea dyeriana (André) Barfuss & W. Till, nom. prov. ≡ Tillandsia dyeriana André, Énum.
Bromél. 1888: 8 (13 Dec 1888).—Type: André 4256: K (holo), NY (iso).
= Tillandsia rutschmannii Rauh, Trop. Subtrop. Pflanzenwelt 12: 5, figs. 1–3, [4a], 5
(1974).—Type: Naundorff s.n. = Hort. Bot. Heidelberg 31701: HEID (holo), US (iso).
7. Rothowia Barfuss & W. Till, nom. prov.—Type: Tillandsia wagneriana L.B. Sm.
A genere Tillandsia L. s.str. stigmate obconico typo laminis-convolutis simili, ovulis obtusis vel
subobtusis anguste subcylindrico-claviformibus, seminibus cum endostomio conico vel cylindrico et embryone cum radicula plusminusve distincta differt. Petala caerulea.
– Tillandsia subg. Phytarrhiza (Vis.) Baker p.p., typo excluso.
174
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Plants acaulescent; leaves lingulate, acute, with a distinct leaf-sheath, forming a tank-rosette;
inflorescence pink, once to twice branched, rhachis ± alate, floral bracts glabrous; petals blue;
stamens and style deeply included within the corolla; filaments lingulate, narrowed at the
apex, anthers basifixed; stigma obconic, resembling the convolute-blade type; ovules slenderly
cylindric, obtuse or subobtuse; seeds with a conical or cylindrical endostome (type e, Groß
1988) and embryo with a ± distinct radicule (type b, Groß 1988).
Named after Ronald Thomas Wagner (1939–1962).
Rothowia laxissima (Mez) Barfuss & W. Till, nom. prov. ≡ Tillandsia laxissima Mez, Bull. Herb.
Boissier, sér. 2, 5: 108 (1905).—Type: Bang 2301 p.p.: B (holo), G, GH, LE, M, MO, NY, PH,
US, W, WU (iso).
Rothowia laxissima var. moorei (H. Luther) Barfuss & W. Till, nom. prov. ≡ Tillandsia laxissima
var. moorei H. Luther, Selbyana 20(1): 13 (1999).—Type: Moore s.n.: SEL (holo).
Rothowia platyrhachis (Mez) Barfuss & W. Till, nom. prov. ≡ Tillandsia platyrhachis Mez, in
C.DC., Monogr. phan. 9: 848 (1896).—Type: Kalbreyer 1328: K (holo).
= Tillandsia platyrhachis var. alba Rauh & Hirtz, Trop. Subtrop. Pflanzenwelt 53: 11, figs.
5–7 (1985).—Type: Rauh & Hirtz 37571: HEID (holo).
= Tillandsia platyrhachis var. magnifica Rauh & von Bismarck, Trop. Subtrop. Pflanzenwelt
53: 6, figs. 2–5 (1985).—Type: Rauh & von Bismarck 66107: HEID (holo).
Rothowia wagneriana (L.B. Sm.) Barfuss & W. Till, nom. prov. ≡ Tillandsia wagneriana
L.B. Sm., Phytologia 9: 254, pl. 4, figs. 1–3 (1963).—Type: Moore 310: US (holo).
8. Tillandsia L., Sp. pl.: 286 (1753).—Type: Tillandsia utriculata L.
Meso- to xerophytic, acaulescent to caulescent; petals free, usually not appendaged; filaments
and anthers free; pollen aperture diffuse, with insulae or operculum, or of the Alcantarea type;
stigma simple-erect or conduplicate-spiral, rarely of the convolute-blade type, seeds of various
endostome types (Groß, 1988: e–n) and embryo types (Groß, 1988: b–g).
8.1. Tillandsia subg. Anoplophytum (Beer) Baker, J. Bot. 25: 212 (1887) ≡ Anoplophytum Beer,
Flora 37: 346 (1854) ≡ Tillandsia sect. Anoplophytum (Beer) Griseb., Fl. Brit. W. I.: 597
(1864).—Type: Tillandsia stricta Sol. ex Sims.
= Tillandsia sect. Eriophyllum K. Koch, Index Seminum Hort. Bot. Berol. "1873", App. 4: 1
(1874), as “Eriophorum”.—Lectotype (proposed): Tillandsia selloa K. Koch. (= Tillandsia
linearis Vell.).
8.2. Tillandsia subg. Diaphoranthema (Beer) Baker, J. Bot. 16: 236 (1878) ≡ Diaphoranthema
Beer, Flora 37: 349 (1854) ≡ Tillandsia sect. Diaphoranthema (Beer) K. Koch, Index Seminum Hort. Bot. Berol. "1873", App. 4: 1 (1874).—Type: Renealmia recurvata L.
= Dendropogon Raf., Neogenyton: 3 (1825) ≡ Tillandsia sect. Strepsia Nutt., Gen. N. Amer.
pl. 1: 208 (1818) ≡ Strepsia (Nutt.) Steud., Nomencl. Bot. ed. 2.2: 645 (1841), nom. illeg.—
Type: Renealmia usneoides L.
8.3. Tillandsia subg. Phytarrhiza (Vis.) Baker, J. Bot. 25: 212, 214 (1887) ≡ Phytarrhiza Vis.,
Mem. R. Ist. Venet. Sci. 5: 340, pl. (1855) ≡ Tillandsia sect. Phytarrhiza (Vis.) K. Koch, Index
Seminum Hort. Bot. Berol. "1873", App. 4: 1 (1874).—Type: Tillandsia duratii Vis.
175
BARFUSS, M.H.J.
NEW CLASSIFICATION
8.4. Tillandsia subg. Pseudalcantarea Mez, in Engl., Pflanzenr. 4.32: 437, 455 (1935).—Type:
Platystachys viridiflora Beer.
8.5. Tillandsia subg. Pseudovriesea Barfuss & W. Till, nom. prov.—Type: Tillandsia tequendamae André.
Folia anguste triangularia, xeromorphica, dense lepidota, petala plerumque biappendiculata,
saepe bicoloria, rariter marginibus crenulatis, stigma laminis conduplicato-spiralibus.
= Tillandsia sect. Trianisandra André, Bromel. Andr.: 63 (1889).—Type: Tillandsia heterandra André.
− Tillandsia subg. Allardtia A. Dietr. p.p., typo excluso.—studied taxa: T. myriantha Baker,
T. barthlottii Rauh.
− Tillandsia subg. Tillandsia p.p., typo excluso.—studied taxa: T. spathacea Mez & Sodiro.
− Vriesea Lindl. p.p., typo excluso.—studied taxa: xerophytic, grey-leaved Vriesea spp. (see
Appendix for Figure 2 and supplementary data for all spp. included).
Leaves narrowly triangular, xeromorphic, densely lepidote, petals usually with two basal appendages, often two-colored, sometimes with crenulated margins, stigma usually conduplicate-spiral, rarely conduplicate-erect, pollen with diffuse sulcus area; seeds with an elongatecylindric endostome (type f, Groß, 1988), embryo of type (b–) or ± f (Groß, 1988).
8.6. Tillandsia subg. Tillandsia ≡ Caraguata Plum. ex Adans., Fam. pl. 2: 67, 532 (Jul–Aug
1763), nom. illeg. (Art. 52.1).—Type: Tillandsia utriculata L.
= Renealmia L., Sp. pl.: 286 (1753), nom. rejic. vs. Renealmia L. f. (1782).—Type: Renealmia paniculata L.
= Bonapartea Ruiz & Pav., Fl. peruv. 3: 38, pl. 262 (1802) ≡ Misandra F. Dietr., Nachtr.
vollst. Lex. Gärtn. 5: 102 (1819) non Comm. ex Juss. (1789: Haloragaceae), nom. illeg. ≡
Acanthospora Spreng., Syst. veg. 2: 25 (1825), nom. illeg. ≡ Buonapartea Sweet, Hort. brit.
ed. 3: 706 (1839), orth. var.—Type: Bonapartea juncea Ruiz & Pav.
= Allardtia A. Dietr., Berliner Allg. Gartenzeitung 20: 241 (1852) ≡ Platystachys K. Koch, Index Seminum Hort. Bot. Berol. "1854", App.: 11 (1855), nom. illeg. ≡ Tillandsia sect. Allardtia (A. Dietr.) E. Morren, Belgique Hort. 27: 272 (1877) ≡ Tillandsia subg. Allardtia (A.
Dietr.) Baker, J. Bot. 26: 40 (1888).—Type: Allardtia cyanea A. Dietr. (≡ Tillandsia guatemalensis L.B. Sm.).
= Platystachys Beer, Fam. Bromel.: 18, 80 (1856), nom. illeg. (Art. 53, Note 1) ≡ Tillandsia
sect. Platystachys (K. Koch) Benth. & Hook. f., Gen. pl. 3(2): 670 (1883) ≡ Tillandsia subg.
Platystachys (K. Koch) Baker, J. Bot. 25: 212, 236 (1887).—Lectotype (Smith & Downs,
1977: 668): Tillandsia setacea Sw.
= Pityrophyllum Beer, Fam. Bromel.: 17, 79 (1856) ≡ Tillandsia sect. Pityrophyllum (Beer) K.
Koch, Index Seminum Hort. Bot. Berol. "1873", App. 4: 1 (1874) ≡ Tillandsia subg. Pityrophyllum (Beer) Baker, J. Bot. 26: 39 (1888).—Type: Tillandsia ionantha Planch.
= Vriesea sect. Cylindrostachys Wittm., in Engl. & Prantl, Nat. Pflanzenfam. II.4: 59 (1888)
≡ Vriesea subg. Cylindrostachys (Wittm.) Harms, in Engl., Nat. Pflanzenfam. ed. 2, 15a: 124
(1930).—Type: Vriesea malzinei E. Morren.
− Tillandsia subg. Pseudalcantarea Mez p.p., typo excluso.—studied taxa: T. paniculata (L.)
L.
− Vriesea sect. Xiphion (E. Morren) Wawra ex Wittm. p.p., typo excluso.—studied taxa: V.
malzinei E. Morren.
176
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
8.7. Tillandsia subg. Viridantha (Espejo) Barfuss & W. Till, nom. prov. ≡ Viridantha Espejo, Acta
Bot. Mex. 60: 27 (2002).—Type: Tillandsia plumosa Baker.
= Viridantha sect. Caulescens Espejo, Acta Bot. Mex. 60: 31 (2002).—Type: Tillandsia tortilis Klotzsch ex Baker.
− Tillandsia subg. Allardtia A. Dietr. p.p., typo excluso.—studied taxa: Tillandsia plumosa
complex, Tillandsia tectorum complex (see Appendix for Figure 2 and supplementary data
for all spp. included).
− Tillandsia subg. Anoplophytum p.p., typo excluso.—studied taxa: T. heteromorpha Mez.
− Tillandsia subg. Tillandsia p.p., typo excluso.—studied taxa: T. lepidosepala L.B Sm.
8.8. Biflora clade.
Name giving taxon: Tillandsia biflora Ruiz & Pav.
− Tillandsia subg. Allardtia A. Dietr. p.p. majore, typo excluso (see Appendix for Figure 2
and supplementary data for all spp. included).
− Tillandsia subg. Pseudalcantarea Mez p.p., typo excluso.—studied taxa: T. baliophylla
Harms.
– Tillandsia subg. Anoplophytum (Beer) Baker p.p., typo excluso.—studied taxa: T. macbrideana L.B. Sm., T. pseudomacbrideana Rauh.
− Tillandsia L. subg. Tillandsia p.p., typo excluso.—studied taxa: T. deppeana Steud., T.
imperialis E. Morren ex Roezl, T. multicaulis Steud.
8.9. Gardneri clade.
Name giving taxon: Tillandsia gardneri Lindl.
– Tillandsia subg. Anoplophytum (Beer) Baker p.p., typo excluso.—studied taxa: T. brachyphylla Baker, T. gardneri Lindl., T. globosa Wawra.
8.10. Purpurea clade.
Name giving taxon: Tillandsia purpurea Ruiz & Pav.
– Tillandsia subg. Phytarrhiza (Vis.) Baker p.p., typo excluso.—studied taxa: T. aurea Mez,
T. cacticola L.B. Sm., T. purpurea Ruiz & Pav., T. straminea Kunth.
8.11. Rauhii clade.
Name giving taxon: Tillandsia rauhii L.B. Sm.
− Tillandsia L. subg. Tillandsia p.p., typo excluso.—studied taxa: T. ecarinata L.B. Sm., T.
ferreyrae L.B. Sm., T. rauhii L.B. Sm., T. teres L.B. Sm..
8.12. Xiphioides clade.
Name giving taxon: Tillandsia xiphioides Ker Gawl.
– Tillandsia subg. Anoplophytum (Beer) Baker p.p., typo excluso.—studied taxa: xerophytic, Andean spp. (see Appendix for Figure 2 and supplementary data for all spp. included).
− Tillandsia subg. Allardtia A. Dietr. p.p., typo excluso.—studied taxa: xerophytic, Andean
spp. (see Appendix for Figure 2 and supplementary data for all spp. included).
8.13. Incertae sedis.
The following taxa are currently unclassified within Tillandsia L.: T. albertiana Verv., T. australis
Mez, T. disticha Kunth, T. edithae Rauh, T. esseriana Rauh & L.B. Sm., T. nana Baker, T. pseudomicans Rauh, T. sphaerocephala Baker.
177
BARFUSS, M.H.J.
NEW CLASSIFICATION
Tribe Vrieseeae W. Till & Barfuss, Amer. J. Bot. 92: 348 (2005).—Type: Vriesea Lindl.
Ovary for a small part inferior, capsule septicidal; petal appendages usually present; stigma
mainly of the convolute-blade and cupulate types, less often of the tubolaciniate, conduplicate-erect, conduplicate-patent, conduplicate-spiral, or simple-erect types; seeds with a flight
apparatus of pseudohairs at the micropylar end; pollen mainly with exine fragments at the
aperture (insulae type) or with solid aperture margins (Alcantarea type), less often with a diffuse aperture.
Subtribe Cipuropsidinae Barfuss & W. Till, nom. prov.—Type: Cipuropsis Ule.
A subtribu typica ovulis obtusis vel subobtusis et stigmatibus simplicibus-erectis, condplicatospialibus vel cupulatis papillosisque differt.
Ovules obtuse or subobtuse, stigma of the simple-erect, conduplicate-spiral, or cupulate types
with papillae (Brown & Gilmartin 1989b).
9. Cipuropsis Ule, Verh. Bot. Vereins Prov. Brandenburg 48: 148 (1907).—Type: Cipuropsis
subandina Ule.
– Tillandsia subg. Allardtia A. Dietr. p.p., typo excluso.—studied taxa: T. amicorum I. Ramírez & Bevil.
− Vriesea sect. Xiphion (E. Morren) Wawra ex Wittm. p.p., typo excluso.—studied taxa: V.
zamorensis (L.B. Sm.) L.B. Sm.
Petals connate for 25% into a common tube, filaments connate for 5 mm with the petals like
the petals themselves, petal appendages present; seeds with an endostome of type c (Groß,
1988) and embryo of type a (Groß, 1988).
Cipuropsis amicorum (I. Ramírez & Bevil.) Barfuss & W. Till, nom. prov. ≡ Tillandsia amicorum
I. Ramírez & Bevil., Acta Bot. Venez. 15: 149, pl. 1 (1989).—Type: Rutkis 452: VEN (holo);
Steyermark, Bu[n]ting & Wessels-Boer 100258: MO, VEN (para); Carreño s.n.: VEN (para);
Steyermark & Wessels-Boer 100448: MO, VEN (para).
Cipuropsis zamorensis (L.B. Sm.) Barfuss & W. Till, nom. prov. ≡ Tillandsia zamorensis L.B. Sm.,
Phytologia 4: 213, pl. 1, figs. 3–5 (1953) ≡ Vriesea zamorensis (L.B. Sm.) L.B. Sm., Phytologia 20: 174 (1970).—Type: Scolnik 1500: NY (holo).
10. Mezobromelia L.B. Sm., Proc. Amer. Acad. Arts 70: 151 (1935).—Type: Mezobromelia bicolor L.B. Sm.
– Tillandsia subg. Allardtia A. Dietr. p.p., typo excluso.—studied taxa: ?T. buseri Mez, T.
schimperiana Wittm.
– Vriesea sect. Xiphion (E. Morren) Wawra ex Wittm. p.p., typo excluso.—studied taxa: V.
rubrobracteata Rauh.
Mesophytic, acaulescent; flower arrangement distichous or polystichous; petals connate/conglutinate and appendaged; filaments free, anthers connate; pollen with diffuse aperture or inaperturate; stigma of the simple-erect type.
178
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Mezobromelia schimperiana (Wittm.) Barfuss & Manzan, nom. prov. ≡ Tillandsia schimperiana
Wittm., Bot. Jahrb. Syst. 11: 67 (1889).—Type: Lehmann XXVI: G (holo).
= Vriesea rubrobracteata Rauh, Trop. Subtrop. Pflanzenwelt 27: 13, Fig. 5 (1979).—Type:
Rauh 37412: HEID (holo).
?= Tillandsia buseri Mez, Bull. Herb. Boissier, sér. 2, 3: 145 (1903).—Type: Langlassé 102a:
G (holo), B, K (iso).
11. Werauhia J.R. Grant, Trop. Subtrop. Pflanzenwelt 91: 28 (1995) ≡ Tillandsia sect. Xiphion
E. Morren, Belgique Hort. 24: 292 (1874), Lectotype (Smith & Downs, 1977: 1069) ≡
Vriesea sect. Xiphion (E. Morren) Wawra ex Wittm., in Engl. & Prantl, Nat. Pflanzenfam.
II.4: 59 (1888).—Type: Tillandsia gladioliflora H. Wendl.
= Werauhia sect. Jutleya J.R. Grant, Trop. Subtrop. Pflanzenwelt 91: 28, 39.—Type:
Thecophyllum pedicellatum Mez & Wercklé.
– Tillandsia subg. Allardtia A. Dietr. p.p., typo excluso.—studied taxa: T. insignis Mez.
Mesophytic, usually acaulescent; petals free, mostly appendaged; filaments and anthers free;
pollen of the insulae type; stigma cupulate without papillae; seeds with an endostome of type
d (Groß, 1988) and embryo of type a, rarely b (Groß, 1988).
12. Chrysostachys clade.
Name giving taxon: Vriesea chrystostachys E. Morren.
– Vriesea sect. Xiphion (E. Morren) Wawra ex Wittm. p.p., typo excluso.—studied taxa: V.
chrystostachys E. Morren, V. ospinae H. Luther.
13. Singularis clade.
Name giving taxon: Tillandsia singularis Mez & Wercklé.
– Tillandsia subg. Allardtia p.p., typo excluso.—studied taxa: T. asplundii L.B. Sm., T. singularis Mez & Wercklé.
14. Splendens clade.
Name giving taxon: Vriesea splendens (Brongn.) Lem.
– Vriesea sect. Vriesea p.p., typo excluso.—studied taxa: V. splendens (Brongn.) Lem..
– Vriesea sect. Xiphion (E. Morren) Wawra ex Wittm. p.p., typo excluso.—studied taxa: V.
glutinosa Lindl.
15. Incertae sedis.
The following taxon is currently unclassified within the Cipuropsis-Mezobromelia clade: Vriesea
tuerckheimii (Mez) L.B. Sm.
Subtribe Vrieseinae Barfuss & W. Till, nom. prov.—Type: Vriesea Lindl.
Ovula distincte appendiculata in chalaza, stigmata laminis convolutis, conduplicatis vel
tubolaciniatis.
Ovules distinctly appendaged at the chalaza, stigma of the convolute-blade (Brown & Gilmartin
1989b) or tubolaciniate type (Leme & Brown, 2004: Vriesea plurifolia) in Vriesea, and of the
conduplicate-patent (Versieux & Wanderley, 2007; Leme 2007) or rarely of the conduplicateerect (Leme, 2009: Alcanatrea roberto-kautskyi; Leme, unpubl. data: Alcantarea cerosa) in
Alcantarea.
179
BARFUSS, M.H.J.
NEW CLASSIFICATION
16. Alcantarea (E. Morren ex Mez) Harms, Notizbl. Bot. Gart. Berlin-Dahlem 10: 802 (1929) ≡
Vriesea subg. Alcantarea E. Morren ex Mez, in Mart., Eichler & Urban, Fl. bras. 3(3): 516
(1894), non Alcantara Glaz. (1909: Asteraceae), nom nud., nec Alcantara Glaz. ex Baroso
(1969: Asteraceae), Lectotype (Grant & Zijlstra, 1998: 93) ≡ Vriesea Gruppe „Reginae"
Wawra, Itin. Princip. S. Coburgi 1: 158 (1883) ≡ Vriesea sect. Reginae (Wawra) Wittm., in
Engl. & Prantl, Nat. Pflanzenfam. II.4: 58 (1888).—Type: Tillandsia regina Vell.
= Tillandsia sect. Macrocyathus K. Koch, Index Seminum Hort. Bot. Berol. „1873",App. 4: 1,
6 (1874).—Type: Tillandsia gigantea sensu K. Koch (1874) (= Vriesea glazioviana Lem.).
Mesophytic, acaulescent; petals free and appendaged; filaments and anthers free; pollen of
the Alcantarea type; stigma conduplicate-patent, rarely conduplicate-erect.
17. Vriesea Lindl., Edwards’s Bot. Reg. 29: sub pl. 10 (1843), as "Vriesia", nom. conserv. vs.
Hexalepis Raf., Fl. tellur. 4: 24 (1838) as well as vs. Vriesea Hassk., Flora 25 (Beibl.): 27
(1842: Scrophulariaceae) ≡ Hexalepis Raf., Fl. tellur. 4: 24 (1838), nom. rejic. vs. Vriesea
Lindl. ≡ Tillandsia sect. Vriesea (Lindl.) Griseb., Nachr. Königl. Ges. Wiss. Georg-AugustUniv. [1]: 17 (13 Jan 1864) ≡ Vriesea ?sect. Psittacinae Wawra, Itin. princ. S. Coburgi 1: 158
(1883), nom. illeg. ≡ Tillandsia subg. Vriesea (Lindl.) Baker, J. Bot. 26: 47 (1888) ≡ Vriesea
subg. Euvriesea Mez, in Mart., Eichler & Urban, Fl. bras. 3(3): 513 (1894) ≡ Vriesea sect.
Genuinae Mez, in Mart., Eichler & Urban, Fl. bras. 3(3): 513 (1894) ≡ Neovriesia Britton ex
Britton & P. Wilson, Bot. Porto Rico 5: 141 (1923), nom. illeg.—Type: Tillandsia psittacina
Hook.
= Tillandsia sect. Synandra K. Koch, Index Seminum Hort. Bot. Berol. "1873", App. 4: 1
(1874).—Type: Vriesea corallina Regel.
= Tillandsia sect. Cyathophora K. Koch, Index Seminum Hort. Bot. Berol. "1873", App. 4: 1
(1874).—Type: Encholirion saundersii Carrière.
= Vriesea sect. Platzmanniae E. Morren, Belgique Hort. 25: 349 (1875).—Type: Vriesea
platzmannii E. Morren.
= Vriesea sect. Brachystachyae Wawra, Itin. princ. S. Coburgi 1: 158 (1883).—Type:
Vriesea carinata Wawra.
= Vriesea sect. Macrostachyae Wawra, Itin. princ. S. Coburgi 1: 158 (1883).—Type: Vriesea
conferta Gaudich.
– Vriesea subg. Conostachys (Griseb.) Mez, in Mart., Eichler & Urban, Fl. bras. 3(3): 516
(1894), typo excluso.—studied taxa: V. poenulata (Baker) E. Morren ex Mez, V. rubida E.
Morren ex Mez (= V. ventricosa (Wawra) Mez).
– Vriesea sect. Xiphion (E. Morren) Wawra ex Wittm. p.p. majore, typo excluso (see Appendix for Figure 2 and supplementary data for all spp. included).
Mesophytic to semi-mesophytic, usually acaulescent; petals free and appendaged; filaments
and anthers free; pollen of the insulae type; stigma of the convolute-blade type or tubolaciniate.
180
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Key to the genera of Tillandsioideae
This key must remain provisional as the resolution in the molecular trees is not sufficient in
some groups and morphological features are lacking for numerous taxa. However, it demonstrates that the molecular phylogeny can be supported in most cases using ovary, seed, stigma,
anther, and petal morphology. The Cipuropsis-Mezobromelia clade and Tillandsia continue to
be critical groups, either because of missing taxa, low phylogenetic resolution, or uncertain
application of morphological characters. Therefore, informal clades of the CipuropsisMezobromelia clade and subgenera and informal clades of Tillandsia are usually not apportioned, except for cases, where differentiating characters have to be utilized for the discrimination of a genus from a subgeneric unit, informal clade or species.
1
1*
2
2*
3
3*
4
4*
Ovary ½–⅔ inferior.—Seeds long appendaged on both ends, chalazal appendage very
long, filiform, undivided, micropylar appendage plumose. Flowers polystichous. Pollen
with diffuse aperture. Stigma lobes with undulate margins (convolute-blade type).—
Lesser Antilles extending to northeastern Venezuela............................... Glomeropitcairnia
Ovary max. ⅓ inferior to completely superior.—Seeds usually long appendaged only on
one end, but chalazal appendage sometimes well developed (e.g., Alcantarea, Tillandsia
p.p., Vriesea p.p., Vriesea tuerckheimii). Flowers polystichous or distichous .......................2
Seeds with an undivided micropylar appendage, chalazal multicellular hairs folded at maturity and forming the flight apparatus.—Ovary max. ⅛ inferior to completely superior.
Pollen with sharp cut aperture margins (Catopsis type). Stigma with erect lobes (simpleerect type), rarely somewhat twisted (tending towards conduplicate-spiral type). Petals
usually white and forming a campanulate corolla, rarely yellow and spreading.—Central
America and Antilles extending to northern South America and Southeastern Brazil ............
.................................................................................................................................... Catopsis
Seeds with a divided micropylar appendage forming a coma (flight apparatus), chalazal
appendage undivided (very rarely divided) and short, or lacking, rarely long and occasionally somewhat divided (Alcantarea), not folded at maturity .................................................3
Micropylar appendage rather short, about equaling the seed proper, chalazal appendage
distinctly larger than the seed proper, sometimes somewhat divided. Petals long, recurved, exposing the long exserted stamens and style.—Pollen with solid aperture margins. Stigma lobes conduplicate and spreading (conduplicate-patent type), rarely not
spreading (conduplicate-erect type: Alcantarea cerosa, Alcantarea roberto-kautskyi), often somewhat twisted around their longitudinal axis.—Endemic to Southeastern Brazil ......
............................................................................................................................... Alcantarea
Micropylar appendage distinctly longer than the seed proper, chalazal appendage lacking
to about half as long as the seed proper, nearly always undivided. Petals forming a tubular
or campanulate corolla, at least in the proximal half ............................................................4
Petals conglutinated/connate into a distinct tube (distinctly longer than 25%).—Petals
white, yellow, or green. Seeds without a distinct chalazal appendage .................................5
Petals not conglutinated/connate into a distinct tube, completely or at least ¾ free from
each other but sometimes connate at the base (max. 25 % of entire length).—Petals violet, pink, red, orange, yellow, green, white, and rarely bicolored. Seeds sometimes with a
distinct chalazal appendage up to the length of the seed proper, rarely longer ...................6
181
BARFUSS, M.H.J.
5
5*
6
6*
7
7*
8
8*
9
9*
10
10*
182
NEW CLASSIFICATION
Petals with basal appendages and anthers forming a tube around the stigma.—Stigma
with erect lobes (simple-erect type). Pollen with a diffuse aperture or nearly inaperturate.—Northern Andes extending to the Antilles, the Guianas, and Bolivia .... Mezobromelia
Petals without basal appendages or anthers not forming a tube around the stigma.—
Stigma with erect lobes (simple-erect type) or infundibuliform with undulate margins
(convolute-blade type). Pollen with a diffuse aperture or inaperturate.—Andean and Central America, extending to the Antilles, the Guianas, and Eastern Brazil ................Guzmania
Stigma lobes fused and forming a cup, margins entire (cupulate type) and without papillae.—Plants mostly forming impounding rosettes. Corolla tubular or campanulate, sometimes zygomorph (stamens then asymmetrically arranged. Pollen with irregular aperture
margins and exine fragments in the aperture (insulae type). Seeds with no or only very
short chalazal appendage.—Central America extending to Ecuador (and Bolivia) . Werauhia
Stigma lobes not of the cupulate type without papillae, but rarely resembling cupulate
with crenulate (tubolaciniate type) (Vriesea p.p.) or papillate (Vriesea tuerckheimii) margins ......................................................................................................................................... 7
Stigma with undulate and papillate margins (convolute-blade type), rarely with crenulate
margins (tubolaciniate type), plants meso- or semi-mesophytic, petal appendages present.—Corolla tubular or campanulate, stamens and style exserted or included. Petals yellow (often with green tips), cream, or brownish (–red), rarely white, free or rarely short
connate into a common tube with the filament bases. Pollen with irregular aperture margins and exine fragments in the aperture (insulae type). Seeds with a distinct chalazal appendage.—Eastern Brazil extending to Venezuela and Peru ...................................... Vriesea
Stigma lobes usually not of the convolute-blade or tubolaciniate types, if rarely resembling convolute-blade then plants xerophytic and petal appendages absent ...................... 8
Filaments connate among each other at least at the base but sometimes for nearly their
whole length, free from the petals. Stigma coralliform.—Leaves narrowly triangular. Petals
white ore yellowish. Ovules clavate, obtuse. Seeds without chalazal appendage.—Central
America to Peru extending to eastern Venezuela and the Guianas ................... Lemeltonia
Filaments free from each other, but sometimes connate/agglutinated to the petals (e.g.,
Cipuropsis and Racinaea). Stigma usually not of the coralliform type, if rarely resembling
the coralliform type then filaments free from each other (Racinaea subg. Pseudophytarrhiza and Racinaea dyeriana) ............................................................................................ 9
Stigma pinnatisect.—Inflorescence undivided, distichous, flat, lanceolate-elliptic in outline. Petals violet, their blades usually strongly enlarged. Leaf bases mostly longitudinally
red striped. Ovules slenderly cylindric, obtuse. Seeds without chalazal appendage.—
Ecuador and Peru, Josemania anceps extending to Guatemala, eastern Venezuela, the
Guianas, and northern Brazil .................................................................................. Josemania
Stigma not pinnatisect ......................................................................................................... 10
Stigma obconical and resembling the convolute-blade type, mesophytic.—Leaves lingulate, forming impounding rosettes. Inflorescence compound, pink, rhachis usually alate.
Flowers distichous, petals with enlarged blade, violet. Seeds with a minute chalazal appendage only.—Andean, Ecuador to Bolivia ........................................................... Rothowia
Stigma not obconical and not resembling the convolute-blade type, if rarely of the convolute-blade type, then plants xerophytic and not forming impounding rosettes (Tillandsia
plumosa complex)................................................................................................................ 11
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
11 Sepals distinctly asymmetric, stigma simple-erect or slightly conduplicate-spiral (both
reduction forms of the coralliform type), if subsymmetric then stigma resembling the coralliform type and leaves lingulate (Racinaea subg. Pseudophytarrhiza and Racinaea dyeriana). Seeds without chalazal appendage.—Flowers small and inconspicuous in most
species, petals white to yellow, rarely blue (Racinaea hamaleana).—Andean, mainly Ecuador, extending to Central America, Bolivia, the Greater Antilles and the Guianas, and
Southeastern Brazil .................................................................................................. Racinaea
11* Sepals symmetric, stigma usually simple-erect or conduplicate-spiral, rarely convolutebladed (Tillandsia subg. Viridantha p.p.) or resembling cupulate with papillate margins
(Vriesea tuerckheimii). Seeds usually with a distinct chalazal appendage.......................... 12
12 Petals without basal appendages ........................................................................................ 13
12* Petals with basal appendages ............................................................................................. 14
13 Petals and filaments basally connate or leaf sheaths drying silver-gray.—Petals white or
yellow ............................................................................................................. Singularis clade
13* Petals and filaments basally not connate and leaf sheaths not drying silver-gray.—From
the southern United Stated to the Antilles, central Argentina and Uruguay with centers of
diversity in northern Central America and the northern and central Andes (not further
keyed out except for taxa of Tillandsia subg. Pseudovriesea bearing petal appendages).......
.................................................................................................................................. Tillandsia
14 Leaves narrowly triangular, xeromorphic, densely lepidote.—Petals usually violet, often
bicolored with green, sometimes with crenulated margins.—Northern and central Andes
extending to Bolivia, Mesoamerica, the Antilles, and eastern Venezuela ...............................
........................................................................................ Tillandsia subg. Pseudovriesea p.p.
14* Leaves lingulate, usually mesomorphic, not densely lepidote.—Corolla tubular, stamens
and style usually included, rarely exserted. Petals white or yellow, rarely red. Pollen with a
diffuse aperture ................................................................................................................... 15
15 Plants Andean extending to Venezuela, Trinidad, and the Guianas ........................................
.............................. Cipuropsis, Chrysostachys clade, Splendens clade, Vriesea tuerckheimii
15* Plant from Mexico ..................................................................................... Tillandsia malzinei
183
BARFUSS, M.H.J.
NEW CLASSIFICATION
Conclusions
Much progress has been achieved in exploring phylogenetic relationships of Tillandsioideae by
adding nuclear DNA information to the already existing and published plastid DNA data
(Barfuss & al., 2005). Most accepted genera are well differentiated by both DNA and morphological data in their actual circumscriptions (Alcantarea, Catopsis, Glomeropitcairnia, Josemania, Lemeltonia, Racinaea, Rothowia, Vriesea, Werauhia). Guzmania in its present circumscription is holophyletic, but whether the corolla tube formation (Leins & Erbar, 2010) is a synapomorphic character for all Guzmania and different to that of Mezobromelia needs to be
further tested. Generic definitions of the Cipuropsis-Mezobromelia clade need further attention, mostly in adding missing taxa and more DNA data as well as in evaluating morphological
characters for defining clades and assessing their phylogenetic position relative to the preliminarily accepted genera Cipuropsis and Mezobromelia. Tillandsia as presently defined is holophyletic according to DNA data, but morphologically still very diverse, which is also demonstrated by the numerous subgenera and informal clades. Although several questions concerning relationships of species of former Tillandsia subg. Phytarrhiza and T. subg. Pseudalcantarea
are solved, in other subgenera statistical support for their holophyletic origin is still missing.
Relationships between clades and subgenera are also mostly unclear and lack statistical support. A solution has to be found for the unclassified species of Tillandsia. To get a fully resolved
picture of relationships within Tillandsioideae, more nuclear DNA markers and a careful revision of morphological characters already identified as useful for discriminating taxa are still
needed.
Acknowledgments
The authors thank Tod F. Stuessy for providing lab space; many botanical gardens and individual people for providing plant material (see "Taxon selection"); the "Botanischer Garten der
Universität Wien" (HBV) for cultivating the majority of samples used; Jason Grant for valuable
discussions; Eva Maria Mayr for helping with plant collections in Ecuador (2006); and Anton
Russell for critically reviewing the manuscript. Financial support was partly provided by the
Austrian Science Foundation (FWF) to W. Till (grant no. P13690-BIO), by the Commission for
Interdisciplinary Ecological Studies (KIÖS) at the Austrian Academy of Sciences (ÖAW) to W. Till
and M.H.J. Barfuss (2007-02), and by the Dept. of Systematic and Evolutionary Botany (Universität Wien).
184
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
References
Altekar, G., Dwarkadas, S., Huelsenbeck, J.P. & Ronquist, F. 2004. Parallel Metropolis-coupled
Markov chain Monte Carlo for Bayesian phylogenetic inference. Bioinformatics 20: 407–
415.
Baker, J.G. 1878. A synopsis of the species of Diaphoranthema. J. Bot. 16: 236–241.
Baker, J.G. 1887. Synopsis of Tillandsieae. J. Bot. 25: 52–55, 115–118, 171–177, 211–215, 234–
246, 277–281, 303–306, 344–347.
Baker, J.G. 1888. A synopsis of Tillandsieae. J. Bot. 26: 12–17, 39–50, 79–82, 104–111, 137–
144, 167–172.
Baker, J.G. 1889. Handbook of the Bromeliaceae. London: G. Bell & Sons.
Barfuss, M., Samuel, M.R. & Till, W. 2004. Molecular phylogeny in subfamily Tillandsioideae
(Bromeliaceae) based on six cpDNA markers: an update. J. Bromeliad Soc. 54: 9–17, 48.
Barfuss, M.H.J., Samuel, R., Till, W. & Stuessy, T.F. 2005. Phylogenetic relationships in subfamily Tillandsioideae (Bromeliaceae) based on DNA sequence data from seven plastid regions. Amer. J. Bot. 92: 337–351.
Beaman, R.S. 1989. Systematics of Tillandsia subgenus Pseudalcantarea (Bromeliaceae) and
cladistic relationships of the Bromelioideae, Pitcairnioideae, and Tillandsioideae. Dissertation, University of Florida.
Beaman, R.S. & Judd, W.S. 1996. Systematics of Tillandsia subgenus Pseudalcantarea (Bromeliaceae). Brittonia 48: 1–19.
Benzing, D.H. 2000. Bromeliaceae: Profile of an adaptive radiation. Cambridge: Cambridge
University Press.
Bergsten, J. 2005. A review of long-branch attraction. Cladistics 21: 163–193.
Betancur, J. & Miranda-Esquivel, D.R. 1999. ¿Existe Sodiroa? Revista Acad. Colomb. Ci. Exact.
23: 189–194.
Böhme, S. 1988. Bromelienstudien III. Vergleichende Untersuchungen zu Bau, Lage und systematischer Verwertbarkeit der Septalnektarien von Bromelien. Trop. Subtrop. Pflanzenwelt 62: 125–274.
Borsch, T. & Quant, D. 2009. Mutational dynamics and phylogenetic utility of noncoding chloroplast DNA. Pl. Syst. Evol. 282: 169–199.
Brongniart, A. 1864. Note sur un nouveau genre de la famille des Broméliacées. Ann. Sci. Nat.,
Bot. 1: 325–329.
Brown, G.K. & Gilmartin, A.J. 1984. Stigma structure and variation in Bromeliaceae—
neglected taxonomic characters. Brittonia 36: 364–374.
Brown, G.K. & Gilmartin, A.J. 1989a. Chromosome numbers in Bromeliaceae. Amer. J. Bot. 76:
657–665.
Brown, G.K. & Gilmartin, A.J. 1989b. Stigma types in Bromeliaceae—a systematic survey. Syst.
Bot. 14: 110–132.
Brown, G.K. & Terry, R.G. 1992. Petal appendages in Bromeliaceae. Amer. J. Bot. 79: 1051–
1071.
Chen, X., Yu, T., Xiong, J., Zhang, Y., Hua, Y., Li, Y. & Zhu, Y. 2004. Molecular cloning and expression analysis of rice phosphoribulokinase gene that is regulated by environmental
stresses. Molec. Biol. Rep. 31: 249–255.
185
BARFUSS, M.H.J.
NEW CLASSIFICATION
Chew, T., De Luna, E. & González, D. 2010. Phylogenetic relationships of the pseudobulbous
Tillandsia species (Bromeliaceae) inferred from cladistic analyses of ITS 2, 5.8S ribosomal
RNA gene, and ETS sequences. Syst. Bot. 35: 86–95.
Crayn, D.M., Winter, K. & Smith, J.A.C. 2004. Multiple origins of crassulacean acid metabolism
and the epiphytic habit in the Neotropical family Bromeliaceae. Proc. Natl. Acad. Sci.
U.S.A. 101: 3703–3708.
Denton, A.L., McConaughy, B.L. & Hall, B.D. 1998. Usefulness of RNA Polymerase II coding
sequences for estimation of green plant phylogeny. Molec. Biol. Evol. 15: 1082–1085.
Doyle, J.J. & Doyle, J.L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf
tissue. Phytochem. Bull. Bot. Soc. Amer. 19: 11–15.
Edgar, R.C. 2004a. MUSCLE: A multiple sequence alignment method with high accuracy and
high throughput. Nucl. Acids Res. 32: 1792–1797. http://www.drive5.com/muscle/
Edgar, R.C. 2004b. MUSCLE: A multiple sequence alignment method with reduced time and
space complexity. B.M.C. Bioinf. 5: 113.
Ehlers, R. 1997 ("1996"). Die rotblühenden brasilianischen Tillandsien/The red-flowered tillandsias from Brazil. Bromelie Sonderheft 3: 1–68.
Ehlers, R. 2009. Die grünblühenden, kleinen, grauen Tillandsien Mexikos/The green-blooming
small, grey tillandsias from Mexico. Bromelie Sonderheft 6: 1–144.
Espejo-Serna, A. 2002. Viridantha, un género nuevo de Bromeliaceae (Tillandsioideae) endémico de México. Acta Bot. Mex. 60: 25–35.
Espejo-Serna, A., López-Ferrari, A.R., Martínez-Correa, N. & Pulido-Esparza, V.A. 2007. Bromeliad flora of Oaxaca, Mexico: richness and distribution. Acta Bot. Mex. 81: 71–147.
Evans, T.M. & Brown, G.K. 1989. Plicate staminal filaments in Tillandsia subgenus Anoplophytum (Bromeliaceae). Amer. J. Bot. 76: 1478–1485.
Farris, J.S., Källersjö, M., Kluge, A.G. & Bult, C. 1994. Testing significance of congruence. Cladistics 10: 315–319.
Farris, J.S., Källersjö, M., Kluge, A.G. & Bult, C. 1995. Constructing a significance test for incongruence. Syst. Biol. 44: 570–572.
Ford, V.S., Lee, J., Baldwin, B.G. & Gottlieb, L.D. 2006. Species divergence and relationships in
Stephanomeria (Compositae): PGIC phylogeny compared to prior biosystematic studies.
Amer. J. Bot. 93: 480–490.
Gardner, C.S. 1982. A systematic study of Tillandsia subgenus Tillandsia. Dissertation, Texas
A&M University.
Gardner, C.S. 1986a. Inferences about pollination in Tillandsia (Bromeliaceae). Selbyana 9: 76–
87.
Gardner, C.S. 1986b. Preliminary Classification of Tillandsia based on floral characters. Selbyana 9: 130–146.
Gilmartin, A.J. 1983. Evolution of mesic and xeric habits in Tillandsia and Vriesea (Bromeliaceae). Syst. Bot. 8: 233–242.
Gilmartin, A.J. & Brown, G.K. 1986. Cladistic tests of hypotheses concerning evolution of xerophytes and mesophytes within Tillandsia subg. Phytarrhiza (Bromeliaceae). Amer. J. Bot.
73: 387–397.
Gilmartin, A.J., Brown, G.K., Varadarajan, G.S. & Neighbors, N. 1989. Status of Glomeropitcairnia within evolutionary history of Bromeliaceae. Syst. Bot. 14: 339–348.
186
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Givnish, T.J., Barfuss, M.H.J., Van Ee, B., Riina, R., Schulte, K., Horres, R., Gonsiska, P.A.,
Jabaily, R.S., Crayn, D.M., Smith, J.A.C., Winter, K., Brown, G.K., Evans, T.M., Holst, B.K.,
Luther, H.E., Till, W., Zizka, G., Berry, P.E. & Sytsma, K.J. 2011. Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: Insights from an eight-locus plastid
phylogeny. Amer. J. Bot. 98: 872–895.
Givnish, T.J., Millam, K.C., Berry, P.E. & Sytsma, K.J. 2007. Phylogeny, adaptive radiation, and
historical biogeography of Bromeliaceae inferred from ndhF sequence data. Pp. 3–26 in:
Columbus, J.T., Friar, E.A., Porter, J.M., Prince, L.M. & Simpson M.G. (eds.), Monocots:
Comparative Biology and Evolution—Poales. Claremont: Rancho Santa Ana Botanic Garden (RSABG).
Givnish, T.J., Millam, K.C., Evans, T.M., Hall, J.C., Pires, J.C., Berry, P.E. & Sytsma, K.J. 2004.
Ancient vicariance or recent long-distance dispersal? Inference about phylogeny and
South American-African disjunctions in Rapateaceae and Bromeliaceae based on ndhF sequence data. Int. J. Plant Sci. 165: S35–S54.
Górniak, M., Paun, O. & Chase, M.W. 2010. Phylogenetic relationships within Orchidaceae
based on a low-copy nuclear coding gene, XDH: Congruence with organellar and nuclear
ribosomal DNA results. Molec. Phylog. Evol. 56: 784–795.
Gortan, G. 1991. Narbenformen bei Bromeliaceen: Variationsmöglichkeiten und Überlegungen
zu systematisch-taxonomischen Korrelationen. Dissertation, Universität Wien.
Granados Mendoza, C.G. 2008. Sistemática del complejo Tillandsia macdougallii (Bromeliaceae). Dissertation, Universidad Nacional Autónoma de México.
Grant, J.R. 1993a. New combinations in Mezobromelia and Racinaea (Bromeliaceae: Tillandsioideae). Phytologia 74: 428–430.
Grant, J.R. 1993b. True tillandsias misplaced in Vriesea (Bromeliaceae: Tillandsioideae). Phytologia 75: 170–175.
Grant, J.R. 1994a. The Tillandsia adpressa assemblage: A review and new combinations in Racinaea (Bromeliaceae: Tillandsioideae). Novon 4: 362–364.
Grant, J.R. 1994b. The reduction of Platyaechmea under Hoplophytum, and a new name in
Tillandsia (Bromeliaceae). Phytologia 77: 99–101.
Grant, J.R. 1995a. Bromelienstudien. The resurrection of Alcantarea and Werauhia, a new
genus. Trop. Subtrop. Pflanzenwelt 91: 1–57.
Grant, J.R. 1995b. New combinations and new taxa in the Bromeliaceae. Phytologia 79: 254–
256.
Grant, J.R. 2005 ("2004"). New combinations and names in Andean Pitcairnia, Tillandsia, and
Werauhia (Bromeliaceae). Vidalia 2: 23–25.
Grant, J.R. & Zijlstra, G. 1998. An annotated catalogue of the generic names of the Bromeliaceae. Selbyana 19: 91–1.
Grisebach, A.H.R. 1864. Ueber die von Fendler in Venezuela gesammelten Bromeliaceen. Nachr. Königl. Ges. Wiss. Georg-August-Univ. [1]: 1–21.
Gross, E. 1988. Bromelienstudien IV. Zur Morphologie der Bromeliaceen-Samen unter
Berücksichtigung systematisch-taxonomischer Aspekte. Trop. Subtrop. Pflanzenwelt 64: 1–
215.
Halbritter, H. 1988. Bromeliaceae: Pollenmorphologie und Systematik. Die Entwicklung des
Pollens von Tillandsia sinuosa L.B. Smith. Dissertation, Universität Wien.
Halbritter, H. 1992. Morphologie und systematische Bedeutung des Pollens der Bromeliaceae.
Grana 31: 197–212.
187
BARFUSS, M.H.J.
NEW CLASSIFICATION
Hall, T.A. 1999. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser., 41: 95–98.
Harms, H. 1929. Bromeliaceae novae III. Notizbl. Bot. Gart. Berlin-Dahlem 10: 784–805.
Harms, H. 1930. Bromeliaceae. Vol. 15a. Pp. 65–159 in: Engler, A. & Prantl, K. (eds.), Die natürlichen Pflanzenfamilien, 2nd edition. Leipzig: W. Engelmann.
Hörandl, E. & Stuessy, T.F. 2010. Paraphyletic groups as natural units of biological classification. Taxon 59: 1641–1653.
Horres, R., Zizka, G., Kahl, G. & Weising, K. 2000. Molecular phylogenetics of Bromeliaceae:
evidence from trnL (UAA) intron sequences of the chloroplast genome. Pl. Biol. 2: 306–
315.
Howarth, D.G. & Baum, D.A. 2002. Phylogenetic utility of a nuclear intron from nitrate reductase for the study of closely related plant species. Molec. Phylog. Evol. 23: 525–528.
Hromadnik, L. 2005. Der Verwandtschaftskreis um Tillandsia tectorum/The Tillandsia tectorum
family. Bromelie Sonderheft 5: 1–120.
Huelsenbeck, J.P. & Ronquist, F. 2001. MrBayes: Bayesian inference of phylogenetic trees.
Bioinformatics 17: 754–755.
Jabaily, R.S. & Sytsma, K.J. 2010. Phylogenetics of Puya (Bromeliaceae): Placement, major
lineages, and evolution of Chilean species. Amer. J. Bot. 97: 337–356.
Kelchner, S.A. 2000. The evolution of noncoding chloroplast DNA and its application in plant
systematics. Ann. Missouri Bot. Gard. 87: 482–498.
Kessler, M. 2002. Environmental patterns and ecological correlates of range size among bromeliad communities of Andean forests in Bolivia. Bot. Rev. (Lancaster). 68: 100–127.
Krömer, T., Kessler, M., Lohaus, G. & Schmidt-Lebuhn, A.N. 2008. Nectar sugar composition
and concentration in relation to pollination syndromes in Bromeliaceae. Pl. Biol. 10: 502–
511.
Lee, M.S.Y. 2001. Uninformative characters and apparent conflict between molecules and
morphology. Molec. Biol. Evol. 18: 676–680.
Leins, P. & Erbar, C. 2010. Flower and fruit: morphology, ontogeny, phylogeny, function and
ecology. Stuttgart: E. Schweizerbart'sche Verlagsbuchhandlung.
Leme, E.M.C. 2007. Improving taxa and character sampling to support generic and infrageneric
status of Alcantarea. J. Bromeliad Soc. 57: 208–215.
Leme, E.M.C. 2009. Notes on Alcantarea: a new medium-sized species and additions to
A. roberto-kautskyi. J. Bromeliad Soc. 59: 19–27.
Leme, E.M.C. & Brown, G.K. 2004. Four new lithophytic Vriesea species (Tillandsioideae) from
southeastern Brazil. Vidalia 2: 3–11.
León, B. & Sagástegui, A. 2008. General overview of Tillandsia subgenus Tillandsia in Peru: The
three-pinnate species and the case of two endemic species/Sinopsis de Tillandsia subgenus Tillandsia en el Peru: las especies tri-pinnadas y el caso de dos especies endémicas.
Revista Peruana Biol. 15: 25–30.
Lewis, C.E. & Doyle, J.J. 2001. Phylogenetic utility of the nuclear gene malate synthase in the
palm family (Arecaceae). Molec. Phylogen. Evol. 19: 409–420.
Lewis, C.E. & Doyle, J.J. 2002. A phylogenetic analysis of tribe Areceae (Arecaceae) using two
low-copy nuclear genes. Pl. Syst. Evol. 236: 1–17.
Lindley, J. 1843. Edwards’s Botanical Register 29: pl. 1–66.
Linné, C. 1753. Species Plantarum. Stockholm: Laurentius Salvius.
188
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
López-Ferrari, A.R. & Espejo-Serna, A. 2009. Nuevas combinaciones en monocotiledóneas
mexicanas IV (Bromeliaceae, Orchidaceae). Acta Bot. Mex. 89. 43–46.
Luther, H.E. 1998. Miscellaneous new taxa of Bromeliaceae (XII). Selbyana 19: 83–90.
Luther, H.E. 2001. De Rebus Bromeliacearum III. Selbyana 22: 34–67.
Luther, H.E. 2008. An alphabetical list of bromeliad binomials, 11th edition. The Bromeliad Society International.
http://selby.org/research/papers/alphabetical-list-bromeliad-binomials.
Luther, H.E. 2010. An alphabetical list of bromeliad binomials, 12th edition. Pp. I–IV, 1–45, Rabinowitz, L. & Holst, B.K. (eds.). Sarasota: The Sarasota Bromeliad Society & Marie Selby Botanical Gardens (MSBG).
Luther, H.E. & Rabinowitz, L. 2010. De Rebus Bromeliacearum IV. Selbyana 30: 147–189.
Luther, H.E. & Sieff, E. 1994. De Rebus Bromeliacearum I. Selbyana 15: 9–93.
Luther, H.E. & Sieff, E. 1997. De Rebus Bromeliacearum II. Selbyana 18: 103–140.
Manzanares, J.M. & Gouda, E.J. 2010. Four new species of the genus Racinaea (Bromeliaceae)
from Ecuador. Phytotaxa 3: 1–18.
Mathews, S. & Donoghue, M.J. 1999. The root of angiosperm phylogeny inferred from duplicate phytochrome genes. Science 286: 947–950.
McNeill, J., Barrie, F.R., Burdet, H.M., Demoulin, V., Hawksworth, D.L., Marhold, K., Nicolson,
D.H., Prado, J., Silva, P.C., Skog, J.E., Wiersema, J.H. & Turland, N.J. (eds.) 2006. International code of botanical nomenclature (Vienna Code): Adopted by the Seventeenth International Botanical Congress Vienna, Austria, July 2005. Regnum Vegetabile 146. Ruggell:
Gantner.
Mez, C. 1891–94. Bromeliaceae. Pp. 173–634, pl. 51–114 in: Martius, C.F.P., Eichler, A.G., Urban, I. (eds.), Flora brasiliensis 3 (3). Leipzig: F. Fleischer.
Mez, C. 1896. Bromeliaceae, vol. 9. Pp. 1–990 in: de Candolle, C. (ed.), Monographiae phanerogamarum. Paris: Masson & Cie.
Mez, C. 1934–35. Bromeliaceae. Pp. 1–667 in: Endler, A. (ed.), Das Pflanzenreich IV. 32. Leipzig:
W. Engelmann.
Morren, E. 1870. Description du Tillandsia (Wallisia) hamaleana Ed. Mn. Belgique Hort. 20: 97–
102, pl. s.n.
Morren, E. 1871. Notice sur le Tillandsia lindeni, var. luxurians. Belgique Hort. 21: 289–290, pl.
20–21.
Nickrent, D.L., Malécot, V., Vidal-Russell, R. & Der, J.P. 2010. A revised classification of Santalales. Taxon 59: 538–558.
Nylander, J.A.A. 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University.
Oxelman, B., Yoshikawa, N., McConaughy, B.L., Luo, J., Denton, A.L. & Hall, B.D. 2004. RPB2
gene phylogeny in flowering plants, with particular emphasis on asterids. Molec. Phylogen. Evol. 32: 462–479.
Palací, C.A. 1997. A systematic revision of the genus Catopsis (Bromeliaceae). Dissertation,
University of Wyoming, Laramie.
Palma-Silva, C., dos Santos, D.G., Kaltchuk-Santos, E. & Bodanese-Zanettini, M.H. 2004.
Chromosome numbers, meiotic behavior, and pollen viability of species of Vriesea and
Aechmea genera (Bromeliaceae) native to Rio Grande do Sul, Brazil. Amer. J. Bot. 91: 804–
807.
189
BARFUSS, M.H.J.
NEW CLASSIFICATION
Ranker, T.A., Soltis, D.E., Soltis, P.S. & Gilmartin, A.J. 1990. Subfamilial phylogenetic relationships of the Bromeliaceae: evidence from chloroplast DNA restriction site variation. Syst.
Bot. 15: 425–434.
Rauh, W. 1973. Bromelienstudien I. Neue und wenig bekannte Arten aus Peru. Trop. Subtrop.
Pflanzenwelt 3: 167–203.
Read, R.W. 1968. A new combination in Vriesea (Bromeliaceae). Phytologia 16: 457–458.
Reeves, G.M., Chase, M.W., Goldblatt, P., De Chies, T., Lejeune, B., Fay, M.F., Cox, A.V. &
Ruddall, P.J. 2001. Molecular systematics of Iridaceae: evidence from four plastid DNA regions. Amer. J. Bot. 88: 2074–2087.
Regel, E. 1869. VI. Annotationes botanicae. Index Seminum Hort. Bot. Petrop. 1868: 77–92.
Ronquist, F. & Huelsenbeck, J.P. 2003. MrBayes 3: Bayesian phylogenetic inference under
mixed models. Bioinformatics 19: 1572–1574.
Ruiz, H. & Pavón, J. 1802. Flora peruviana, et chilensis. 3: i–xxiv, 1–95, pl. 223–325. [Madrid]:
Gabriél de Sancha.
Russell, A., Samuel, R., Rupp, B., Barfuss, M.H.J., Safran, M., Besendorfer, V. & Chase, M.W.
2010a. Phylogenetics and cytology of a pantropical orchid genus Polystachya (Polystachyinae; Vandeae; Orchidaceae): evidence from plastid DNA sequence data. Taxon 59: 389–
404.
Russell, A., Samuel, R., Klejna, V., Barfuss, M.H.J., Rupp, B. & Chase, M.W. 2010b. Reticulate
evolution in diploid and tetraploid species of Polystachya (Orchidaceae) as shown by plastid DNA sequences and low-copy nuclear genes. Ann. Bot. (Oxford) 106: 37–56.
Samuel, R., Kathriarachchi, H., Hoffmann, P., Barfuss, M.H.J., Wurdack, K.J., Davis, C.C. &
Chase, M.W. 2005. Molecular phylogenetics of Phyllanthaceae: Evidence from plastid
matK and nuclear PHYC sequences. Amer. J. Bot. 92:132–141.
Schill, R., Dannenbaum, C. & Jentzsch, E.-M. 1988. Untersuchungen an Bromeliennarben/Investigations on the stigma of Bromeliaceae. Beitr. Biol. Pflanzen 63: 221–252.
Schmidt-Lebuhn, A.N., Kessler, M. & Hensen, I. 2007. Hummingbirds as drivers of plant speciation? Trends Pl. Sci. 12: 329–331.
Schulte, K., Barfuss, M.H.J. & Zizka, G. 2009. Phylogeny of Bromelioideae (Bromeliaceae) inferred from nuclear and plastid DNA loci reveals the evolution of the tank habit within the
subfamily. Molec. Phylogen. Evol. 51: 327–339.
Smith, L.B. 1935. Studies in the Bromeliaceae - VI. Proc. Amer. Acad. Arts 70: 147–224.
Smith, L.B. 1951. Studies in the Bromeliaceae - XVI. Contr. U.S. Natl. Herb. 29: 429–520.
Smith, L.B. & Downs, R.J. 1974. Pitcairnioideae (Bromeliaceae). Fl. Neotrop. Monogr. 14 (1): 1–
660.
Smith, L.B. & Downs, R.J. 1977. Tillandsioideae (Bromeliaceae). Fl. Neotrop. Monogr.14 (2):
661–1492.
Smith, L.B. & Downs, R.J. 1979. Bromelioideae (Bromeliaceae). Fl. Neotrop. Monogr.14 (3):
1493–2142.
Smith, L.B. & Till, W. 1998. Bromeliaceae. Pp. 74–99 in: Kubitzki, K. (ed.), The families and genera of vascular plants, vol. 4. Berlin: Springer.
Spencer, M.A. & Smith, L.B. 1993. Racinaea, a new genus of Bromeliaceae (Tillandsioideae).
Phytologia 74: 151–160.
Stefano, M., Papini, A. & Brighigna, L. 2008. A new quantitative classification of ecological
types in the bromeliad genus Tillandsia (Bromeliaceae) based on trichomes. Revista Biol.
Trop. 56: 191–203.
190
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
Swofford, D.L. 2003. PAUP*: Phylogenetic analysis using parsimony (*and other methods),
version 4.0b10. Sunderland, Massachusetts: Sinauer.
Tardivo, R.C. 2002. Revisão taxonômica de Tillandsia L. subgênero Anoplophytum (Beer) Baker
(Bromeliaceae). Dissertation, Universidade de São Paulo.
Tel-Zur, N., Abbo, S., Myslabodski, D. & Mizrahi, Y. 1999. Modified CTAB procedure for DNA
isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Pl.
Molec. Biol. Reporter 17: 249–254.
Terry, R.G. & Brown, G.K. 1996. A study of evolutionary relationships in Bromeliaceae based
on comparison of DNA sequences from the chloroplast gene ndhF. J. Bromeliad Soc. 46:
107–112, 123.
Terry, R.G., Brown, G.K. & Olmstead, R.G. 1997a. Examination of subfamilial phylogeny in
Bromeliaceae using comparative sequencing of the plastid locus ndhF. Amer. J. Bot. 84:
664–670.
Terry, R.G., Brown, G.K. & Olmstead, R.G. 1997b. Phylogenetic relationships in subfamily Tillandsioideae (Bromeliaceae) using ndhF sequences. Syst. Bot. 22: 333–345.
Thomas, M.M., Garwood, N.C., Baker, W.J., Henderson, S.A., Russell, S.J., Hodel, D.R. &
Bateman, R.M. 2006. Molecular phylogeny of the palm genus Chamaedorea, based on the
low-copy nuclear genes PRK and RPB2. Molec. Phylogen. Evol. 38: 398–415.
Till, W. 1984. Sippendifferenzierung innerhalb Tillandsia subgenus Diaphoranthema in
Südamerika mit besonderer Berücksichtigung des Andenostrandes und der angrenzenden
Gebiete. Dissertation, Universität Wien.
Till, W. 1992. Systematics and evolution of the tropical-subtropical Tillandsia subgenus Diaphoranthema (Bromeliaceae). Selbyana 13: 88–94.
Till, W. 2000a. Tillandsioideae. Pp. 555–572 in: Benzing, D.H. (ed.), Bromeliaceae: profile of an
adaptive radiation. Cambridge: Cambridge University Press.
Till, W. 2000b. Tillandsia and Racinaea. Pp. 573–586 in: Benzing, D.H. (ed.), Bromeliaceae:
profile of an adaptive radiation. Cambridge: Cambridge University Press.
Till, W. & Barfuss, M.H.J. 2006. Progress towards a new classification of Tillandsioideae. J.
Bromeliad Soc. 56: 253–259.
Till, W., Halbritter, H. & Gortan, G. 1997. Some notes on the remarkable bromeliad genus
Glomeropitcairnia. J. Bromeliad Soc. 47: 65–72.
Ule, E. 1907. II. Beiträge zur Flora der Hylaea nach den Sammlungen von Ule’s Amazonas Expedition. Verh. Bot. Vereins Prov. Brandenburg 48: 129–150.
Utley, J.F. 1978. A new combination in Guzmania (Bromeliaceae). Phytologia 40: 55–57.
Utley, J.F. 1983. A revision of the Middle American thecophylloid Vriesea (Bromeliaceae).
Tulane Stud. Zool. Bot. 24: 1–81.
Utley, J.F. & Luther, H.E. 1991. Studies in Middle American Bromeliaceae II. Ann. Missouri Bot.
Gard. 78: 270.
Versieux, L.M. 2009. Sistemática, filogenia e morfologia de Alcantarea (Bromeliaceae). Dissertation, Universidade de São Paulo.
Versieux, L.M., Barbará, T., Wanderley, M.d.G.L., Calvente, A., Fay, M.F. & Lexer, C. 2012.
Molecular phylogenetics of the Brazilian giant bromeliads (Alcantarea, Bromeliaceae): implications for morphological evolution and biogeography. Molec. Phylog. Evol. 64: 177–
189.
191
Versieux, L.M., Elbl, P.M., Wanderley, M.G.L., de Menezes, N.L. 2010. Alcantarea (Bromeliaceae) leaf anatomical characterization and its systematic implications. Nordic J. Bot. 28:
385–397.
Versieux, L.M. & Wanderleym M.G.L. 2007. A new species of Alcantarea (E. Morren ex Mez)
Harms, Bromeliaceae. Hoehnea 34: 409–413.
Weber, W. & Smith, L.B. 1983. A new combination in Mezobromelia. J. Bromeliad Soc. 33:
121–122.
Whitten, W.M., Williams, N.H. & Chase, M.W. 2000. Subtribal and generic relationships of
Maxillarieae (Orchidaceae) with emphasis on Stanhopeinae: combined molecular evidence. Amer. J. Bot. 87: 1842–1856.
Wittmack, L. 1888. Bromeliaceae. Pp. 32–58 in: Engler, A. & Prantl, K. (eds.), Die natürlichen
Pflanzenfamilien II. 4. Leipzig: W. Engelmann.
Yoder, A.D., Irwin, J.A. & Payseur, B.A. 2001. Failure of the ILD to determine data combinability for slow Ioris phylogeny. Syst. Biol. 50: 408–424.
Appendix
Figure 2. (p. 193) Selected equally most-parsimonious phylogram found in total combined analysis supplemented
with BP and PP mostly below branches, and branch length is given above branches. A small arrowhead below
branches indicates BP and PP positions where space was insufficient. Alternative topologies of terminal branches
and backbone relationships from Bayesian analysis are displayed in boxes mostly in front of the corresponding
clades or on the left side. Their positions within the complete tree are highlighted by capital letters (A–P: terminals,
Q–S: backbones). Relevant type species for each taxonomic unit and name-giving taxa for informal clades are underlined. For samples which are similar to a already described species (aff.), with a vague identification (cf.), or undetermined species (sp.), as well as for clearly polyphyletic species voucher details are given in addition to the DNA
reference number. Alternative tree topologies from Bayesian analysis are (1) terminals: A = Catopsis p.p., B =
Werauhia p.p., C = Mezobromelia, D and E = Alcantarea p.p., F = Guzmania p.p., G = Racinaea p.p., H = Rothowia, I
and J = T. subg. Tillandsia p.p., K = T. subg. Viridantha p.p., L = Purpurea clade, M = Biflora clade p.p., N = T. subg.
Anoplophytum p.p., O = T. subg. Diaphoranthema p.p., P = Xiphioides clade; (2) backbones: Q = CipuropsisMezobromelia clade, R = Tillandsieae, S = T. subg. Anoplophytum s.l. plus T. subg. Phytarrhiza and T. subg. Diaphoranthema.
Supplementary Data. (pp. 195–205) Accessions included in this study arranged in hierarchical, followed by alphabetical order (according to the chapter "Classification system of Tillandsioideae"). Attributes ofamplified fragments
of PHYC and PRK are listed. SNP = single nucleotide polymorphism, SSR = single sequence repeat (homopolymers).
192
1
1
*Tillandsia bermejoensis B0034
*Tillandsia bermejoensis B0034
2
86/100
Tillandsia diaguitensis B0429
Tillandsia diaguitensis B0429
Tillandsia
koehresiana
B0371
Tillandsia
koehresiana B0371
1
1
6
Tillandsia cochabambae B0392
Tillandsia lorentziana B0374
6
-/61
Tillandsia
lorentziana
B0374
Tillandsia aff. arequitae W. Till 6068 B0387
2 1
Tillandsia cardenasii B0409
Tillandsia cochabambae B0392
4
Tillandsia aff. arequitae W. Till 6068 B0387
Tillandsia cardenasii B0409
1
5
4
Tillandsia gerdae B0411
Tillandsia xiphioides subsp. prolata B0418
27
1
-/93
4
Usneoides clade
Tillandsia xiphioides subsp. prolata B0418
4
Tillandsia muhriae B0438
1
99/100
-/93
-/79
3
60/97
1
Tillandsia muhriae B0438
Tillandsia aff. lotteae P. Lechner 20-451 B0808
6
1
2
-/94 97/100
Tillandsia yuncharaensis B0417
Tillandsia aff. lotteae P. Lechner 20-451 B0808
1
2
*Tillandsia argentina B0087
*Tillandsia xiphioides subsp. xiphioides B0040
12
-/76
2
2
-/65
-/72
Tillandsia argentina B0797
Tillandsia yuncharaensis B0417
1
Tillandsia rosarioae B0403
Tillandsia gerdae B0411
3
9
3
62/89
Tillandsia markusii B0399
Tillandsia argentina B0087
22
60/97
6
10
4
Tillandsia zecheri var. cafayatensis B0361
Tillandsia
argentina B0797
-/54
-/72
100/100
9
4
*Tillandsia xiphioides subsp. xiphioides B0040
Tillandsia rosarioae B0403
1
62/89
Tillandsia aff. lotteae P. Lechner 20-451 B0808
Tillandsia zecheri var. cafayatensis B0361
-/100
5
97/100 Tillandsia aff. lotteae P. Lechner 20-451 B0809
Tillandsia markusii B0399
5
91/100
4
1
Tillandsia ramellae B0430
Tillandsia ramellae B0430
11
2
Tillandsia aff. vernicosa W. Till 6045 B0355
6
83/100
1 89/100 Tillandsia buchlohii B0395
18
-/96
1
Tillandsia recurvata
11
Tillandsia rosacea B0405
100/100
67/98
Xiphioides clade
3
2
*Tillandsia caulescens B0071
2
64/92
*Tillandsia didisticha B0038
10
98/100
Tillandsia comarapaensis B0407
*Tillandsia usneoides B0109
71/96 2 *Tillandsia usneoides B0109
2
71/96
6
6
Tillandsia usneoides B0451
Tillandsia usneoides B0451
92/100 2
92/100 2
*Tillandsia usneoides B0083
*Tillandsia usneoides B0083
27
22
8
1
99/100
Capillaris
clade
26
Tillandsia mollis B0206
Tillandsia landbeckii subsp. andina B0423
6
-/99
3
9
99/100
8
91/100
Tillandsia cf. usneoides W. Till 21024 B0539
Tillandsia landbeckii subsp. andina B0423
3
-/54
1
9
62/81
Tillandsia mollis B0206
Tillandsia cf. usneoides W. Till 21024 B0539
1 Tillandsia duratii B0737
2
-/76
1
2
Tillandsia duratii B0740
62/88
7
1
1
*Tillandsia duratii B0088
56/99
63/100
1
3
Tillandsia
aff. streptocarpa L. Hromadnik 20056 B0226
23
5
23
-/100
Tillandsia paleacea B0404
99/100
100/100
3
Tillandsia kirschnekii B0384
4
Tillandsia recurvata B0529
18
1
100/100
Tillandsia recurvata B0540
Tillandsia virescens B0516
4
90/100
1
13
Tillandsia capillaris B0518
100/100
24
Tillandsia kuehhasii B0396
1
4
17
-/99
-/64
Tillandsia esseriana
9
*Tillandsia funebris B0089
99/100
*Tillandsia albertiana B0033
67/92 1
1
7
Tillandsia albertiana B0225
2
92/100
6
Tillandsia albertiana B0212
Tillandsia
edithae B0425
8
100/100
Tillandsia edithae B0436
16
*Tillandsia
esseriana B0069
9
15
2
84/100
3
Tillandsia edithae
99/100
Tillandsia esseriana B0200
-/56 100/100
2
Tillandsia araujei B0394
2
14
1
Tillandsia aff. araujei E.C.M. Leme 491 B0422
7
4
4
Tillandsia recurvifolia var. subsecundifolia B0401
63/100
4
*Tillandsia
tenuifolia B0026
5
4
*Tillandsia bergeri B0097
91/100 3
10
*Tillandsia bergeri B0097
5
2
91/100
*Tillandsia bergeri cv. Alba B0110
3
6
4
*Tillandsia bergeri cv. Alba B0110
7
5
5
*Tillandsia tenuifolia B0026
-/53
1
Tillandsia albertiana
-/90
Tillandsia jonesii B0389
6
92/100
Tillandsia recurvifolia var. subsecundifolia B0401
12
*Tillandsia ixioides B0043
5
7
*Tillandsia pohliana B0080
2
22
5
15
-/71
84/100
*Tillandsia pohliana B0080
22
16
-/55
4
Tillandsia aff. araujei E.C.M. Leme 491 B0422
2
99/100
Tillandsia burle-marxii B0393
-/61
1
12
4
*Tillandsia
ixioides B0043
2
1
Tillandsia aff. tenuifolia W. Till 11082 B0414
-/90
2
-/76
Tillandsia
araujei
B0394
5
12
**Tillandsia stricta s.l. B0081 & B0410
1
-/77
Tillandsia jonesii B0389
6
6
Tillandsia neglecta B0339
19
7
Gardneri clade
*Tillandsia brachyphylla B0082
13
100/100
4
84/100
16
Tillandsia globosa B0419
18
100/100
Gardneri clade
*Tillandsia gardneri B0041
Tillandsia australis B0203
13
100/100
Tillandsia australis B0759
1 Tillandsia lajensis B0242
1 Tillandsia lajensis B0242
60/99
60/99
Tillandsia lajensis B0546
Tillandsia lajensis B0546
5
3
3
86/100
86/100
Tillandsia lajensis B0554
Tillandsia lajensis B0554
-/55
2
Tillandsia lajensis B0555
Tillandsia lajensis B0555
4
Tillandsia buseri var. nubicola B0561
Tillandsia buseri var. nubicola B0561
7
7
-/80
6
2
100/100
100/100 Tillandsia cf. superba J.M. Manzanares 7787 B0602
Tillandsia cf. superba J.M. Manzanares 7787 B0602
-/80
62/96
5
5
Tillandsia orbicularis B0375
Tillandsia orbicularis B0375
2
2
2
1
-/63
Tillandsia sp. B0634
Tillandsia sp. J.M. Manzanares 8146 B0634
3
72/98
72/98
-/80
Tillandsia cf. buseri var. nubicola R. Wülfinghoff s.n. B0441
2 Tillandsia cf. buseri var. nubicola R. Wülfinghoff s.n. B0441
7
2
2
2
2
Tillandsia cf. buseri var. nubicola R. Wülfinghoff s.n. B0442
Tillandsia cf. buseri var. nubicola R. Wülfinghoff s.n. B0442
57/100
68/99
8
8
11
Tillandsia fendleri W. Till s.n. B0445
Tillandsia fendleri W. Till s.n. B0445
11
57/100
8
5
-/63
Tillandsia glauca B0618
Tillandsia
glauca
B0618
6
-/63
90/100
-/85
Tillandsia fosteri B0639
Tillandsia fosteri B0639
4
3
7
4
*Tillandsia demissa B0075
*Tillandsia demissa B0075
15
12
*Tillandsia fendleri H. & L. Hromadnik 2052 B0009
*Tillandsia fendleri H. & L. Hromadnik 2052 B0009
1
5
-/57
9
6
-/57
*Tillandsia
baliophylla B0101
*Tillandsia
baliophylla
B0101
37
3
2
2
*Tillandsia brevilingua B0056
Tillandsia confinis B0587
3
8
100/100
1
-/93 2 Tillandsia rudolfii B0654
34
100/100
Tillandsia brevilingua B0252
100/100
6
6
Tillandsia brenneri B0236
*Tillandsia brevilingua B0056
8
100/100 1 Tillandsia brevilingua B0252
31
Tillandsia
deppeana
B0457
-/87
5
16
99/100
6
100/100
5
15
Tillandsia deppeana B0458
Tillandsia brenneri B0236
-/55
5
99/100
7
1
*Tillandsia
heterophylla
B0047
Tillandsia
deppeana
B0457
-/63
5
99/100
17
8
16
*Tillandsia multicaulis B0107
Tillandsia deppeana B0458
13
90/100
1
7
99/100
1
Tillandsia
imperialis
B0292
*Tillandsia
heterophylla
B0047
-/63
61/100
1
16
6
2
*Tillandsia kauffmannii B0074
*Tillandsia multicaulis B0107
-/55
4
99/100
90/100 1
2
4
Tillandsia roezlii B0764
Tillandsia imperialis B0292
92/78
8
1
11
Tillandsia subconcolor B0227
18
*Tillandsia kauffmannii B0074
4
99/100
3
-/62
2
6
Tillandsia
confinis B0587
100/100
Tillandsia roezlii B0764
2
8
92/78
-/93 1 Tillandsia rudolfii B0654
10
Tillandsia subconcolor B0227
4
3
11
Tillandsia
stenoura
B0635
Tillandsia stenoura B0635
21
Tillandsia somnians B0301
93/100
13
95/100
100/100
4
Tillandsia somnians B0312
-/93
6
Tillandsia
pastensis B0521
3
-/88
1
5
Tillandsia maculata B0574
1
2
5
-/55
Tillandsia
tovarensis
B0605
71/100
14
*Tillandsia biflora B0090
3
23
1 -/50
Tillandsia ionochroma B0600
31
1
Tillandsia complanata B0562
7
100/100
12
2 80/100
Tillandsia complanata B0563
-/68
4
Tillandsia complanata B0244
1
18
8
5
Biflora clade
Tillandsia denudata var. vivipara B0750
1
5
100/100
-/96
*Tillandsia macbrideana B0070
3
4
6
1
Tillandsia krahnii B0235
2
6
*Tillandsia pseudomacbrideana B0036
17
80/100 3
Tillandsia incarnata B0522
5
81/100
Tillandsia latifolia var. leucophylla B0228
62/98
3
-/52
2
9
Tillandsia latifolia var. latifolia B0433
2
58/100
1
*Tillandsia latifolia var. divaricata B0068
4
Tillandsia
confertiflora
B0614
2
-/81
13
1
Tillandsia turneri B0650
11
Tillandsia floribunda B0351
14
7
*Tillandsia rauhii B0092
11
68/100
2
100/Tillandsia teres B0201
13
Rauhii clade
4
57/Tillandsia ecarinata B0237
9
8
100/84
Tillandsia ferreyrae B0241
3
3
Tillandsia purpurea B0246
Tillandsia purpurea B0246
8
8
2
2
99/100
3
99/100
3
Tillandsia straminea B0247
Tillandsia straminea B0247
82/99
82/99
2
Purpurea
clade
2
2
11
14
*Tillandsia cacticola B0044
*Tillandsia cacticola B0044
66/94
5
100/100
100/100
4
4
25
24
Tillandsia aurea B0250
Tillandsia aurea B0250
-/72
3
57/11
Tillandsia nana B0343
Tillandsia sphaerocephala B0366
9
4
99/100
Tillandsia
pseudomicans
B0347
18
*Tillandsia tortilis B0049
3
94/100
85/100
2
5
Tillandsia lepidosepala B0219
72/100
1
Tillandsia mauryana B0238
1
3
4
*Tillandsia plumosa B0086
90/100
8
82/98
6
59/99
Tillandsia atroviridipetala B0215
5
2
Tillandsia ignesiae B0222
71/100
2
4
Tillandsia oblivata B0205
Tillandsia oblivata B0205
3
4
63/100
63/100
16
6
4
Tillandsia balsasensis B0221
Tillandsia balsasensis B0221
1
2
-/97
-/51
Tillandsia heteromorpha B0224
51/100 2 Tillandsia reducta B0209
3
1
73/100 4
Tillandsia stellifera B0239
Tillandsia chusgonensis B0210
9
23
51/100
1
73/100
Tillandsia heteromorpha B0224
4
2 Tillandsia reducta B0209
97/100
Singularis clade
8
3
99/100
1
Tillandsia chusgonensis B0210
Tillandsia stellifera B0239
97/100
-/93
5
95/100
3
3
2
Tillandsia tomekii B0218
Tillandsia tomekii B0218
2
-/99
Tillandsia
tectorum var. tectorum f. tectorum B0249
Tillandsia tectorum var. tectorum f. tectorum B0249
3
Tillandsia
tectorum
var.
globosa
B0248
14
4
92/100
6
1
7
Tillandsia tectorum var. tectorum f. gigantea B0253
66/100
100/100
7
14
*Tillandsia rupicola B0039
64/98
10
7
Tillandsia tectorum var. viridula B0245
Chrysostachys clade
2
12
85/100
Tillandsia sphaerocephala B0366
Tillandsia hitchcockiana B0468
2
72/100
Tillandsia hitchcockiana B0629
4
96/100
Tillandsia cereicola B0134
1
Tillandsia cereicola B0476
2
14
1
-/100
Tillandsia espinosae B0143
6
22
-/88
97/100 1 Tillandsia espinosae B0462
4
Vriesea tuerckheimii B0148
2
1
-/98
89/100
Tillandsia andreettae B0373
3
3
80/100
Tillandsia cf. werneriana W. Rauh 40226 B0514
6
Tillandsia cf. werneriana BGBM 109-40-74-83 B0144
1
6
Tillandsia fragrans B0632
-/86
3
Tillandsia spathacea B0565
1
-/52
6
3
Tillandsia tequendamae B0569
-/100
14
2
2
Tillandsia strobeliae B0198
-/88
3
Splendens clade
7
-/92
100/100
*Tillandsia werneriana B0067
1
-/93 Tillandsia peruviana B0470
3
17
**Tillandsia barclayana B0028 & B0142
*Tillandsia appenii B0066
2
9
-/70
2
77/100
Tillandsia appenii B0464
10
3
Tillandsia seleriana B0413
1
*Tillandsia barthlottii B0035
21
-/53
6
5
2
Tillandsia ehlersiana B0431
99/100
Tillandsia barthlottii B0716
-/56
7
4
3
3
*Tillandsia ionantha B0084
-/97
Tillandsia cf. porphyrocraspeda W. Till 13080 B0350
68/99
1
21
8
Tillandsia aff. caput-medusae HBV B00B81-1 B0424
2
Tillandsia
myriantha
B0760
3
-/100
-/90
10
8
1
Tillandsia praschekii B0364
Tillandsia frank-hasei B0315
6
2
7
Tillandsia mitlaensis var. tulensis B0402
Tillandsia
petraea
B0630
5
-/94
4
89/100
9
Tillandsia pueblensis B0398
6
Tillandsia arpocalyx B0552
5
4
77/100
Tillandsia schatzlii B0416
*Tillandsia ionantha B0084
5
1
6
21
-/60
Tillandsia guerreroensis B0349
Tillandsia aff. caput-medusae HBV B00B81-1 B0424
2
-/97
14
10
Tillandsia variabilis B0308
2
Tillandsia seleriana B0413
2
1
13
6
-/70
Tillandsia cryptopoda B0787
-/53
Tillandsia ehlersiana B0431
3
7
3
-/57
10
Tillandsia cf. rhomboidea W. Till 19009 B0306
-/100
3
Tillandsia praschekii B0364
90/100
3
5
2
Tillandsia cf. rhomboidea W. Till 15048 B0372
1
-/100
Tillandsia mitlaensis var. tulensis B0402
3
4
1
Tillandsia cf. beutelspacheri R. Ehlers M890701 B0360
-/55
Tillandsia
pueblensis B0398
5
5
4
9
89/100
5
Tillandsia exserta B0390
77/100
Tillandsia schatzlii B0416
-/99
2
5
6
*Tillandsia fasciculata B0076
Tillandsia
guerreroensis
B0349
4
-/60
5
14
-/58
Tillandsia glabrior B0340
3
Tillandsia variabilis B0308
2
-/60
9
13
4
Tillandsia baileyi B0421
-/70
Tillandsia
cryptopoda
B0787
3
10
-/79
7
4
Tillandsia durangensis B0365
Tillandsia cf. rhomboidea W. Till 19009 B0306
3
90/100
2 -/99 2
3
-/83
Tillandsia tricolor B0785
2
8
Tillandsia
cf.
rhomboidea
W.
Till 15048 B0372
-/100
12
3
-/69
5
5
*Tillandsia caput-medusae B0046
Tillandsia cf. beutelspacheri R. Ehlers M890701 B0360
4
-/81
8
1
Tillandsia plagiotropica B0386
7
*Tillandsia fasciculata B0076
1
8
8
98/100
Tillandsia velutina B0427
Tillandsia plagiotropica B0386
7
4
8
*Tillandsia remota B0072
98/100
5
12
Tillandsia velutina B0427
8
14
98/100
Tillandsia pseudosetacea B0333
*Tillandsia caput-medusae B0046
6
1
5
*Tillandsia guatemalensis HBV B260/96 B0008
2
Tillandsia glabrior B0340
3
-/67
-/60
6
9
11
Tillandsia selleana B0243
3
Tillandsia baileyi B0421
-/79
6
-/72
10
35
Tillandsia leiboldiana B0323
2
12
Tillandsia durangensis B0365
-/99
91/100
2
89/100
3
*Tillandsia guatemalensis H. & L. Hromadnik 14257 B0103
5
13
Tillandsia tricolor B0785
8
5
97/100
*Tillandsia guatemalensis L. Hromadnik 15127 B0104
*Tillandsia remota B0072
8
22
7
*Tillandsia punctulata B0061
98/100
Tillandsia pseudosetacea B0333
2
-/58
11
1
*Tillandsia andrieuxii B0063
*Tillandsia guatemalensis HBV B260/96 B0008
2
2
4
-/67
6
8
Tillandsia moronesensis B0380
11
2
Tillandsia selleana B0243
-/75
9
-/72
6
Tillandsia achyrostachys B0408
Tillandsia leiboldiana B0323
9
76/98
6
2
89/100
Tillandsia huajuapanensis B0713
*Tillandsia guatemalensis H. & L. Hromadnik 14257 B0103
13
5
8
Tillandsia juerg-rutschmannii B0715
97/100
*Tillandsia guatemalensis L. Hromadnik 15127 B0104
4
2
Tillandsia moronesensis B0380
76/98 2
12
9
2
Tillandsia achyrostachys B0408
91/100
60/6
11
Tillandsia huajuapanensis B0713
3
3
6
*Tillandsia andrieuxii B0063
-/75
19
*Tillandsia punctulata B0061
5
Tillandsia juerg-rutschmannii B0715
8
2
Tillandsia exserta B0390
Tillandsia aff. utriculata J. Lautner 95/48 B0743
7
Tillandsia aff. utriculata J. Lautner 95/48 B0747
100/100
1
20
Tillandsia utriculata B0805
100/100
*Tillandsia utriculata B0100
4
6
2
99/100
62/96
Tillandsia utriculata B0807
17
1
Tillandsia filifolia B0788
89/100
6
1
8
80/100
Tillandsia filifolia B0790
15
100/100
5
5
Tillandsia fuchsii B0391
67/94
12
Tillandsia aff. cucaensis W. Rauh 70802 B0732
4
Tillandsia propagulifera B0310
1
-/57
1
4
Tillandsia cf. mima var. mima W. Rauh 53543 B0709
-/96
Tillandsia spiraliflora B0762
3
81/100
74/100
1 Tillandsia mima var. chiletensis B0708
4
Tillandsia mima var. chiletensis B0710
6
4
79/100
-/70
Tillandsia extensa B0712
19
Tillandsia adpressiflora B0597
5
11
55/77
Tillandsia hildae B0763
Tillandsia secunda B0527
Tillandsia secunda B0528
Tillandsia secunda B0549
Tillandsia secunda B0528
20
17
1
100/100
100/100
3
Tillandsia secunda B0551
Tillandsia secunda B0549
35
1
26
Tillandsia secunda B0527
Tillandsia secunda B0551
-/75
6
17
12
Tillandsia malzinei B0145
*Tillandsia paniculata B0102
31
5
3
100/100
Tillandsia malzinei B0492
90/100
17
*Tillandsia paniculata B0102
*Tillandsia disticha B0048
2
94/99
52
Tillandsia disticha B0202
9
100/100
Tillandsia disticha B0233
Tillandsia macropetala B0742
7
100/100
9
Tillandsia macropetala B0748
11
99/100
*Tillandsia viridiflora B0006
21
2
99/100
Tillandsia grandis B0124
6
99/100 Tillandsia grandis B0125
1 Lemeltonia triglochinoides B0723
78/94
2
1
10
Lemeltonia triglochinoides B0725
85/100
2
3
Lemeltonia triglochinoides B0724
-/97
3
11
Lemeltonia scaligera HBV B128/96 B0327
70/100
3
9
Lemeltonia scaligera L. Hromadnik 17033 B0234
67/100
4
1
Lemeltonia monadelpha B0745
-/86
1
Lemeltonia
acosta-solisii B0626
10
36
1
100/100
Lemeltonia cornuta B0744
3
100/100
*Lemeltonia dodsonii B0016
9
6
100/100
Lemeltonia dodsonii B0721
*Rothowia wagneriana B0058
*Rothowia wagneriana B0058
5
5
100/100
100/100
Rothowia wagneriana B0217
Rothowia wagneriana B0217
1
1
Rothowia platyrhachis allele 2 B0753
Rothowia platyrhachis allele 2 B0753
-/86
7
20
Rothowia laxissima B0294
Rothowia platyrhachis allele 1 B0753
9
17
100/100
81/100
Rothowia laxissima B0757
Rothowia
laxissima
B0294
81/100
8
6
100/100
Rothowia platyrhachis allele 1 B0753
Rothowia laxissima B0757
1
Racinaea pallidoflavens B0369
8
3
98/100
2
Racinaea diffusa B0480
-/76
7
Racinaea tetrantha var. tetrantha B0367
Racinaea tetrantha var. aurantiaca B0496
Racinaea tetrantha var. aurantiaca B0496
9
9
2
2
100/100
100/100
4
4
Racinaea pseudotetrantha B0624
Racinaea pseudotetrantha B0624
1
-/56
-/84 2
-/84 2
*Racinaea seemannii B0024
*Racinaea seemannii B0024
4
4
1
2
-/88
*Racinaea
elegans
B0051
*Racinaea
elegans B0051
7
57/95 1
5
4
10
57/95
7
1
Racinaea riocreuxii B0440
Racinaea riocreuxii B0440
-/79
9
8
1
-/79
Racinaea
tetrantha
var.
caribaea
B0448
Racinaea tetrantha var. caribaea B0448
93/100
4
71/100
2
2
20
*Racinaea ropalocarpa B0057
*Racinaea ropalocarpa B0057
4
4
74/100
74/100
-/81
1
1
21
1
Racinaea parviflora B0335
Racinaea parviflora B0335
74/97
74/97 3
3
5
Racinaea tillii B0572
Racinaea tillii B0572
55/98
3
Racinaea pugiformis B0613
5
8
76/100
Racinaea sinuosa B0617
3
51/91
9
Racinaea pectinata B0520
2
*Racinaea spiculosa B0099
9
2
100/100
Racinaea spiculosa B0304
Racinaea multiflora var. multiflora B0426
8
13
100/100
61/100
9
Racinaea multiflora var. decipiens B0478
100/100
3
Racinaea multiflora var. tomensis B0437
4
2
84/99
Racinaea insularis B0494
2
64/100
2
7
Racinaea fraseri J.M. Manzanares 7815 B0616
4
96/100 1
53/100
Racinaea fraseri W. Till 21049a B0547
13
1
Racinaea dyeriana B0151
20
100/100
100/100
Racinaea dyeriana B0456
1
Racinaea venusta B0729
1
69/87 2
16
Racinaea venusta B0731
100/100
1
*Racinaea venusta B0007
21
2
95/100
Racinaea hamaleana B0251
7
5
Racinaea hamaleana B0730
98/100
Racinaea hamaleana B0749
1
**Josemania lindenii B0023 & B0746
4 1
95/100
Josemania cyanea B0780
4
Josemania cyanea B0782
85/100
Josemania anceps B0741
5
3
66/99
99/100 Josemania anceps B0781
30
6
Josemania pretiosa B0231
3
100/100
Josemania umbellata B0216
10
99/100
Josemania umbellata B0758
2
*Guzmania donnell-smithii B0053
11
2
6
100/100
Guzmania donnell-smithii B0126
9
4 67/100
Guzmania flagellata B0475
-/64
5
Guzmania rauhiana B0297
5
12
Guzmania lingulata var. lingulata B0444
2
-/69
-/71
8
1
Guzmania lingulata var. minor B0778
7
6
Guzmania glomerata B0131
97/100 3
Guzmania conglomerata B0314
7
1
18
-/72
*Guzmania patula B0011
3
90/100
8
1
100/100
*Guzmania rhonhofiana B0096
55/93
3
Guzmania sphaeroidea B0506
10
13
Guzmania roezlii B0286
2
58/77
2
87/100
5
1
Guzmania strobilantha B0504
-/74
8
Guzmania coriostachya B0584
3
*Guzmania musaica var. musaica B0014
6
1
3 97/100
Guzmania musaica var. discolor B0489
8
6
*Guzmania herrerae B0010
91/100
10
Guzmania cf. scherzeriana W. Till 15011 B0295
1
*Guzmania multiflora B0094
1
-/50
1
1
*Guzmania multiflora B0094
3
3
Guzmania multiflora B0606
-/50
1
4
1
Guzmania multiflora B0606
Guzmania polycephala B0651
12
3
*Guzmania acorifolia B0052
2 2
*Guzmania condensata B0055
-/62
4
2
3
6
*Guzmania condensata B0055
Guzmania
roseiflora
B0291
-/75
1
-/96
3
-/66
6
3
Guzmania roseiflora B0291
7
2
-/75
Guzmania squarrosa B0607
4
5
-/77 2 Guzmania polycephala B0651
-/75
*Guzmania acorifolia B0052
8
7
9
Guzmania squarrosa B0607
-/75
Guzmania retusa B0498
9
10
Guzmania retusa B0498
Guzmania
brasiliensis
B0303
2
1
-/57
2
2
Guzmania marantoidea B0487
-/56
1
1
Guzmania cf. killipiana J.M. Manzanares 8127 B0627
-/64
4
2
Guzmania farciminiformis B0796
-/87
6
1 1
Guzmania tarapotina B0591
-/67
4
Guzmania conifera B0138
9
Guzmania acuminata B0300
3
5
Guzmania ekmanii B0447
4
90/100
4
Guzmania virescens B0718
11
Guzmania xanthobracteata B0623
2
6
*Guzmania graminifolia allele 1 B0120
3
83/100
57/96
7
16
*Guzmania graminifolia allele 2 B0120
13
67/100
Guzmania pearcei B0610
6
Guzmania danielii B0510
2
-/68
10
8
Guzmania sp. J.M. Manzanares 8131 B0628
3
-/72
8
*Guzmania wittmackii B0012
91/74
14
6
**Guzmania variegata B0015 & B0646
7
-/50
9
Guzmania calamifolia B0483
1
11
51/74
-/69
Guzmania mosquerae B0582
3
Guzmania sanguinea var. sanguinea B0495
13
5
100/100
Guzmania sanguinea var. brevipedicellata B0500
52/79
9
21
Guzmania fusispica B0289
64/91
12
13
17
*Guzmania angustifolia B0093
94/100
39
68/100
3
*Guzmania monostachia B0022
67/61
4
*Guzmania melinonis B0032
22
28
5
100/100
Guzmania melinonis B0593
100/100
10
Guzmania nicaraguensis B0479
36
*Guzmania hutchisonii B0003
2
3
Alcantarea imperialis B0135
Alcantarea imperialis B0135
2
2 Alcantarea imperialis B0154
1
Alcantarea imperialis B0154
-/99
32
Alcantarea regina B0136
*Alcantarea imperialis B0001
3
1
94/100
1
100/100
Alcantarea cf. geniculata L.F. Carvalho 5 B0751
Alcantarea edmundoi B0171
3
90/100
1
3
3
*Alcantarea imperialis B0001
90/100
Alcantarea edmundoi B0161
3
Alcantarea
edmundoi B0161
Alcantarea
regina
B0136
13
4
1
1
Alcantarea edmundoi B0171
73/100
94/100 Alcantarea cf. geniculata L.F. Carvalho 5 B0751
2
Alcantarea nevaresii E.M.C. Leme 2299 B0169
2
12
2
Alcantarea
nevaresii
E.M.C.
Leme
2299
B0169
1
73/100
Alcantarea geniculata B0153
1
-/58 2
Alcantarea geniculata B0153
2
66/99 3
Alcantarea nevaresii E.M.C. Leme 2227 B0163
3
66/99
Alcantarea nevaresii E.M.C. Leme 2227 B0163
2
-/78
1
*Alcantarea duarteana B0059
3
96/100
1
Alcantarea
duarteana
B0165
-/73
9
7
Alcantarea benzingii B0166
7
Alcantarea roberto-kautskyi B0158
Alcantarea nahoumii B0156
1 Alcantarea nahoumii B0156
69/13
1
-/88
Alcantarea sp. E.M.C. Leme 3660 B0160
Alcantarea sp. E.M.C. Leme 3660 B0160
3
1
2
100/100
-/81
Alcantarea aff. extensa E.M.C. Leme 1942 B0164
Alcantarea burle-marxii B0159
3
2
2
59/-/78
Alcantarea glazioviana B0167
Alcantarea aff. extensa E.M.C. Leme 1942 B0164
7
4
8
Alcantarea burle-marxii B0159
Alcantarea sp. E.M.C. Leme 4803 B0168
91/100
91/100
1
5
1
Alcantarea sp. E.M.C. Leme 4803 B0168
Alcantarea glazioviana B0167
-/62
Alcantarea heloisae B0155
7
2
4
88/100
52/90
Alcantarea odorata B0157
3
Alcantarea farneyi B0170
1
*Vriesea jonghei B0065
6
8
2 96/100
Vriesea platynema B0146
12
Vriesea pabstii B0357
3
4
Vriesea
flava B0322
6
3
10
51/99
Vriesea maxoniana B0490
7
4
29
*Vriesea correia-araujoi B0095
8
Vriesea procera var. tenuis B0296
7
100/100
5
69/100
Vriesea neoglutinosa B0311
5
6
*Vriesea psittacina allele 1 B0020
3
-/96
2
3
*Vriesea psittacina allele 2 B0020
-/96
6
2
Vriesea erythrodactylon B0309
-/97
8
4
12
Vriesea sucrei B0302
5
-/100
Vriesea scalaris B0147
4
60/96 1 Vriesea simplex B0505
10
3
*Vriesea saundersii B0004
Vriesea unilateralis B0316
18
100/100
2
Vriesea roethii B0511
-/59
11
1
1
100/100
Vriesea ruschii B0719
7
Vriesea sparsiflora B0484
20
4
Vriesea longicaulis B0290
8
89/100
Vriesea plurifolia B0376
5
89/100
9
6
Vriesea aff. croceana G. Croce s.n. B0801
11
66/100
Vriesea croceana B0802
1
Mezobromelia sp. J.M. Manzanares 7640 B0194
Mezobromelia schimperiana B0589
1
3
-/61
1
Mezobromelia schimperiana B0611
Mezobromelia schimperiana B0612
3
1
3
-/93
Mezobromelia
schimperiana
B0334
Mezobromelia aff. schimperiana J.M. Manzanares 7714 B0197
1
Mezobromelia bicolor B0578
Mezobromelia bicolor B0583
17
2
1
57/72
Mezobromelia
schimperiana
B0589
Mezobromelia
schimperiana B0334
1
7
8
2
93/100
Mezobromelia aff. schimperiana J.M. Manzanares 7714 B0197
Mezobromelia schimperiana B0611
99/100
99/100
2 2
2
Mezobromelia schimperiana B0612
Mezobromelia sp. J.M. Manzanares 7640 B0194
1
2
2
2
Mezobromelia bicolor B0583
66/94
66/94
Mezobromelia cf. pleiosticha J.M. Manzanares 7766 B0330
2
Mezobromelia cf. pleiosticha J.M. Manzanares 7766 B0330
Mezobromelia bicolor B0578
1
1
Mezobromelia pleiosticha B0274
Mezobromelia pleiosticha B0274
5
5
19
99/100 2 Mezobromelia pleiosticha B0649
99/100 2
85/100
Mezobromelia pleiosticha B0649
1
*Cipuropsis zamorensis B0045
5
93/100
3
4
1
71/100
*Cipuropsis cv. Elan B0117
82/98 1
Cipuropsis amicorum B0318
14
4
*Tillandsia singularis B0064
22
17
99/100
Tillandsia asplundii B0588 Singularis clade
14
18
Vriesea
tuerckheimii
B0148
66/100
17
*Vriesea chrysostachys B0005
10
6
32
85/100
*Vriesea ospinae B0054 Chrysostachys clade
-/88
3
*Vriesea splendens B0037
14
10
100/100
Vriesea glutinosa B0130 Splendens clade
2
Werauhia hygrometrica B0598
10
100/100
2
Werauhia hygrometrica B0621
72/98
11
4
Werauhia haltonii B0580
67/93
4
*Werauhia ororiensis B0025
6
17
8
69/100
20
Werauhia sintenisii B0450
-/99
16
26
Werauhia pedicellata B0752
100/100
100/100
24
*Werauhia insignis B0017
2
11
Werauhia viridiflora B0285
5
89/100
2
1
2
Werauhia viridiflora B0325
Werauhia viridiflora B0285
5
89/100
3
3
Werauhia ampla B0132
10
Werauhia viridiflora B0325
7
19
5
99/100
4
Werauhia gladioliflora B0149
-/80
13
100/100
Werauhia gladioliflora B0149
8
11
4
5
98/100
Werauhia gladioliflora B0509
98/100
Werauhia gladioliflora B0509
99/100
21
17
3
*Werauhia ringens B0019
21
Werauhia ampla B0132
9
60/99
100/100
Werauhia guadelupensis B0313
16
*Werauhia tarmaensis B0018
18
12
99/100
Werauhia werckleana B0497
10
*Catopsis nutans B0002
6
9
19
82/100
Catopsis delicatula B0461
52/100
9
2
Catopsis sessiliflora B0459
18
4
*Catopsis subulata B0105
10
*Catopsis nutans B0002
12
11
6
Catopsis nitida B0463
9
70/100
4
82/100
Catopsis delicatula B0461
14
52/100
4
*Catopsis juncifolia B0029
9
17
Catopsis
sessiliflora B0459
-/88
5
-/55
*Catopsis morreniana B0106
27
13
11
Catopsis nitida B0463
2
11
96/100
99/100
Catopsis morreniana B0488
22
99
70/100
*Catopsis
subulata
B0105
7
Catopsis minimiflora B0467
17
*Catopsis juncifolia B0029
100/100
26
44
Catopsis paniculata B0507
2
*Glomeropitcairnia erectiflora B0030
19
100/100
1
83
100/100
Glomeropitcairnia erectiflora B0128
9
100/100
*Glomeropitcairnia penduliflora B0013
S
10
83/100
Xiphioides clade
86/100
2
P
Tillandsia subg. Phytarrhiza
P
O
O
Tillandsia subg. Anoplophytum
Tillandsia subg. Phytarrhiza
&
Tillandsia subg. Diaphoranthema
Tillandsia subg. Anoplophytum
N
N
S
M
Tillandsia subgg./clades
R
Tillandsia subg.
Pseudovriesea
Lemeltonia
M
Racinaea subg. Racinaea
Racinaea subg.
Pseudophytarrhiza
Josemania
Rothowia
Guzmania
L
Mezobromelia
Q
L
Tillandsia subg. Viridantha
Cipuropsis
K
K
Tillandsia subg. Pseudovriesea
J
J
Tillandsia subg. Tillandsia
Tillandsia
I
I
Tillandsia subg. Pseudalcantarea
Lemeltonia
H
Rothowia
H
G
Core Tillandsieae
G
Racinaea
Racinaea subg. Racinaea
Racinaea subg. Pseudophytarrhiza
Josemania
Tillandsieae
R
F
F
Guzmania
Core Tillandsioideae
E
E
Alcantarea
D
D
Vrieseinae
Vriesea
Mezobromelia
Tillandsioideae
C
Vrieseeae
CipuropsisMezobromelia
clade
C
Cipuropsis
Q
Cipuropsidinae
Werauhia
B
B
Catopsis
A
Pogospermeae
A
Glomeropitcairnia
5 changes
Glomeropitcairnieae
PHYC
PRK
postulated
ploidy level
Geno
types
Length
Glomeropitcairnia erectiflora Mez
B0030
1
2
1177
Glomeropitcairnia erectiflora Mez
B0128
0
1
1177
0
0
0
1
1198
1198
1198
2n = 2x
Glomeropitcairnia penduliflora (Griseb.) Mez
B0013
0
1
1177
0
0
0
1
1205
1205
1205
2n = 2x
Catopsis delicatula L.B. Sm.
B0461
3
2
1177
0
0
0
1
927
927
927
2n = 2x
Catopsis juncifolia Mez & Wercklé ex Mez
B0029
0
1
1177
0
0
0
1
975
975
975
2n = 2x
Indels
SSR
Consensus
length
Allele 2
length
(a2)
SNPs
SNPs
Geno
types
Allele 1
length
(a1)
DNA no.
Taxon
Bromeliaceae tribe Glomeropitcairnieae Harms
Glomeropitcairnia (Mez) Mez
7
0
0
2
1198
1198
1198
2n = 2x
Bromeliaceae tribe Pogospermeae Brongn.
Catopsis Griseb.
Catopsis minimiflora Matuda
B0467
0
1
1177
7
1
0
2
1005
1005
1004
2n = 2x
Catopsis morreniana Mez
B0106
0
1
1177
3
0
0
2
969
969
969
2n = 2x
Catopsis morreniana Mez
B0488
0
1
1177
4
0
0
2
969
969
969
2n = 2x
Catopsis nitida (Hook.) Griseb.
B0463
0
1
1177
0
0
0
1
968
968
968
2n = 2x
Catopsis nutans (Sw.) Griseb.
B0002
0
1
1177
0
0
0
1
927
927
927
2n = 2x
Catopsis paniculata E. Morren
B0507
0
1
1177
0
0
0
1
892
892
892
2n = 2x
Catopsis sessiliflora (Ruiz & Pav.) Mez
B0459
1
2
1177
6
0
0
2
927
927
927
2n = 2x
Catopsis subulata L.B. Sm.
B0105
2
2
1177
6
0
0
2
969
969
969
2n = 2x
Bromeliaceae tribe Tillandsieae Rchb.
Guzmania Ruiz & Pav.
Guzmania acorifolia (Griseb.) Mez
B0052
0
1
1192
0
0
0
1
1021
1021
1021
2n = 2x
Guzmania acuminata L.B. Sm.
B0300
0
1
1177
1
0
0
2
992
992
992
2n = 2x
Guzmania angustifolia (Baker) Wittm.
B0093
1
2
1177
9
2
0
2
1050
1050
1018
2n = 2x
Guzmania brasiliensis Ule
B0303
0
1
1177
0
0
0
1
1037
1037
1037
2n = 2x
Guzmania calamifolia André ex Mez
B0483
1
2
1177
1
0
0
2
1033
1033
1033
2n = 2x
Guzmania condensata Mez & Wercklé
B0055
0
1
1192
0
0
0
1
918
918
918
2n = 2x
Guzmania conglomerata H. Luther
B0314
0
1
1177
8
0
0
2
967
967
967
2n = 2x
Guzmania conifera (André) André ex Mez
B0138
2
2
1177
0
0
0
1
993
993
993
2n = 2x
Guzmania coriostachya (Griseb.) Mez
B0584
1
2
1177
5
1
0
2
926
920
926
2n = 2x
Guzmania danielii L.B. Sm.
B0510
1
2
1177
2
0
0
2
1056
1056
1056
2n = 2x
Guzmania donnell-smithii Mez
B0053
0
1
1177
0
0
0
1
1040
1040
1040
2n = 2x
Guzmania donnell-smithii Mez
B0126
0
1
1177
0
0
0
1
1040
1040
1040
2n = 2x
Guzmania ekmanii (Harms) Harms ex Mez
B0447
0
1
1177
0
0
0
1
1026
1026
1026
2n = 2x
Guzmania farciminiformis H. Luther
B0796
1
2
1177
3
0
0
2
1008
1008
1008
2n = 2x
Guzmania flagellata S. Pierce & J.R. Grant
B0475
0
1
1177
0
0
0
1
1035
1035
1035
2n = 2x
Guzmania fusispica Mez & Sodiro
B0289
0
1
1177
0
0
0
1
1050
1050
1050
2n = 2x
Guzmania glomerata Mez & Wercklé
B0131
0
1
1177
0
0
0
1
967
967
967
2n = 2x
Guzmania graminifolia (André ex Baker) L.B. Sm.
B0120
2
2
1177
9
4
1
2
n.u.
1209!
1185!
2n = 2x
Guzmania herrerae H. Luther & W.J. Kress
B0010
1
2
1177
13
1
1
2
1220
1220
1219
2n = 2x
Guzmania hutchisonii (L.B. Sm.) Barfuss & W. Till
B0003
1
2
1177
0
0
0
1
1129
1129
1129
2n = 2x
Guzmania cf. killipiana L.B. Sm.
B0627
0
1
1177
8
0
0
2
1020
1020
1020
2n = 2x
Guzmania lingulata (L.) Mez var. lingulata
B0444
0
1
1177
0
0
0
1
1003
1003
1003
2n = 2x
Guzmania lingulata var. minor (Mez) L.B. Sm. &
Pittendr.
B0778
0
1
1177
0
0
0
1
949
949
949
2n = 2x
Guzmania marantoidea (Rusby) H. Luther
B0487
6
2
1177
6
2
0
2
1022
1018
939
2n = 2x
Guzmania melinonis Regel
B0032
0
1
1177
0
0
0
1
1132
1132
1132
2n = 2x
Guzmania melinonis Regel
B0593
1
2
1177
0
0
1
1
1154
1154
1154
2n = 2x
Guzmania monostachia (L.) Rusby ex Mez
B0022
0
1
1177
0
0
0
1
1005
1005
1005
2n = 2x
195
BARFUSS, M.H.J.
NEW CLASSIFICATION
PHYC
PRK
postulated
ploidy level
SNPs
Geno
types
Length
Guzmania mosquerae (Wittm.) Mez
B0582
0
1
1180
0
0
0
1
1055
1055
1055
2n = 2x
Guzmania multiflora (André) André ex Mez
B0094
0
1
1177
0
0
0
1
926
926
926
2n = 2x
Guzmania multiflora (André) André ex Mez
B0606
0
1
1177
0
0
1
1
1098
1098
1098
2n = 2x
Guzmania musaica var. discolor H. Luther
B0489
0
1
1177
4
0
1
2
1225
1225
1225
2n = 2x
Guzmania musaica (Linden & André) Mez var. musaica
B0014
1
2
1177
1
0
1
2
1225
1225
1225
2n = 2x
Guzmania nicaraguensis Mez & C.F. Baker
B0479
0
1
1177
0
0
0
1
1127
1127
1127
2n = 2x
Guzmania patula Mez & Wercklé
B0011
0
1
1177
0
0
0
1
831
831
831
2n = 2x
Guzmania pearcei (Baker) L.B. Sm.
B0610
0
1
1177
0
0
1
1
1232
1232
1232
2n = 2x
Guzmania polycephala Mez & Wercklé ex Mez
B0651
0
1
1177
0
0
0
1
917
917
917
2n = 2x
Indels
SSR
Consensus
length
Allele 2
length
(a2)
DNA no.
SNPs
Geno
types
Allele 1
length
(a1)
Taxon
Guzmania rauhiana H. Luther
B0297
0
1
1177
0
0
0
1
1325
1325
1325
2n = 2x
Guzmania retusa L.B. Sm.
B0498
1
2
1177
0
0
0
1
995
995
995
2n = 2x
Guzmania rhonhofiana Harms
B0096
0
1
1177
0
0
0
1
831
831
831
2n = 2x
Guzmania roezlii (E. Morren) Mez
B0286
0
1
1177
0
0
0
1
884
884
884
2n = 2x
Guzmania roseiflora Rauh
B0291
6
2
1177
1
1
0
2
934
934
928
2n = 2x
Guzmania sanguinea var. brevipedicellata Gilmartin
B0500
0
1
1177
3
0
0
2
1050
1050
1050
2n = 2x
Guzmania sanguinea (André) André ex Mez var.
sanguinea
B0495
2
2
1177
4
0
0
2
1050
1050
1050
2n = 2x
Guzmania cf. scherzeriana Mez
B0295
0
1
1177
0
0
1
1
1218
1218
1218
2n = 2x
Guzmania sphaeroidea (André) André ex Mez
B0506
0
1
1177
1
0
0
2
838
838
838
2n = 2x
Guzmania squarrosa (Mez & Sodiro) L.B. Sm. &
Pittendr.
B0607
0
1
1177
0
0
0
1
929
929
929
2n = 2x
Guzmania strobilantha (Ruiz & Pav.) Mez
B0504
0
1
1177
0
0
0
1
880
880
880
2n = 2x
Guzmania tarapotina Ule
B0591
7
2
1177
14
2
0
2
1038
?
?
2n = 2x
Guzmania variegata L.B. Sm.
B0015
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Guzmania variegata L.B. Sm.
B0646
8
2
1177
0
0
0
1
1015
1015
1015
2n = 2x
Guzmania virescens (Hook.) Mez
B0718
5
2
1177
3
1
0
2
1035
1035
1029
2n = 2x
Guzmania wittmackii (André) André ex Mez
B0012
0
1
1177
0
0
0
1
1030
1030
1030
2n = 2x
Guzmania xanthobractea Gilmartin
B0623
0
1
1177
0
0
0
1
1033
1033
1033
2n = 2x
Guzmania sp.
B0628
1
2
1177
2
0
0
2
1043
1043
1043
2n = 2x
Josemania anceps (Lodd.) Barfuss & W. Till
B0741
0
1
1177
0
0
0
1
1048
1048
1048
2n = 2x
Josemania anceps (Lodd.) Barfuss & W. Till
B0781
0
1
1177
0
0
0
1
1048
1048
1048
2n = 2x
Josemania cyanea (Linden ex K. Koch) Barfuss & W. Till
B0780
2
2
1177
4
1
0
2
1049
1049
1048
2n = 2x
Josemania cyanea (Linden ex K. Koch) Barfuss & W. Till
B0782
5
2
1177
6
0
0
2
1049
1049
1049
2n = 2x
Josemania Barfuss & W. Till
Josemania lindenii (Regel) Barfuss & W. Till
B0023
n.d
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Josemania lindenii (Regel) Barfuss & W. Till
B0746
0
1
1177
7
0
0
2
1049
1049
1049
2n = 2x
Josemania pretiosa (Mez) Barfuss & W. Till
B0231
3
2
1177
0
0
0
1
1049
1049
1049
2n = 2x
Josemania umbellata (André) Barfuss & W. Till
B0216
1
2
1177
9
1
1
2
1071
1071
1070
2n = 2x
Josemania umbellata (André) Barfuss & W. Till
B0758
5
2
1177
14
1
1
2
1071
1071
1070
2n = 2x
Lemeltonia acosta-solisii (Gilmartin) Barfuss & W. Till
B0626
0
1
1177
0
0
1
1
1103
1103
1103
2n = 2x
Lemeltonia cornuta (Mez & Sodiro) Barfuss & W. Till
B0744
0
1
1177
0
0
1
1
1103
1103
1103
2n = 2x
Lemeltonia dodsonii (L.B. Sm.) Barfuss & W. Till
B0016
3
2
1177
0
0
1
1
1137
1137
1137
2n = 2x
Lemeltonia dodsonii (L.B. Sm.) Barfuss & W. Till
B0721
1
2
1177
0
0
1
1
1116
1116
1116
2n = 2x
Lemeltonia monadelpha (E. Morren) Barfuss & W. Till
B0745
0
1
1177
0
0
1
1
1120
1120
1120
2n = 2x
Lemeltonia scaligera (Mez & Sodiro) Barfuss & W. Till
B0234
0
1
1177
9
0
1
2
1119
1119
1119
2n = 2x
Lemeltonia scaligera (Mez & Sodiro) Barfuss & W. Till
B0327
1
2
1177
4
0
1
2
1120
1120
1120
2n = 2x
Lemeltonia triglochinoides (C. Presl) Barfuss & W. Till
B0723
5
2
1177
7
0
1
2
1118
1118
1118
2n = 2x
Lemeltonia Barfuss & W. Till
196
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
PHYC
PRK
postulated
ploidy level
SNPs
Geno
types
Length
Lemeltonia triglochinoides (C. Presl) Barfuss & W. Till
B0724
3
2
1177
0
0
1
1
1126
1126
1126
2n = 2x
Lemeltonia triglochinoides (C. Presl) Barfuss & W. Till
B0725
3
2
1177
4
1
1
2
1125
?
?
2n = 2x
Racinaea hamaleana (E. Morren) Barfuss & W. Till
B0251
0
1
1177
2
1
1
2
1122
1122
1121
2n = 2x
Racinaea hamaleana (E. Morren) Barfuss & W. Till
B0730
0
1
1177
6
1
1
2
1118
1118
1119
2n = 2x
Indels
SSR
Consensus
length
Allele 2
length
(a2)
DNA no.
SNPs
Geno
types
Allele 1
length
(a1)
Taxon
Racinaea M.A. Spencer & L.B. Sm.
Racinaea subg. Pseudophytarrhiza Barfuss & W. Till
Racinaea hamaleana (E. Morren) Barfuss & W. Till
B0749
1
2
1177
8
1
1
2
1122
1122
1118
2n = 2x
Racinaea venusta (Mez & Wercklé) Barfuss & W. Till
B0007
0
1
1177
4
0
1
2
1131
1131
1131
2n = 2x
Racinaea venusta (Mez & Wercklé) Barfuss & W. Till
B0729
2
2
1177
2
2
1
2
1129
?
?
2n = 2x
Racinaea venusta (Mez & Wercklé) Barfuss & W. Till
B0731
0
1
1177
2
2
1
2
1129
?
?
2n = 2x
Racinaea diffusa (L.B. Sm.) M.A. Spencer & L.B. Sm.
B0480
2
2
1177
0
0
0
1
844
844
844
2n = 2x
Racinaea dyeriana (André) Barfuss & W. Till
B0151
0
1
1177
0
0
0
1
953
953
953
2n = 2x
Racinaea subg. Racinaea
Racinaea dyeriana (André) Barfuss & W. Till
B0456
0
1
1177
4
0
0
2
953
953
953
2n = 2x
Racinaea elegans (L.B. Sm.) M.A. Spencer & L.B. Sm.
B0051
1
2
1177
0
0
0
1
881
881
881
2n = 2x
Racinaea fraseri (Baker) M.A. Spencer & L.B. Sm.
B0547
0
1
1177
0
0
1
1
1144
1144
1144
2n = 2x
Racinaea fraseri (Baker) M.A. Spencer & L.B. Sm.
B0616
0
1
1177
0
0
1
1
1140
1140
1140
2n = 2x
Racinaea insularis (Mez) M.A. Spencer & L.B. Sm.
B0494
0
1
1177
0
0
0
1
1139
1139
1139
2n = 2x
B0478
1
2
1177
0
0
1
1
1141
1141
1141
2n = 2x
B0426
0
1
1177
0
0
1
1
1141
1141
1141
2n = 2x
Racinaea multiflora var. decipiens (André) M.A.
Spencer & L.B. Sm.
Racinaea multiflora (Benth.) M.A. Spencer & L.B. Sm.
var. multiflora
Racinaea multiflora var. tomensis (L.B. Sm.) M.A.
Spencer & L.B. Sm.
B0437
0
1
1177
7
0
0
2
1142
1142
1142
2n = 2x
Racinaea pallidoflavens (Mez) M.A. Spencer & L.B. Sm.
B0369
1
2
1177
5
0
0
2
838
838
838
2n = 2x
Racinaea parviflora (Ruiz & Pav.) M.A. Spencer & L.B.
Sm.
B0335
4
2
1177
5
0
0
2
885
885
885
2n = 2x
Racinaea pectinata (André) M.A. Spencer & L.B. Sm.
B0520
0
1
1177
0
0
0
1
920
920
920
2n = 2x
Racinaea pseudotetrantha (Gilmartin & H. Luther) J.R.
Grant
B0624
0
1
1177
0
0
0
1
870
870
870
2n = 2x
Racinaea pugiformis (L.B. Sm.) M.A. Spencer & L.B. Sm.
B0613
1
2
1177
0
0
0
1
902
902
902
2n = 2x
Racinaea riocreuxii (André) M.A. Spencer & L.B. Sm.
B0440
3
2
1177
3
0
0
2
885
885
885
2n = 2x
Racinaea ropalocarpa (André) M.A. Spencer & L.B. Sm.
B0057
5
2
1177
9
1
0
2
885
885
881
2n = 2x
Racinaea seemannii (Baker) M.A. Spencer & L.B. Sm.
B0024
1
2
1177
7
3
0
2
869
?
?
2n = 2x
Racinaea sinuosa (L.B. Sm.) M.A. Spencer & L.B. Sm.
B0617
4
2
1177
1
0
0
2
940
940
940
2n = 2x
Racinaea spiculosa (Griseb.) M.A. Spencer & L.B. Sm.
B0099
0
1
1177
0
0
0
1
979
979
979
2n = 2x
Racinaea spiculosa (Griseb.) M.A. Spencer & L.B. Sm.
B0304
0
1
1177
0
0
1
1
1008
1008
1008
2n = 2x
B0496
0
1
1177
0
0
0
1
870
870
870
2n = 2x
B0448
0
1
1177
0
0
0
1
906
906
906
2n = 2x
B0367
2
2
1177
5
1
0
2
906
906
904
2n = 2x
B0572
0
1
1177
3
1
0
2
908
908
906
2n = 2x
Rothowia laxissima (Mez) Barfuss & W. Till
B0294
0
1
1177
0
0
1
1
1144
1144
1144
2n = 2x
Rothowia laxissima (Mez) Barfuss & W. Till
B0757
0
1
1177
0
0
1
1
1144
1144
1144
2n = 2x
Rothowia platyrhachis (Mez) Barfuss & W. Till
B0753
2
2
1177
10
3
1
2
n.u.
1149
1143
2n = 2x
Rothowia wagneriana (L.B. Sm.) Barfuss & W. Till
B0058
0
1
1177
0
0
1
1
1149
1149
1149
2n = 2x
Rothowia wagneriana (L.B. Sm.) Barfuss & W. Till
B0217
0
1
1177
0
0
1
1
1149
1149
1149
2n = 2x
B0394
9
2
1177
6
0
1
2
1293
1293
1293
2n = 2x
Racinaea tetrantha var. aurantiaca (Griseb.) M.A.
Spencer & L.B. Sm.
Racinaea tetrantha var. caribaea (L.B. Sm.) M.A.
Spencer & L.B. Sm.
Racinaea tetrantha (Ruiz & Pav.) M.A. Spencer & L.B.
Sm. var. tetrantha
Racinaea tilli Manzan. & Gouda
Rothowia Barfuss & W. Till
Tillandsia L.
Tillandsia subg. Anoplophytum (Beer) Baker
Tillandsia araujei Mez
197
BARFUSS, M.H.J.
NEW CLASSIFICATION
PHYC
PRK
postulated
ploidy level
SNPs
Geno
types
Length
Tillandsia aff. araujei Mez
B0422
0
1
1177
2
0
1
2
1112
1112
1112
2n = 2x
Tillandsia bergeri Mez
B0097
7
2
1177
0
0
0
1
1058
1058
1058
2n = 2x
Tillandsia bergeri Mez cv. Alba
B0110
2
2
1177
0
0
1
1
1089
1089
1089
2n = 2x
Indels
SSR
Consensus
length
Allele 2
length
(a2)
DNA no.
SNPs
Geno
types
Allele 1
length
(a1)
Taxon
Tillandsia burle-marxii Ehlers
B0393
0
1
1177
0
0
1
1
1133
1133
1133
2n = 2x
Tillandsia ixioides Griseb.
B0043
0
1
1177
1
0
1
2
1129
1129
1129
2n = 2x
Tillandsia jonesii Strehl
B0389
3
2
1177
13
1
1
2
1113
1113
1109
2n = 2x
Tillandsia neglecta E. Pereira
B0339
9
2
1177
3
0
1
2
1120
1120
1120
2n = 2x
Tillandsia pohliana Mez
B0080
0
1
1177
0
0
0
1
990
990
990
2n = 2x
Tillandsia recurvifolia var. subsecundifolia (W. Weber
& Ehlers) W. Till
B0401
1
2
1177
4
0
1
2
1131
1131
1131
2n = 2x
Tillandsia stricta var. albifolia H. Hrom. & Rauh
B0410
1
2
1177
2
0
1
2
1113
1113
1113
2n = 2x
Tillandsia stricta Sol. ex Sims var. stricta
B0081
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d
n.d.
n.d.
n.d.
n.d.
Tillandsia tenuifolia L.
B0026
5
2
1177
10
0
1
2
1112
1112
1112
2n = 2x
Tillandsia aff. tenuifolia L.
B0414
0
1
1177
2
0
1
2
1115
1115
1115
2n = 2x
Tillandsia capillaris Ruiz & Pav.
B0518
11
4
1180
13
2
1
2
1054
?
?
2n = 4x!
Tillandsia funebris A. Cast.
B0089
1
2
1177
9
3
1
2
1141
1141
?
2n = 2x
Tillandsia kuehhasii W. Till
B0396
1
2
1180
7
1
1
2
1053
1053
1051
2n = 2x
Tillandsia landbeckii subsp. andina W. Till
B0423
0
1
1180
0
0
1
1
1131
1131
1131
2n = 2x
Tillandsia mollis H. Hrom. & W. Till
B0206
4
2
1177
8
2
1
2
1133
?
?
2n = 2x
Tillandsia recurvata (L.) L.
B0529
0
1
1177
0
0
0
1
1145
1145
1145
2n = 2x
Tillandsia recurvata (L.) L.
B0540
0
1
1177
0
0
1
1
1145
1145
1145
2n = 2x
Tillandsia usneoides (L.) L.
B0083
4
2
1177
3
1
1
2
1130
1130
1129
2n = 2x
Tillandsia usneoides (L.) L.
B0109
3
2
1177
8
1
1
2
1133
1134
1133
2n = 2x
Tillandsia usneoides (L.) L.
B0451
3
2
1177
9
1
1
2
1133
1133
1132
2n = 2x
Tillandsia cf. usneoides (L.) L.
B0539
0
1
1177
0
0
1
1
1128
1128
1128
2n = 2x
Tillandsia virescens Ruiz & Pav.
B0516
3
4
1180
9
1
1
2
1056
1056
1054
2n = 4x!
Tillandsia duratii Vis.
B0088
5
2
1180
7
0
0
2
1134
1134
1134
2n = 2x
Tillandsia duratii Vis.
B0737
4
2
1180
8
0
1
2
1134
1134
1134
2n = 2x
Tillandsia duratii Vis.
B0740
5
2
1180
9
1
1
2
1135
1135
1134
2n = 2x
Tillandsia kirschnekii Rauh & W. Till
B0384
1
2
1180
4
1
1
2
1134
1134
1133
2n = 2x
Tillandsia subg. Diaphoranthema (Beer) Baker
Tillandsia subg. Phytarrhiza (Vis.) Baker
Tillandsia paleacea C. Presl
B0404
4
2
1180
11
1
1
2
1137
1137
1134
2n = 2x
Tillandsia aff. streptocarpa Baker
B0226
2
2
1180
3
1
1
2
1135
1135
1134
2n = 2x
Tillandsia grandis Schltdl.
B0124
2
2
1177
2
0
1
2
1141
1141
1141
2n = 2x
Tillandsia grandis Schltdl.
B0125
1
2
1177
9
2
1
2
1148
1148
1147
2n = 2x
Tillandsia macropetala Wawra
B0742
2
2
1177
3
3
0
2
1142
1142
1136
2n = 2x
Tillandsia macropetala Wawra
B0748
2
2
1177
1
1
1
2
1139
1139
1136
2n = 2x
Tillandsia viridiflora (Beer) Baker
B0006
0
1
1177
0
1
1
2
1139
1139
1138
2n = 2x
B0373
0
1
1177
0
0
0
1
1136
1136
1136
2n = 2x
Tillandsia subg. Pseudalcantarea Mez
Tillandsia subg. Pseudovriesea Barfuss & W. Till
Tillandsia andreettae (Rauh) J.R. Grant
Tillandsia appenii (Rauh) J.R. Grant
B0066
0
1
1177
4
0
1
2
1133
1133
1133
2n = 2x
Tillandsia appenii (Rauh) J.R. Grant
B0464
3
2
1177
1
1
1
2
1115
1115
1114
2n = 2x
Tillandsia arpocalyx André
B0552
0
1
1177
1
1
1
2
1176
1176
1132
2n = 2x
Tillandsia barclayana Baker
B0028
n.d.
n.d.
n.d.
n.d.
n.d
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Tillandsia barclayana Baker
B0142
3
2
1177
4
2
1
2
1137
1137
1134
2n = 2x
198
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
PHYC
PRK
postulated
ploidy level
SNPs
Geno
types
Length
Tillandsia barthlottii Rauh
B0035
3
2
1177
0
1
1
2
1133
1133
1130
2n = 2x
Tillandsia barthlottii Rauh
B0716
1
2
1177
3
1
1
2
1134
1134
1133
2n = 2x
Tillandsia cereicola Mez
B0134
1
2
1177
7
1
1
2
1137
1138
1137
2n = 2x
Tillandsia cereicola Mez
B0476
0
1
1177
2
1
1
2
1142
1142
1136
2n = 2x
Tillandsia espinosae L.B. Sm.
B0143
0
1
1177
0
0
0
1
1135
1135
1135
2n = 2x
Tillandsia espinosae L.B. Sm.
B0462
0
1
1177
2
0
1
2
1136
1136
1136
2n = 2x
Indels
SSR
Consensus
length
Allele 2
length
(a2)
DNA no.
SNPs
Geno
types
Allele 1
length
(a1)
Taxon
Tillandsia fragrans André
B0632
2
2
1177
10
1
0
2
1132
1132
1131
2n = 2x
Tillandsia frank-hasei J.R. Grant
B0315
0
1
1177
0
0
1
1
1131
1131
1131
2n = 2x
Tillandsia hitchcockiana L.B. Sm.
B0468
0
1
1177
2
0
0
2
1137
1137
1137
2n = 2x
Tillandsia hitchcockiana L.B. Sm.
B0629
2
2
1177
0
0
0
1
1137
1137
1137
2n = 2x
Tillandsia myriantha Baker
B0760
0
1
1177
0
0
0
1
1102
1102
1102
2n = 2x
Tillandsia peruviana J.R. Grant
B0470
0
1
1177
2
0
1
2
1136
1136
1136
2n = 2x
Tillandsia petraea L.B. Sm.
B0630
1
2
1177
0
0
0
1
1110
1110
1110
2n = 2x
Tillandsia cf. porphyrocraspeda J.R. Grant
B0350
0
1
1177
3
2
1
2
1132
1132
1132
2n = 2x
Tillandsia spathacea Mez & Sodiro
B0565
0
1
1177
0
0
0
1
1130
1130
1130
2n = 2x
Tillandsia strobeliae (Rauh) J.R. Grant
B0198
2
2
1177
4
1
1
2
1131
1131
1130
2n = 2x
Tillandsia tequendamae André
B0569
0
1
1177
0
0
0
1
1131
1131
1131
2n = 2x
Tillandsia werneriana J.R. Grant
B0067
0
1
1177
2
2
1
2
1137
1137
1135
2n = 2x
Tillandsia cf. werneriana J.R. Grant
B0144
2
2
1177
2
1
0
2
1149
1149
1148
2n = 2x
Tillandsia cf. werneriana J.R. Grant
B0514
3
2
1177
7
0
0
2
1136
1136
1136
2n = 2x
Tillandsia achyrostachys E. Morren ex Baker
B0408
6
2
1177
4
1
0
2
1103
1103
1099
2n = 2x
Tillandsia adpressiflora Mez
B0597
2
2
1180
0
0
1
1
1139
1139
1139
2n = 2x
Tillandsia andrieuxii (Mez) L.B. Sm.
B0063
1
2
1177
6
1
0
2
1098
1098
1080
2n = 2x
Tillandsia baileyi Rose ex Small
B0421
5
2
1180
8
1
0
2
1140
1140
1139
2n = 2x
Tillandsia cf. beutelspacheri Matuda
B0360
2
2
1177
9
2
0
2
1138
1138
1137
2n = 2x
Tillandsia caput-medusae E. Morren
B0046
0
1
1180
0
0
0
1
1142
1142
1142
2n = 2x
Tillandsia aff. caput-medusae E. Morren
B0424
5
2
1180
1
0
2
2
1162
1162
1162
2n = 2x
Tillandsia subg. Tillandsia
Tillandsia cryptopoda L.B. Sm.
B0787
0
1
1177
0
0
1
1
1133
1133
1133
2n = 2x
Tillandsia aff. cucaensis Wittm.
B0732
15
2
1177
0
0
0
1
1135
1135
1135
2n = 2x
Tillandsia durangensis Rauh & Ehlers
B0365
6
2
1180
0
0
1
1
1140
1140
1140
2n = 2x
Tillandsia ehlersiana Rauh
B0431
1
2
1180
2
0
1
2
1140
1140
1140
2n = 2x
Tillandsia exserta Fernald
B0390
1
2
1177
5
0
0
2
1135
1135
1135
2n = 2x
Tillandsia extensa Mez
B0712
1
2
1177
9
1
1
2
1130
1131
1130
2n = 2x
Tillandsia fasciculata Sw.
B0076
2
2
1177
18
3
0
2
1136
1131!
1134!
2n = 2x
Tillandsia filifolia Schltdl. & Cham.
B0788
8
2
1177
9
3
1
2
1121
1121
1120
2n = 2x
Tillandsia filifolia Schltdl. & Cham.
B0790
5
2
1177
12
2
1
2
1123
1123
1121
2n = 2x
Tillandsia fuchsii W. Till
B0391
3
2
1177
11
1
0
2
1120
1120
1118
2n = 2x
Tillandsia glabrior (L.B. Sm.) López-Ferr., Espejo & I.
Ramírez
B0340
5
2
1180
2
2
0
2
1138
1138
1137
2n = 2x
Tillandsia guatemalensis L.B. Sm.
B0008
4
2
1177
11
1
0
2
1129
1129
1128
2n = 2x
Tillandsia guatemalensis L.B. Sm.
B0103
3
2
1177
7
0
0
2
1128
1128
1128
2n = 2x
Tillandsia guatemalensis L.B. Sm.
B0104
8
2
1177
1
0
0
2
1129
1129
1129
2n = 2x
Tillandsia guerreroensis Rauh
B0349
6
2
1177
8
3
1
2
1137
?
?
2n = 2x
Tillandsia hildae Rauh
B0763
2
2
1180
7
1
0
2
1130
1130
1116
2n = 2x
Tillandsia huajuapanensis Ehlers & Lautner
B0713
3
2
1177
6
1
0
2
1130
1130
1129
2n = 2x
Tillandsia ionantha Planch.
B0084
3
2
1180
7
0
2
2
1136
1136
1136
2n = 2x
199
BARFUSS, M.H.J.
NEW CLASSIFICATION
PHYC
PRK
postulated
ploidy level
SNPs
Geno
types
Length
Tillandsia juerg-rutschmannii Rauh
B0715
1
2
1177
4
1
0
2
1137
1137
1136
2n = 2x
Tillandsia leiboldiana Schltdl.
B0323
4
2
1177
0
1
0
2
1116
1116
1106
2n = 2x
Tillandsia malzinei (E. Morren) Baker
B0145
0
1
1177
0
0
0
1
1125
1125
1125
2n = 2x
Indels
SSR
Consensus
length
Allele 2
length
(a2)
DNA no.
SNPs
Geno
types
Allele 1
length
(a1)
Taxon
Tillandsia malzinei (E. Morren) Baker
B0492
0
1
1177
3
1
0
2
1125
1125
1110
2n = 2x
Tillandsia mima var. chiletensis Rauh
B0708
2
2
1177
1
0
1
2
1130
1130
1130
2n = 2x
Tillandsia mima var. chiletensis Rauh
B0710
2
2
1177
1
0
1
2
1130
1130
1130
2n = 2x
Tillandsia cf. mima L.B. Sm. var. mima
B0709
5
2
1177
0
0
0
1
1130
1130
1130
2n = 2x
Tillandsia mitlaensis var. tulensis Lautner & Ehlers
B0402
0
1
1177
8
0
1
2
1141
1141
1141
2n = 2x
Tillandsia moronesensis Ehlers
B0380
0
1
1177
1
0
0
2
1137
1137
1137
2n = 2x
Tillandsia paniculata (L.) L.
B0102
0
1
1177
0
0
1
1
1130
1130
1130
2n = 2x
Tillandsia plagiotropica Rohweder
B0386
0
1
1177
0
0
0
1
1116
1116
1116
2n = 2x
Tillandsia praschekii Ehlers & Willinger
B0364
0
1
1177
3
2
1
2
1140
1142
1140
2n = 2x
Tillandsia propagulifera Rauh
B0310
2
2
1177
8
2
1
2
1131
1130
1130
2n = 2x
Tillandsia pseudosetacea Ehlers & Rauh
B0333
0
1
1177
0
0
0
1
1135
1135
1135
2n = 2x
Tillandsia pueblensis L.B. Sm.
B0398
2
2
1180
8
2
1
2
1144
1146
1144
2n = 2x
Tillandsia punctulata Schltdl.& Cham.
B0061
0
1
1177
0
0
0
1
1138
1138
1138
2n = 2x
Tillandsia remota Wittm.
B0072
4
2
1177
4
0
0
2
1135
1135
1135
2n = 2x
Tillandsia cf. rhomboidea André
B0306
0
1
1177
0
0
0
1
1134
1134
1134
2n = 2x
Tillandsia cf. rhomboidea André
B0372
0
1
1177
0
0
1
1
1134
1134
1134
2n = 2x
Tillandsia schatzlii Rauh
B0416
0
1
1177
0
0
1
1
1144
1144
1144
2n = 2x
Tillandsia secunda Kunth
B0527
0
1
1177
3
0
1
2
1129
1129
1129
2n = 2x
Tillandsia secunda Kunth
B0528
0
1
1177
3
1
1
2
1130
1129
1128
2n = 2x
Tillandsia secunda Kunth
B0549
3
2
1177
2
1
1
2
1130
1130
1129
2n = 2x
Tillandsia secunda Kunth
B0551
3
2
1177
3
0
1
2
1129
1129
1129
2n = 2x
Tillandsia seleriana Mez
B0413
5
2
1180
0
0
1
1
1144
1144
1144
2n = 2x
Tillandsia selleana Harms
B0243
0
1
1177
0
0
0
1
1128
1128
1128
2n = 2x
Tillandsia spiraliflora Rauh
B0762
4
2
1177
8
0
1
2
1130
1130
1130
2n = 2x
Tillandsia tricolor Schltdl. & Cham.
B0785
3
2
1180
4
1
0
2
1131
1131
1114
2n = 2x
Tillandsia utriculata L.
B0100
0
1
1180
0
0
1
1
1143
1143
1143
2n = 2x
Tillandsia utriculata L.
B0805
0
1
1180
0
0
1
1
1143
1143
1143
2n = 2x
Tillandsia utriculata L.
B0807
0
1
1180
0
0
1
1
1140
1140
1140
2n = 2x
Tillandsia aff. utriculata L.
B0743
0
1
1180
0
0
1
1
1143
1143
1143
2n = 2x
Tillandsia aff. utriculata L.
B0747
0
1
1180
0
0
1
1
1143
1143
1143
2n = 2x
Tillandsia variabilis Schltdl.
B0308
0
1
1180
0
0
0
1
1129
1129
1129
2n = 2x
Tillandsia velutina Ehlers
B0427
0
1
1177
4
2
0
2
1123
?
?
2n = 2x
Tillandsia atroviridipetala Matuda
B0215
0
1
1177
3
0
1
2
1082
1082
1082
2n = 2x
Tillandsia balsasensis Rauh
B0221
0
1
1177
0
0
1
1
1127
1127
1127
2n = 2x
Tillandsia chusgonensis L. Hrom.
B0210
5
2
1177
5
1
1
2
1085
1085
1084
2n = 2x
Tillandsia heteromorpha Mez
B0224
0
1
1177
0
0
0
1
1081
1081
1081
2n = 2x
Tillandsia subg. Viridantha (Espejo) Barfuss & W. Till
Tillandsia ignesiae Mez
B0222
7
2
1177
7
1
1
2
1084
1084
1081
2n = 2x
Tillandsia lepidosepala L.B. Sm.
B0219
1
2
1177
0
0
1
1
1083
1083
1083
2n = 2x
Tillandsia mauryana L.B. Sm.
B0238
3
2
1177
2
2
1
2
1084
1084
1082
2n = 2x
Tillandsia oblivata L. Hrom.
B0205
2
2
1177
0
1
1
2
1081
1081
1067
2n = 2x
Tillandsia plumosa Baker
B0086
2
2
1177
2
1
1
2
1082
1082
1081
2n = 2x
Tillandsia reducta L.B. Sm.
B0209
0
1
1177
4
1
1
2
1084
1084
1080
2n = 2x
200
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
PHYC
Taxon
DNA no.
SNPs
Geno
types
PRK
Length
SNPs
Indels
SSR
Geno
types
Consensus
length
Allele 1
length
(a1)
Allele 2
length
(a2)
postulated
ploidy level
Tillandsia rupicola Baker
B0039
0
1
1177
0
0
0
1
1096
1096
1096
2n = 2x
Tillandsia stellifera L. Hrom.
B0239
0
1
1177
0
0
0
1
1081
1081
1081
2n = 2x
Tillandsia tectorum var. globosa L. Hrom.
B0248
0
1
1177
2
0
0
2
1094
1094
1094
2n = 2x
Tillandsia tectorum var. tectorum f. gigantea L. Hrom.
B0253
4
2
1177
2
0
0
2
1097
1097
1097
2n = 2x
Tillandsia tectorum E. Morren var. tectorum f.
tectorum
B0249
0
1
1177
4
3
0
2
1131
1131
1127
2n = 2x
Tillandsia tectorum var. viridula L. Hrom.
B0245
0
1
1177
1
0
1
2
1127
1127
1127
2n = 2x
Tillandsia tomekii L. Hrom.
B0218
1
2
1177
6
1
1
2
1128
1128
1127
2n = 2x
Tillandsia tortilis Klotzsch ex Baker
B0049
2
2
1177
1
0
1
2
1087
1087
1087
2n = 2x
Tillandsia baliophylla Harms
B0101
0
1
1177
0
0
1
1
1108
1108
1108
2n = 2x
Tillandsia biflora Ruiz & Pav.
B0090
0
1
1177
0
0
1
1
1641
1641
1641
2n = 2x
Tillandsia brenneri Rauh
B0236
0
1
1180
0
0
1
1
1122
1122
1122
2n = 2x
Tillandsia brevilingua Mez ex Harms
B0056
0
1
1180
0
1
0
2
1124
1124
1123
2n = 2x
Tillandsia brevilingua Mez ex Harms
B0252
1
2
1180
0
1
0
2
1124
1124
1123
2n = 2x
Tillandsia buseri var. nubicola Gilmartin
B0561
0
1
1177
1
0
1
2
1101
1101
1101
2n = 2x
Tillandsia cf. buseri var. nubicola Gilmartin
B0441
1
2
1177
0
0
1
1
1124
1124
1124
2n = 2x
Biflora clade
Tillandsia cf. buseri var. nubicola Gilmartin
B0442
4
2
1177
1
0
1
2
1117
1117
1117
2n = 2x
Tillandsia complanata Benth.
B0244
4
2
1177
1
1
0
2
1131
1131
1129
2n = 2x
Tillandsia complanata Benth.
B0562
1
2
1177
8
1
0
2
1133
1133
1129
2n = 2x
Tillandsia complanata Benth.
B0563
3
2
1177
6
1
0
2
1129
1129
1128
2n = 2x
Tillandsia confertiflora André
B0614
4
2
1177
0
0
1
1
1119
1119
1119
2n = 2x
Tillandsia confinis L.B. Sm.
B0587
2
2
1177
0
2
1
2
1134
1134
1127
2n = 2x
Tillandsia demissa L.B. Sm.
B0075
3
2
1177
0
0
0
1
1124
1124
1124
2n = 2x
Tillandsia denudata var. vivipara Rauh
B0750
2
2
1177
1
0
0
2
1135
1135
1135
2n = 2x
Tillandsia deppeana Steud.
B0457
0
1
1177
3
0
0
2
1111
1111
1111
2n = 2x
Tillandsia deppeana Steud.
B0458
0
1
1177
2
1
0
2
1111
1111
1109
2n = 2x
Tillandsia fendleri Griseb.
B0009
0
1
1177
0
2
0
2
1116
1116
1114
2n = 2x
Tillandsia fendleri Griseb.
B0445
0
1
1177
0
0
1
1
1117
1117
1117
2n = 2x
Tillandsia floribunda Kunth
B0351
1
2
1180
4
1
1
2
1068
1068
1067
2n = 2x
Tillandsia fosteri Gilmartin
B0639
3
2
1177
12
2
1
2
1123
1123
1117
2n = 2x
Tillandsia glauca L.B. Sm.
B0618
1
2
1177
0
0
1
1
1117
1117
1117
2n = 2x
Tillandsia heterophylla E. Morren
B0047
0
1
1177
0
0
0
1
1110
1110
1110
2n = 2x
Tillandsia imperialis E. Morren ex Roezl
B0292
1
2
1177
8
1
1
2
1113
1113
1111
2n = 2x
Tillandsia incarnata Kunth
B0522
0
1
1177
0
0
1
1
1113
1113
1113
2n = 2x
Tillandsia ionochroma André ex Mez
B0600
0
1
1177
0
0
1
1
1692
1692
1692
2n = 2x
Tillandsia kauffmannii Ehlers
B0074
0
1
1177
0
0
1
1
1114
1114
1114
2n = 2x
Tillandsia krahnii Rauh
B0235
0
1
1177
0
0
0
1
1120
1120
1120
2n = 2x
Tillandsia lajensis André
B0242
0
1
1177
1
0
1
2
1120
1120
1120
2n = 2x
Tillandsia lajensis André
B0546
0
1
1177
0
0
1
1
1120
1120
1120
2n = 2x
Tillandsia lajensis André
B0554
0
1
1177
1
0
1
2
1120
1120
1120
2n = 2x
Tillandsia lajensis André
B0555
0
1
1177
0
0
1
1
1120
1120
1120
2n = 2x
Tillandsia latifolia var. divaricata (Benth.) Mez
B0068
2
2
1177
7
2
0
2
1115
1116!
1100!
2n = 2x
Tillandsia latifolia Meyen var. latifolia
B0433
3
2
1177
4
1
1
2
1115
1115
1098
2n = 2x
Tillandsia latifolia var. leucophylla Rauh
B0228
1
2
1177
6
1
1
2
1115
1115
1098
2n = 2x
Tillandsia macbrideana L.B. Sm.
B0070
2
2
1177
13
2
1
2
1113
?
?
2n = 2x
Tillandsia maculata Ruiz & Pav.
B0574
4
2
1177
1
1
0
2
1124
1124
1116
2n = 2x
201
BARFUSS, M.H.J.
NEW CLASSIFICATION
PHYC
Taxon
DNA no.
SNPs
Geno
types
PRK
Length
SNPs
Indels
SSR
Geno
types
Consensus
length
Allele 1
length
(a1)
Allele 2
length
(a2)
postulated
ploidy level
Tillandsia multicaulis Steud.
B0107
0
1
1177
0
0
0
1
1115
1115
1115
2n = 2x
Tillandsia orbicularis L.B. Sm.
B0375
0
1
1177
0
0
1
1
1085
1085
1085
2n = 2x
Tillandsia pastensis André
B0521
0
1
1177
0
0
0
1
1095
1095
1095
2n = 2x
Tillandsia pseudomacbrideana Rauh
B0036
3
2
1177
8
1
1
2
1129
1129
1116
2n = 2x
Tillandsia roezlii E. Morren
B0764
1
2
1177
1
0
1
2
1115
1115
1115
2n = 2x
Tillandsia rudolfii E. Gross & Hase
B0654
0
1
1177
7
2
1
2
1113
1113
1113
2n = 2x
Tillandsia somnians L.B. Sm.
B0301
1
2
1177
8
0
1
2
1100
1100
1100
2n = 2x
Tillandsia somnians L.B. Sm.
B0312
0
1
1177
0
0
1
1
1100
1100
1100
2n = 2x
Tillandsia stenoura Harms
B0635
0
1
1177
10
2
1
2
1114
1114
1109
2n = 2x
Tillandsia subconcolor L.B. Sm.
B0227
0
1
1177
0
0
1
1
1115
1115
1115
2n = 2x
Tillandsia cf. superba Mez & Sodiro
B0602
3
2
1177
0
0
1
1
1101
1101
1101
2n = 2x
Tillandsia tovarensis Mez
B0605
1
2
1177
0
0
0
1
1104
1104
1104
2n = 2x
Tillandsia turneri Baker
B0650
0
1
1177
0
0
1
1
1081
1081
1081
2n = 2x
Tillandsia sp.
B0634
6
2
1177
8
0
1
2
1088
1088
1088
2n = 2x
Tillandsia brachyphylla Baker
B0082
2
2
1177
2
0
1
2
1097
1097
1097
2n = 2x
Tillandsia gardneri Lindl.
B0041
0
1
1177
0
0
1
1
1092
1092
1092
2n = 2x
Tillandsia globosa Wawra
B0419
5
2
1177
10
2
0
2
1084
?
?
2n = 2x
Gardneri clade
Purpurea clade
Tillandsia aurea Mez
B0250
0
1
1177
6
0
0
2
1096
1096
1096
2n = 2x
Tillandsia cacticola L.B. Sm.
B0044
0
1
1177
1
0
0
2
1096
1096
1096
2n = 2x
Tillandsia purpurea Ruiz & Pav.
B0246
0
1
1177
0
0
0
1
1095
1095
1095
2n = 2x
Tillandsia straminea Kunth
B0247
3
2
1177
0
0
0
1
1096
1096
1096
2n = 2x
Tillandsia ecarinata L.B. Sm.
B0237
1
2
1180
5
1
1
2
1134
1134
1132
2n = 2x
Tillandsia ferreyrae L.B. Sm.
B0241
1
2
1177
2
2
1
2
1132
1132
1130
2n = 2x
Tillandsia rauhii L.B. Sm.
B0092
0
1
1177
0
0
0
1
1128
1128
1128
2n = 2x
Tillandsia teres L.B. Sm.
B0201
3
2
1177
8
0
0
2
1128
1128
1128
2n = 2x
Tillandsia aff. arequitae (André) André ex Mez
B0387
0
1
1177
0
0
1
1
1132
1132
1132
2n = 2x
Tillandsia argentina C.H. Wright
B0087
2
2
1177
9
3
1
2
1129
1127!
1125!
2n = 2x
Tillandsia argentina C.H. Wright
B0797
3
2
1177
4
0
1
2
1129
1129
1129
2n = 2x
Tillandsia bermejoensis H. Hrom.
B0034
1
2
1177
0
0
1
1
1130
1130
1130
2n = 2x
Tillandsia buchlohii Rauh
B0395
5
2
1177
4
0
1
2
1129
1129
1129
2n = 2x
Tillandsia cardenasii L.B. Sm.
B0409
0
1
1177
4
1
1
2
1138
1138
1136
2n = 2x
Tillandsia caulescens Brongn. ex Baker
B0071
0
1
1177
0
0
1
1
1130
1130
1130
2n = 2x
Tillandsia cochabambae E. Gross & Rauh
B0392
6
2
1177
8
1
1
2
1132
1132
1131
2n = 2x
Tillandsia comarapaensis H. Luther
B0407
5
2
1177
2
0
1
2
1129
1129
1129
2n = 2x
Tillandsia diaguitensis A. Cast.
B0429
7
2
1177
8
0
1
2
1129
1129
1129
2n = 2x
Tillandsia didisticha (E. Morren) Baker
B0038
1
2
1177
0
0
1
1
1130
1130
1130
2n = 2x
Rauhii clade
Xiphioides clade
Tillandsia gerdae Ehlers
B0411
6
2
1177
8
3
1
2
1131
?
?
2n = 2x
Tillandsia koehresiana Ehlers
B0371
1
2
1177
5
2
1
2
1131
1131
1127
2n = 2x
Tillandsia lorentziana Griseb.
B0374
0
1
1177
4
3
1
2
1139
?
?
2n = 2x
Tillandsia aff. lotteae H. Hrom.
B0808
0
1
1177
0
0
1
1
1131
1131
1131
2n = 2x
Tillandsia aff. lotteae H. Hrom.
B0809
0
1
1177
0
0
1
1
1131
1131
1131
2n = 2x
Tillandsia markusii L. Hrom.
B0399
1
2
1177
7
3
1
2
1129
1129
1126
2n = 2x
202
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
PHYC
PRK
postulated
ploidy level
SNPs
Geno
types
Length
Tillandsia muhriae W. Weber
B0438
0
1
1177
6
0
1
2
1130
1130
1130
2n = 2x
Tillandsia ramellae W. Till & S. Till
B0430
0
1
1177
0
2
1
2
1138
?
?
2n = 2x
Tillandsia rosacea L. Hrom. & W. Till
B0405
4
2
1177
1
0
1
2
1128
1128
1128
2n = 2x
Indels
SSR
Consensus
length
Allele 2
length
(a2)
DNA no.
SNPs
Geno
types
Allele 1
length
(a1)
Taxon
Tillandsia rosarioae L. Hrom.
B0403
5
2
1177
5
2
1
2
1129
1129
1127
2n = 2x
Tillandsia aff. vernicosa Baker
B0355
0
1
1177
11
0
1
2
1129
1129
1129
2n = 2x
Tillandsia xiphioides subsp. prolata H. Luther
B0418
1
2
1177
6
2
1
2
1130
?
?
2n = 2x
Tillandsia xiphioides Ker Gawl. subsp. xiphioides
B0040
3
2
1177
8
1
1
2
1130
1130
1129
2n = 2x
Tillandsia yuncharaensis W. Till
B0417
0
1
1177
7
0
1
2
1129
1129
1129
2n = 2x
Tillandsia zecheri var. cafayatensis Palací & G.K. Br.
B0361
5
2
1177
11
3
1
2
1138
?
?
2n = 2x
Tillandsia albertiana F. Verv.
B0033
2
2
1177
0
0
1
1
1138
1138
1138
2n = 2x
Tillandsia albertiana F. Verv.
B0212
0
1
1177
0
0
1
1
1136
1136
1136
2n = 2x
Tillandsia albertiana F. Verv.
B0225
0
1
1177
4
0
1
2
1141
1141
1141
2n = 2x
Tillandsia australis Mez
B0203
0
1
1177
0
0
1
1
1115
1115
1115
2n = 2x
Tillandsia australis Mez
B0759
0
1
1177
0
0
1
1
1115
1115
1115
2n = 2x
Tillandsia disticha Kunth
B0048
0
1
1177
2
1
1
2
1131
1131
1130
2n = 2x
Tillandsia disticha Kunth
B0202
3
2
1177
1
1
1
2
1130
1131
1130
2n = 2x
Tillandsia disticha Kunth
B0233
2
2
1177
0
0
1
1
1134
1134
1134
2n = 2x
Tillandsia edithae Rauh
B0425
2
2
1177
2
0
1
2
1136
1136
1136
2n = 2x
Incertae sedis
Tillandsia edithae Rauh
B0436
0
1
1177
0
0
1
1
1136
1136
1136
2n = 2x
Tillandsia esseriana Rauh & L.B. Sm.
B0069
0
1
1177
7
2
1
2
1157
1157!
1128!
2n = 2x
Tillandsia esseriana Rauh & L.B. Sm.
B0200
3
2
1177
0
0
1
1
1129
1129
1129
2n = 2x
Tillandsia nana Baker
B0343
0
1
1177
1
1
1
2
1133
1133
1132
2n = 2x
Tillandsia pseudomicans Rauh
B0347
3
2
1177
3
0
1
2
1115
1115
1115
2n = 2x
Tillandsia sphaerocephala Baker
B0366
1
2
1177
2
1
0
2
1135
1135
1134
2n = 2x
B0318
1
2
1177
0
0
0
1
903
903
903
2n = 2x
Bromeliaceae tribe Vrieseeae W. Till & Barfuss
Bromeliaceae subtribe Cipuropsidinae Barfuss & W.
Till
Cipuropsis Ule
Cipuropsis amicorum (I. Ramírez & Bevil.) Barfuss & W.
Till
Cipuropsis zamorensis (L.B. Sm.) Barfuss & W. Till
B0045
0
1
1177
4
0
0
2
915
915
915
2n = 2x
Cipuropsis cv. Elan
B0117
5
2
1177
7
1
0
2
918
918
915
2n = 2x
Mezobromelia bicolor L.B. Sm.
B0578
3
2
1177
3
2
0
2
902
?
?
2n = 2x
Mezobromelia bicolor L.B. Sm.
B0583
1
2
1177
6
0
0
2
901
901
901
2n = 2x
Mezobromelia pleiosticha (Griseb.) Utley & H. Luther
B0274
0
1
1177
0
0
0
1
923
923
923
2n = 2x
Mezobromelia L.B. Sm.
Mezobromelia pleiosticha (Griseb.) Utley & H. Luther
Mezobromelia cf. pleiosticha (Griseb.) Utley & H.
Luther
Mezobromelia schimperiana (Wittm.) Barfuss &
Manzan.
Mezobromelia schimperiana (Wittm.) Barfuss &
Manzan.
Mezobromelia schimperiana (Wittm.) Barfuss &
Manzan.
Mezobromelia schimperiana (Wittm.) Barfuss &
Manzan.
Mezobromelia aff. schimperiana (Wittm.) Barfuss &
Manzan.
Mezobromelia sp.
B0649
0
1
1177
0
0
0
1
923
923
923
2n = 2x
B0330
5
2
1177
5
1
0
2
901
901
899
2n = 2x
B0334
2
2
1177
4
1
0
2
901
901
899
2n = 2x
B0589
1
2
1177
8
0
0
2
899
899
899
2n = 2x
B0611
2
2
1177
3
1
0
2
901
901
899
2n = 2x
B0612
0
1
1177
6
1
0
2
901
901
899
2n = 2x
B0197
0
1
1177
0
0
0
1
886
886
886
2n = 2x
B0194
1
2
1177
5
0
0
2
901
901
901
2n = 2x
B0132
4
2
1177
7
0
0
2
1015
1015
1015
2n = 2x
Werauhia J.R. Grant
Werauhia ampla (L.B. Sm.) J.R. Grant
203
BARFUSS, M.H.J.
NEW CLASSIFICATION
PHYC
PRK
postulated
ploidy level
SNPs
Geno
types
Length
Werauhia gladioliflora (H. Wendl.) J.R. Grant
B0149
0
1
1177
0
0
0
1
1026
1026
1026
2n = 2x
Werauhia gladioliflora (H. Wendl.) J.R. Grant
B0509
0
1
1177
0
0
0
1
1026
1026
1026
2n = 2x
Werauhia guadelupensis (Baker) J.R. Grant
B0313
0
1
1177
0
0
0
1
1079
1079
1079
2n = 2x
Indels
SSR
Consensus
length
Allele 2
length
(a2)
DNA no.
SNPs
Geno
types
Allele 1
length
(a1)
Taxon
Werauhia haltonii (H. Luther) J.R. Grant
B0580
0
1
1180
0
0
0
1
975
975
975
2n = 2x
Werauhia hygrometrica (André) J.R. Grant
B0598
0
1
1180
0
0
0
1
975
975
975
2n = 2x
Werauhia hygrometrica (André) J.R. Grant
B0621
0
1
1180
0
0
0
1
975
975
975
2n = 2x
Werauhia insignis (Mez) W. Till, Barfuss & R. Samuel
B0017
0
1
1159
0
0
0
1
1031
1031
1031
2n = 2x
Werauhia ororiensis (Mez) J.R. Grant
B0025
2
2
1180
9
2
0
2
962
962
954
2n = 2x
Werauhia pedicellata (Mez & Wercklé) J.R. Grant
B0752
0
1
1180
0
0
0
1
1016
1016
1016
2n = 2x
Werauhia ringens (Griseb.) J.R. Grant
B0019
0
1
1177
0
0
0
1
1045
1045
1045
2n = 2x
Werauhia sintenisii (Baker) J.R. Grant
B0450
0
1
1180
0
0
0
1
1031
1031
1031
2n = 2x
Werauhia tarmaensis (Rauh) J.R. Grant
B0018
0
1
1177
0
0
0
1
1081
1081
1081
2n = 2x
Werauhia viridiflora (Regel) J.R. Grant
B0285
0
1
1177
0
0
0
1
908
908
908
2n = 2x
Werauhia viridiflora (Regel) J.R. Grant
B0325
7
2
1177
3
0
0
2
908
908
908
2n = 2x
Werauhia werckleana (Mez) J.R. Grant
B0497
3
2
1177
8
0
0
2
1083
1083
1083
2n = 2x
Vriesea chrysostachys E. Morren
B0005
0
1
1177
0
0
0
1
1029
1029
1029
2n = 2x
Vriesea ospinae H. Luther
B0054
0
1
1177
4
1
0
2
1036
1036
1035
2n = 2x
Tillandsia asplundii L.B. Sm.
B0588
0
1
1177
2
2
0
2
921
921
918
2n = 2x
Tillandsia singularis Mez & Wercklé
B0064
0
1
1180
0
0
0
1
914
914
914
2n = 2x
Chrysostachys clade
Singularis clade
Splendens clade
Vriesea glutinosa Lindl.
B0130
0
1
1177
0
0
0
1
1067
1067
1067
2n = 2x
Vriesea splendens (Brongn.) Lem.
B0037
0
1
1177
0
0
1
1
1069
1069
1069
2n = 2x
B0148
0
1
1177
1
0
0
2
1090
1090
1090
2n = 2x
Alcantarea benzingii Leme
B0166
0
1
1177
0
0
0
1
1125
1125
1125
2n = 2x
Alcantarea burle-marxii (Leme) J.R. Grant
B0159
2
2
1177
11
1
1
2
1134
1134
1133
2n = 2x
Alcantarea duarteana (L.B. Sm.) J.R. Grant
B0059
0
1
1177
4
0
0
2
1133
1133
1133
2n = 2x
Alcantarea duarteana (L.B. Sm.) J.R. Grant
B0165
0
1
1177
3
1
0
2
1133
1133
1132
2n = 2x
Alcantarea edmundoi (Leme) J.R. Grant
B0161
2
2
1177
1
0
1
2
1134
1134
1134
2n = 2x
Alcantarea edmundoi (Leme) J.R. Grant
B0171
0
1
1177
0
0
0
1
1135
1135
1135
2n = 2x
Alcantarea aff. extensa (L.B. Sm.) J.R. Grant
B0164
4
2
1177
7
1
1
2
1138
1138
1133
2n = 2x
Alcantarea farneyi (Martinelli & A.F. Costa) J.R. Grant
B0170
0
1
1177
4
0
1
2
1135
1135
1135
2n = 2x
Alcantarea geniculata (Wawra) J.R. Grant
B0153
2
2
1177
0
0
0
1
1133
1133
1133
2n = 2x
Alcantarea cf. geniculata (Wawra) J.R. Grant
B0751
0
1
1177
0
0
1
1
1134
1134
1134
2n = 2x
Alcantarea glazioviana (Lem.) Leme
B0167
0
1
1177
10
2
0
2
1134
?
?
2n = 2x
Alcantarea heloisae J.R. Grant
B0155
9
2
1177
4
2
0
2
1135
?
?
2n = 2x
Alcantarea imperialis (Carrière) Harms
B0001
2
2
1177
5
1
1
2
1135
1134!
1135!
2n = 2x
Alcantarea imperialis (Carrière) Harms
B0135
0
1
1177
0
0
1
1
1135
1135
1135
2n = 2x
Alcantarea imperialis (Carrière) Harms
B0154
4
2
1177
0
2
1
2
1137
?
?
2n = 2x
Incertae sedis
Vriesea tuerckheimii (Mez) L.B. Sm.
Bromeliaceae subtribe Vrieseinae W. Till & Barfuss
Alcantarea (E. Morren ex Mez) Harms
Alcantarea nahoumii (Leme) J.R. Grant
B0156
0
1
1177
0
0
1
1
1133
1133
1133
2n = 2x
Alcantarea nevaresii Leme
B0163
1
2
1177
0
0
0
1
1134
1134
1134
2n = 2x
Alcantarea nevaresii Leme
B0169
1
2
1177
2
0
1
2
1134
1134
1134
2n = 2x
204
OF TILLANDSIOIDEAE (BROMELIACEAE)
PART 3, CHAPTER 5
PHYC
Taxon
DNA no.
SNPs
Geno
types
PRK
Length
SNPs
Indels
SSR
Geno
types
Consensus
length
Allele 1
length
(a1)
Allele 2
length
(a2)
postulated
ploidy level
Alcantarea odorata (Leme) J.R. Grant
B0157
0
1
1177
0
0
0
1
1133
1133
1133
2n = 2x
Alcantarea regina (Vell.) Harms
B0136
0
1
1177
0
0
0
1
1134
1134
1134
2n = 2x
Alcantarea roberto-kautskyi Leme
B0158
2
2
1177
2
1
1
2
1140
1140
1138
2n = 2x
Alcantarea sp.
B0160
0
1
1177
1
1
1
2
1134
1134
1133
2n = 2x
Alcantarea sp.
B0168
5
2
1177
0
0
1
1
1133
1133
1133
2n = 2x
Vriesea correia-araujoi E. Pereira & I.A. Penna
B0095
0
1
1177
12
3
0
2
1118
1118
1116
2n = 2x
Vriesea croceana Leme & G.K. Br.
B0802
1
2
1177
7
0
0
2
1134
1134
1134
2n = 2x
Vriesea aff. croceana Leme & G.K. Br.
B0801
0
1
1177
0
0
1
1
1133
1133
1133
2n = 2x
Vriesea Lindl.
Vriesea erythrodactylon (E. Morren) E. Morren ex Mez
B0309
0
1
1177
0
0
0
1
1133
1133
1133
2n = 2x
Vriesea flava A.F. Costa, H. Luther & Wand.
B0322
4
2
1180
0
0
0
1
999
999
999
2n = 2x
Vriesea jonghei (K. Koch) E. Morren
B0065
2
2
1177
3
2
1
2
1115
1115
1112
2n = 2x
Vriesea longicaulis (Baker) Mez
B0290
1
2
1177
7
2
1
2
1117
1117
1115
2n = 2x
Vriesea maxoniana (L.B. Sm.) L.B. Sm.
B0490
0
1
1180
0
0
0
1
1135
1135
1135
2n = 2x
Vriesea neoglutinosa Mez
B0311
7
2
1177
8
0
0
2
1121
1121
1121
2n = 2x
Vriesea pabstii McWill. & L.B. Sm.
B0357
0
1
1177
0
0
0
1
1115
1115
1115
2n = 2x
Vriesea platynema Gaudich.
B0146
0
1
1183
0
0
0
1
1135
1135
1135
2n = 2x
Vriesea plurifolia Leme
B0376
0
1
1177
0
0
1
1
1132
1132
1132
2n = 2x
Vriesea procera var. tenuis L.B. Sm.
B0296
0
1
1180
0
0
0
1
1138
1138
1138
2n = 2x
Vriesea psittacina (Hook.) Lindl.
B0020
6
2
1177
7
5
0
2
n.u.
1067!
999!
2n = 2x
Vriesea roethii W. Weber
B0511
1
2
1177
2
0
1
2
1117
1117
1117
2n = 2x
Vriesea ruschii L.B. Sm.
B0719
5
2
1177
11
0
0
2
1116
1116
1116
2n = 2x
Vriesea saundersii (Carrière) E. Morren ex Mez
B0004
0
1
1177
0
0
0
1
1133
1133
1133
2n = 2x
Vriesea scalaris E. Morren
B0147
0
1
1177
0
0
0
1
1027
1027
1027
2n = 2x
Vriesea simplex (Vell.) Beer
B0505
4
2
1177
5
1
0
2
1027
1027
1026
2n = 2x
Vriesea sparsiflora L.B. Sm.
B0484
2
2
1177
0
0
0
1
1115
1115
1115
2n = 2x
Vriesea sucrei L.B. Sm. & Read
B0302
1
2
1177
0
0
0
1
1024
1024
1024
2n = 2x
Vriesea unilateralis (Baker) Mez
B0316
2
2
1177
4
0
0
2
1117
1117
1117
2n = 2x
205
Appendix
Abstracts 2005–2012 (Conferences)
Oral presentations and posters
Botany 2012
Adaptive radiation, correlated evolution,
and determinants of net diversification rates
in Bromeliaceae: Test of an a priori model
Thomas J. Givnish 1, Michael H.J. Barfuss 2, Benjamin Van Ee 3,
Ricarda Riina 4, Katharina Schulte 5, Ralf Horres 6,
Philip A. Gonsiska 1, Rachel S. Jabaily 1, Darren M. Crayn 5,
J. Andrew C. Smith 7, Klaus Winter 8, Gregory K. Brown 9,
Timothy M. Evans 10, Bruce K. Holst 11, Harry E. Luther 11,
Walter Till 2, Georg Zizka 12, Paul E. Berry 4
& Kenneth .J. Sytsma 1
1
University of Wisconsin-Madison, Department of Botany, 430 Lincoln Drive, Birge Hall, Madison, WI, 53706, USA; 2 University of Vienna, Faculty of Life Sciences, Department of Systematic
and Evolutionary Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria; 3
Black Hills State University, Spearfish, USA; 4 University of Michigan Herbarium, Ecology and
Evolutionary Biology, 3600 Varsity Dr, Ann Arbor, Michigan, 48108, USA; 5 James Cook University, Australian Tropical Herbarium, Cairns, QLD, 4870, Australia; 6 GenXPro GmbH, Altenhöferallee 3 60438, Frankfurt/Main, Germany; 7 University of Oxford, Department of Plant
Sciences, South Parks Road, Oxford, OX1 3RB, United Kingdom; 8 Smithsonian Tropical Research
Institute, P.O. Box 2072, Balboa, Ancón, Republic of Panama; 9 University of Wyoming, Department of Botany, 3165, 1000 E University Avenue, Laramie, Wyoming, 82071, USA; 10 Grand
Valley State University, Biology Department, 1 Campus Drive, Allendale, Michigan, 49401, USA;
11
Marie Selby Botanic Gardens, 811 South Palm Avenue, Sarasota, Florida, 34236-7726, USA;
12
Research Institute Senckenberg, Department of Botany, Frankfurt/Main, Germany
Author for correspondence: Thomas J. Givnish, givnish@wisc.edu
Oral presentation: (638) in: Botany 2012: The Next Generation (abstracts). Columbus, Ohio:
Greater Columbus Convention Center, 7–11 July 2012.
Abstract
We present an integrative model predicting associations among epiphytism, the tank habit,
entangling seeds, C3 vs. CAM photosynthesis, avian pollinators, life in fertile, moist montane
habitats, and species richness and net rates of species diversification in the monocot family
Bromeliaceae. We test the predictions of this model by overlaying individual character-states
on a molecular phylogeny, relating evolutionary shifts to time and reconstructed shifts in geographic distribution; by quantifying patterns of correlated and contingent evolution among
pairs of traits; and by analyzing the apparent impact of individual traits on diversification within subfamilies. All patterns of correlated evolution among pairs of traits and environmental
conditions predicted by our model were significant. The pattern and timing of shifts in phenotype and expansion of distributions outside the Guayana Shield also generally accorded with
the model's predictions. Patterns of contingent evolution were largely consistent with the
model. Species richness and net rates of species diversification were most closely tied to life in
fertile, moist, geographically extensive cordilleras, with weaker ties to epiphytism, avian pollination, and the tank habit. The highest rates of diversification were seen in the core tillandsioids, associated with the Andes, and especially the tank-epiphyte clade of bromelioids, associated with the Serra do Mar and nearby ranges. Six adaptive radiations and associated speciation - including one clade of CAM epiphytes, one of predominantly C3 epiphytes, three of CAM
terrestrials in arid habitats (in Central America, high elevations in the Andes and low elevations
in the Brazilian Shield), and one of C3 terrestrials in rain- and cloud-forest understories - account for > 80% of total species number in the family. This integrative study is among the first
to test a priori hypotheses about the relationships among phylogeny, phenotypic evolution,
geographic spread, and net species diversification.
210
XVIII IBC 2011
Systematics, evolution, and phylogeography
of Tillandsia (Bromeliaceae) and related genera
Michael H.J. Barfuss, Walter Till & Rosabelle Samuel
University of Vienna, Faculty of Life Sciences, Department of Systematic and Evolutionary
Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria
Author for correspondence: Michael H.J. Barfuss, michael.h.j.barfuss@univie.ac.at
Oral presentation: P. 189 (SYM004) in: XVIII IBC 2011: XVIII International Botanical Congress
(abstracts). Melbourne, Australia: Melbourne Congress and Exhibition Centre, 23–30 July 2011.
Abstract
Tillandsia L. is the largest genus within Bromeliaceae comprising more than 660 accepted species in six recognized subgenera (Tillandsia subgen. Allardtia, T. subgen. Anoplophytum, T.
subgen. Diaphoranthema, T. subgen. Phytarrhiza, T. subgen. Pseudalcantarea, T. subgen. Tillandsia). Eight additional genera have been associated with Tillandsia in subfamily Tillandsioideae, i.e., Alcantarea, Catopsis, Glomeropitcairnia, Guzmania, Mezobromelia, Racinaea,
Vriesea, and Werauhia. The segregation of a group of Tillandsia species occurring in Mexico
(Viridantha) and the transfer of xerophytic Vriesea species to Tillandsia is not accepted by all
bromeliad researchers based on long-established morphological characterizations of traditionally accepted genera. Habit shifts from phytotelms to extreme xerophytes, life form transitions
from terrestrials to epiphytes and lithophytes, shifts in pollination syndromes, and seed and
stigma morphology are key events and characters for the evolution and systematics of genera
and infrageneric units, but they can only be interpreted in the context of DNA sequence data,
since some of these features have evolved independently within different phylogenetic lineages. Therefore we conducted DNA sequence analyses from nuclear genes (i.e., PRK and PHYC,
totaling about 2500 bp) and published chloroplast markers (with about 6500 bp) in combination with a reevaluation of morphological characters. Based on these results three new genera
with a distinctive stigma and seed morphology are segregated from Tillandsia, i.e., Josemania,
Lemeltonia, and Rothowia. Species of all three belonged to the former subgenus Phytarrhiza,
which turned out to be highly polyphyletic. Circumscription of the remaining Tillandsia subgenera also changes significantly and new infrageneric taxa and groups emerge (e.g., T. subgen. Pseudovriesea, T. subgen. Viridantha). DNA data suggest that the subfamily had its origin
in the geologically old parts of northern South America. From there the two earliest diverging
lineages migrated into the Caribbean (Catopsis, Glomeropitcairnia) with some taxa extending
also into the Andes and into Central America (Catopsis). The next diverging clade (Vrieseeae)
splits into two subgroups, one radiating into eastern Brazil (Alcantarea, Vriesea), the other
spreading into the Andes (Cipuropsis, Werauhia). Within Tillandsieae the earliest lineage is
Guzmania with a predominately Andean distribution, but also extending into Central America
and the Caribbean. Early diverging taxa of the core Tillandsieae have their current distribution
mainly in the Andes of northern Peru, Ecuador and Colombia, which seems to be the ancient
area for the whole Tillandsia s.l. complex. Tillandsia s.str. has two centers of diversity, one in
the Northern and Central Andes, the other in the mountain systems of Northern Central America, which proves to have reached Mexico and adjacent areas during at least three independent colonization events from the South (T. subgen. Pseudalcantarea, T. subgen. Tillandsia p.p.,
and T. subgen. Viridantha p.p.).
212
XVIII IBC 2011
New taxonomic implications in Tillandsioideae
(Bromeliaceae) based on DNA data and morphology
Michael H.J. Barfuss, Walter Till & Rosabelle Samuel
University of Vienna, Faculty of Life Sciences, Department of Systematic and Evolutionary
Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria
Author for correspondence: Michael H.J. Barfuss, michael.h.j.barfuss@univie.ac.at
Poster: Pp. 568–569 (P0730) in: XVIII IBC 2011: XVIII International Botanical Congress (abstracts). Melbourne, Australia: Melbourne Congress and Exhibition Centre, 23–30 July 2011.
Abstract
In order to establish a natural and stable classification system for Tillandsioideae we conducted phylogenetic analyses using newly generated single-copy nDNA sequences (i.e., PHYC and
PRK) and published cpDNA data (i.e., atpB-rbcL spacer, matK, rbcL, partial rbcL-accD spacer,
rps16 intron, partial trnK intron, trnL intron and trnL-trnF spacer). Derived phylogenetic units
were then characterized using previously used, neglected, and new morphological characters.
A new classification for Tillandsioideae is urgently needed, since modern bromeliad taxonomists rely on a more than 30 years old monograph. Subsequent taxonomic changes were only
partly summarized in Floras or recent taxonomic treatments of certain groups. Our results
highlight the presence of two new subtribes (Cipuropsidinae, Vrieseinae), three new genera
(Josemania, Lemeltonia, Rothowia), and three new subgenera (Racinaea subg. Pseudophytarrhiza, T. subg. Viridantha, and T. subg. Pseudovriesea). Beside species from newly erected
taxa, several others are also reclassified where initial placement was controversial among
bromeliad researchers. Parsimony analysis revealed the following, mostly well-supported general branching pattern: ((Glomeropitcairnia, Catopsis) (((Alcantarea, Vriesea) (Werauhia,
Cipuropsis–Mezobromelia clade)) (Guzmania, ((Josemania, Racinaea) (Rothowia, (Lemeltonia,
Tillandsia); Bayesian analysis in contrast revealed a slightly different branching pattern within
core Tillandsieae: (Guzmania, ((Josemania, Rothowia) (Racinaea (Lemeltonia, Tillandsia))).
Relationships of subgenera and informal clades within Tillandsia are mostly unsupported, but
most clades themselves receive good statistical support. Although much progress has been
achieved in exploring phylogenetic relationships of Tillandsioideae by combining both cpDNA
and nDNA sequence data, a final and conclusive classification for the whole subfamily cannot
be presented here. The most critical groups that remain to be solved using new molecular
markers are the Cipuropsis–Mezobromelia alliance and the genus Tillandsia.
214
XVIII IBC 2011
Origin, phylogeny, adaptive radiation,
and geographic diversification of Bromeliaceae
Thomas J. Givnish 1, Michael H.J. Barfuss 2, Benjamin Van Ee 3,
Ricarda Riina 4, Katharina Schulte 5, Ralf Horres 6,
Philip A. Gonsiska 1, Rachel S. Jabaily 1, Darren M. Crayn 5,
J. Andrew C. Smith 8, Klaus Winter 9, Bruce K. Holst 10,
Harry E. Luther 10, Walter Till 2, Georg Zizka 11, Paul E. Berry 4,
Ann Arbor 1, & Kenneth .J. Sytsma 1
1
University of Wisconsin-Madison, Department of Botany, 430 Lincoln Drive, Birge Hall, Madison, WI, 53706, USA; 2 University of Vienna, Faculty of Life Sciences, Department of Systematic
and Evolutionary Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria;
3
Black Hills State University, Spearfish, USA; 4 University of Michigan Herbarium, Ecology and
Evolutionary Biology, 3600 Varsity Dr, Ann Arbor, Michigan, 48108, USA; 5 James Cook University, Australian Tropical Herbarium, Cairns, QLD, 4870, Australia; 6 GenXPro GmbH, Altenhöferallee 3, 60438 Frankfurt/Main, Germany; 8 University of Oxford, Department of Plant
Sciences, South Parks Road, Oxford, OX1 3RB, United Kingdom; 9 Smithsonian Tropical Research
Institute, P.O. Box 2072, Balboa, Ancón, Republic of Panama; 10 Marie Selby Botanic Gardens,
811 South Palm Avenue, Sarasota, Florida, 34236-7726, USA; 11 Research Institute Senckenberg, Department of Botany, Frankfurt/Main, Germany
Author for correspondence: Thomas J. Givnish, givnish@wisc.edu
Oral presentation: Pp. 186–187 (SYM004) in: XVIII IBC 2011: XVIII International Botanical Congress (abstracts). Melbourne, Australia: Melbourne Congress and Exhibition Centre, 23–30 July
2011.
Abstract
Sequence variation in eight rapidly evolving plastid regions for 90 bromeliad species from 45 of
56 described genera confirm that the eight bromeliad subfamilies are related to each other in
ladder-like fashion: (Brocchinoidieae, (Lindmanioideae, (Tillandsioideae, (Hechtioideae, (Navioideae, (Pitcairnioideae, (Puyoideae, Bromelioideae). Puya shows a basal split between species
found primarily at higher elevations in the Andes and those found near sea level in Chile. The
earliest-divergent bromelioids are mostly restricted to the southern Andes and Chilean coast.
We calibrated this phylogeny against the ages of fossil monocots using penalized likelihood,
and assessed patterns of biogeographic spread using maximum parsimony, Bayesian inference,
and S-DIVA. Bromeliads appear to have arisen in the Guayana Shield roughly 100 Mya, spread
centrifugally in the New World, and reached tropical West Africa via long-distance dispersal 9.2
Mya. Modern lineages began to diverge from each other 19 Mya, with invasions of drier or
higher peripheral areas in Central America and northern South America beginning 16–13 Mya,
coincident with a major adaptive radiation – the 'bromeliad revolution' – involving the repeated evolution of epiphytism, the tank habit, CAM photosynthesis, and avian pollination, as well
as several features of leaf and trichome anatomy, and an accelerated pace of species diversification. This revolution coincided with the uplift of the northern Andes and its invasion by epiphytic tillandsioids and by ancestors of the pitcairnioids, puyoids, and bromelioids. Bromelioids
invaded the Serra do Mar and nearby mountains in southeastern Brazil, most likely from
southern Chile, starting 9.1 Mya. A major radiation of epiphytic bromelioids in Brazil began 5.7
Mya. Calculations of net rates of species diversification for subfamilies or clades of similar rank
range from 0.16 sp sp-1 My-1 in brocchinioids to 1.12 sp sp-1 My-1 in the tank-epiphytic bromelioids. Acquisition of the epiphytic habit and related traits appears to have accelerated net species diversification by favoring seed traits that increased attachment to epiphytic perches, and
coincidentally increased the ability of tillandsioids and bromelioids to colonize extensive montane regions in the Andes and Central America, and in the Serra do Mar and nearby mountains
in coastal Brazil, permitting geographic speciation to proceed in massively parallel fashion as
epiphytes occupied a cloud-forest landscape dissected by numerous drier, lower valleys that
could act as extrinsic barriers to gene flow. Avian pollination (mainly by hummingbirds)
evolved at least twice, coincident with the invasion of cool, wet montane habitats. Net diversification rates were significantly higher in hummingbird-pollinated clades, perhaps reflecting
the rise of gullet-shaped flowers adapted to such pollinators and the resulting opportunity for
rapid partitioning of another rapidly speciating montane clade based on differences in bill
length. Entomophily is the ancestral condition in bromeliads, and bat pollination appears to
have evolved several times from hummingbird pollination; chiropterophily is associated with
warmer and/or drier conditions then ornithophily.
216
BioSystematics 2011
Conflicting phylogenetic signal within the nuclear
marker PRK highlights the importance of hybridization
events in the diversification of Bromeliaceae
Ingo Michalak 1,2,3, Daniele Silvestro 1,2,3, David Brie 1,2,
Michael H.J. Barfuss 4, Katharina Schulte 5 & Georg Zizka 1,2,3
1
Senckenberg Research Institute, Department of Botany and Molecular Evolution, 253 Senckenberganlage 25, 60325 Frankfurt/Main, Germany
2
Goethe University, Institute of Ecology, Evolution and Diversity, Senckenberganlage 25, 60325
Frankfurt/Main, Germany
3
Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, 60325 Frankfurt/Main, Germany
4
University of Vienna, Faculty of Life Sciences, Department of Systematic and Evolutionary
Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria
5
James Cook University, Australian Tropical Herbarium, Sir Robert Norman Building (E2), PO
Box 6811, Cairns 4870, QLD, Australia
Author for correspondence: Ingo Michalak, imichalak@senckenberg.de; Daniele Silvestro,
dsilvestro@senckenberg.de
Poster: Pp. 252–253 (II-74) in: BioSystematics Berlin 2011: 7th International Congress of Systematic and Evolutionary Biology (ICSEB VII), 12th Annual Meeting of the Society of Biological
Systematics (Gesellschaft für Biologische Systematik, GfBS), 20th International Symposium “Biodiversity and Evolutionary Biology” of the German Botanical Society (DBG) (abstracts). Berlin,
Germany: 21–27 February 2011.
Abstract
The family Bromeliaceae includes 57 genera and over 1700 species which have a predominantly Neotropical distribution (1 species in West Africa), and underwent several major radiation
events over the past 20 million years. Despite the remarkable morphological diversity within
the family only little variation is found in plastid DNA sequences, severely hampering the inference of interspecific relationships based on these markers. Nuclear markers generally yield a
higher proportion of informative sites compared to plastid sequences, but are more difficult to
amplify and problems in distinguishing paralogous and orthologous sequences may arise. We
produced nuclear (PRK) and plastid alignments for representatives of the subfamilies Bromelioideae and Pitcairnioideae s.str. (with focus on the genus Fosterella) and investigated their
contrasting evolutionary histories. Here we show that plastid and nuclear phylogenies yield
incongruent topologies and that complex patterns within the nuclear phylogenetic reconstructions based on the PRK dataset are best explained with the occurrence of paralogous copies
but also with past hybridization events within Bromelioideae as well as within Fosterella. The
importance of hybridization in the rapid radiation of Bromeliaceae is discussed as well as potential strategies to extract phylogenetic information from these and similar data sets.
218
Systematics 2009
The genus Deuterocohnia Mez (Bromeliaceae):
Conflicting data in phylogenetic analysis
Nicole Schuetz 1, Michael H.J. Barfuss 2,
Kurt Weising 1 & Georg Zizka 3
1
Universität Kassel, Institut für Biologie, AG Systematik und Morphologie der Pflanzen, Heinrich-Plett-Straße 40, 34132, Kassel, Germany
2
University of Vienna, Faculty of Life Sciences, Department of Systematic and Evolutionary
Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria
3
Senckenberg Research Institute, Department of Botany and Molecular Evolution, 253 Senckenberganlage 25, 60325 Frankfurt/Main, Germany
Author for correspondence: Nicole Schuetz, nschuetz@uni-kassel.de
Oral presentation: P. 118 in: Systematics 2009, 7th Biennial Conference of the Systematics Association (abstracts). Leiden, The Netherlands: National Herbarium of The Netherlands and
National Museum of Natural History Naturalis, Leiden University Medical Centre, 10–14 August
2009.
220
Systematics 2009
Phylogenetic utility of the nuclear marker PRK
on a low taxonomic level: A case study
in the genus Fosterella (Bromeliaceae)
David Brie 1,2, Katharina Schulte1, Michael H.J. Barfuss 4
& Georg Zizka 1,2,3
1
Senckenberg Research Institute, Department of Botany and Molecular Evolution, 253 Senckenberganlage 25, 60325 Frankfurt/Main, Germany
2
Goethe University, Institute of Ecology, Evolution and Diversity, Senckenberganlage 25, 60325
Frankfurt/Main, Germany
3
Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, 60325 Frankfurt/Main, Germany
4
University of Vienna, Faculty of Life Sciences, Department of Systematic and Evolutionary
Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria
Author for correspondence: D. Brie, dbrie@senckenberg.de
Oral presentation: P. 118 in: Systematics 2009: 7th Biennial Conference of the Systematics Association (abstracts). Leiden, The Netherlands: National Herbarium of The Netherlands and
National Museum of Natural History Naturalis, Leiden University Medical Centre, 10–14 August
2009.
222
Botany & Mycology 2009
Classification, adaptive radiation, and geographic
diversification in Bromeliaceae: Insights from
a new multi-locus phylogeny
Thomas J. Givnish 1, Benjamin Van Ee 2, Michael H.J. Barfuss 3,
Ricarda Riina 4, Katharina Schulte 5, Ralf Horres 6,
Philip A. Gonsiska 1, Rachel .S. Jabaily 1, Darren M. Crayn 7,
J. Andrew C. Smith 8, Klaus Winter 9, Gregory K. Brown 10,
Timothy M. Evans 11, Bruce K. Holst 12, Harry E. Luther 12,
Walter Till 3, Georg Zizka 5, Paul .E. Berry 13 & Kenneth J. Sytsma 8
1
University of Wisconsin-Madison, Department of Botany, 430 Lincoln Drive, Birge Hall, Madison, WI, 53706, USA; 2 Harvard University, Botanical Museum, 26 Oxford St, Cambridge, Massachusetts, 02138, USA; 3 University of Vienna, Faculty of Life Sciences, Department of Systematic and Evolutionary Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria;
4
University of Michigan, Herbarium, 3600 Varsity Drive, Ann Arbor, Michigan, 48108, USA;
5
Research Institute Senckenberg, Department of Botany, Frankfurt/Main, Germany; 6 GenXPro
GmbH, Altenhöferallee 3 60438, Frankfurt/Main, Germany; 7 James Cook University, Australian
Tropical Herbarium, Cairns, QLD, 4870, Australia; 8 University of Oxford, Department of Plant
Sciences, South Parks Road, Oxford, OX1 3RB, United Kingdom; 9 Smithsonian Tropical Research
Institute, P.O. Box 2072, Balboa, Ancón, Republic of Panama; 10 University of Wyoming, Department of Botany, 3165, 1000 E University Avenue, Laramie, Wyoming, 82071, USA; 11 Grand
Valley State University, Biology Department, 1 Campus Drive, Allendale, Michigan, 49401, USA;
12
Marie Selby Botanic Gardens, 811 South Palm Avenue, Sarasota, Florida, 34236-7726, USA; 13
University of Michigan Herbarium, Ecology and Evolutionary Biology, 3600 Varsity Dr, Ann Arbor, Michigan, 48108, USA
Author for correspondence: Thomas J. Givnish, givnish@wisc.edu
Oral presentation: (753) In: Botany & Mycology 2009 (abstracts). Snowbird, Utah, USA: Snowbird Center, 25–29 July 2009.
Abstract
The slow rate of molecular evolution in Bromeliaceae, frequent morphological homoplasy, and
extensive molecular and morphological divergence from the families most closely to it have
hampered progress toward an understanding of evolutionary relationships within the family.
Here we present a molecular phylogeny based on more than 9500 aligned bases from eight
rapidly genes and spacers, for 90 ingroup taxa and three outgroups. The eight-subfamily classification recently advanced by Givnish et al. is supported. Brocchinioideae is sister to all other
extant bromeliads; Lindmanioideae, also endemic to the Guayana Shield, diverged next, followed by Tillandsioideae. It appears that xeromorphic Hechtioideae diverged from the bromeliad spine next, followed by Navioideae (endemic to the Guayana Shield, with one species on
the Brazilian Shield), Pitcairnioideae, with Puyoideae and Bromelioideae being sister to each
other. Calibration of the bromeliad molecular tree against dates corresponding to nonbromeliad fossils indicates that bromeliads began to divergence from other monocots 70 Mya,
and that the extant bromeliad genera began to diverge from each other only in the last 19 My.
Fifty-one million years between the origin of bromeliads and initial divergence of surviving
lineages helps explain the difficulty in identifying their closest relatives. Extant species of Brocchinia began to diverge from each other about 17 Mya, before almost any other genera began
to diverge, helping explain why Brocchinia shows such a wide range of adaptive types. The
pace of diversification accelerated greatly 13 Mya, coincident with the rise of several morphological and physiological adaptations to dry or epiphytic conditions arose, and invasion of areas
peripheral to the Guayana Shield. This “bromeliad revolution” corresponds to the time of multiple origins of CAM photosynthesis, epiphytism, bird pollination, tank habit (following its initial
origin in Brocchinia), and absorptive trichomes. A new model for determinants of bromeliad
diversity is presented and discussed.
224
Monocots IV
Molecular phylogenetics of Tillandsia (Bromeliaceae)
and related genera
Michael H.J. Barfuss, Rosabelle Samuel & Walter Till
University of Vienna, Faculty of Life Sciences, Department of Systematic and Evolutionary
Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria
Author for correspondence: Michael H.J. Barfuss, michael.h.j.barfuss@univie.ac.at
Oral presentation: In: Monocots IV: The 4th international conference on the comparative biology of the monocotyledons & the 5th international symposium on grass systematics and evolution (abstracts). Copenhagen, Denmark: University of Copenhagen, H.C. Ørsted Institute, 11–
15 August 2008.
Abstract
Tillandsia is the most species-rich genus of subfamily Tillandsioideae (Bromeliaceae). Its traditional delimitation and internal classification are the subjects of much debate and conflict with
recent molecular studies. Previously investigated plastid regions have shown little variation
across Tillandsioideae, due to rapid radiation and subsequent genetic isolation/speciation.
Until now it has not been possible to unambiguously infer phylogenetic relationships within
the core Tillandsioideae, especially the Tillandsia s. l. (incl. Racinaea and Viridantha) and
Vriesea s. l. (incl. Alcantarea and Werauhia) backbones. In this study we have analysed additional chloroplast and nuclear DNA markers to obtain more phylogenetic information, with the
particular aim of resolving these backbones better. Results from the difficult ITS (Internal transcribed spacer) region show ITS 1 to be highly variable, but the ITS region as a whole contains
only little phylogenetic signal. Therefore, sequences from the single-copy nuclear genes PRK
(Phosphoribulokinase) and PHYC (Phytochrome C) are here combined with ITS and chloroplast
sequence data to provide a well-supported molecular phylogeny. Only now we can propose a
novel classification for Tillandsia and its closely related genera that were only partially reflected by previous chloroplast studies.
226
Monocots IV
Adaptive radiation and diversification in Bromeliaceae:
Inferences from a new multigene phylogeny
Thomas J. Givnish 1, Ricarda Riina 2, Benjamin Van Ee 3, Paul .E.
Berry 4, Kenneth J. Sytsma 5, Michael H.J. Barfuss 6, Walter Till 6,
Ralf Horres 7, Katharina Schulte 8 & Georg Zizka 8
1
University of Wisconsin-Madison, Department of Botany, 430 Lincoln Drive, Birge Hall, Madison, WI, 53706, USA; 2 University of Michigan, Herbarium, 3600 Varsity Drive, Ann Arbor, Michigan, 48108, USA; 3 Harvard University, Botanical Museum, 26 Oxford St, Cambridge, Massachusetts, 02138, USA; 4 University of Michigan Herbarium, Ecology and Evolutionary Biology,
3600 Varsity Dr, Ann Arbor, Michigan, 48108, USA; 5 University of Oxford, Department of Plant
Sciences, South Parks Road, Oxford, OX1 3RB, United Kingdom; 6 University of Vienna, Faculty
of Life Sciences, Department of Systematic and Evolutionary Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria; 7 GenXPro GmbH, Altenhöferallee 3 60438, Frankfurt/Main, Germany; 8 Research Institute Senckenberg, Department of Botany, Frankfurt/Main,
Germany
Author for correspondence: Thomas J. Givnish, givnish@wisc.edu
Oral presentation: In: Monocots IV: The 4th international conference on the comparative biology of the monocotyledons & the 5th international symposium on grass systematics and evolution (abstracts). Copenhagen, Denmark: University of Copenhagen, H.C. Ørsted Institute, 11–
15 August 2008.
Abstract
Bromeliads show an unusually slow rate of molecular evolution, substantial homoplasy in
morphological character-states, and substantial molecular and morphological divergence from
other monocots, making inference of relationships within the family difficult. Here we present
the most comprehensive analysis of bromeliad phylogeny to date, based on sequencing three
rapidly evolving plastid genes (matK, ndhF, rps16) and three rapidly evolving plastid spacers
(psbA-trnH, rbcL-atpB, trnL-trnF) across nearly 100 bromeliad taxa and outgroups drawn from
six other families of Poales. We use these data to test the monophyly and recently proposed
relationships among the eight subfamilies of Bromeliaceae; trace the evolution of several
characters crucial for the evolution of the epiphytic habit under parsimony, maximum likelihood, and Bayesian frameworks; infer the broad-scale geographic patterns of geographic diversification within the family; and produce a calibrated chronology of evolution within the
family. Together, these findings allow us to identify the timing of the “bromeliad revolution”,
during which several traits crucial to extensive speciation within the family apparently arose,
accompanying its breakout from the exceedingly humid, infertile environments of the Guayana
Shield.
228
Systematics 2008
The backbone problem: Hunting for informative
characters in DNA sequences of Tillandsioideae
(Bromeliaceae) for reconstructing a
well-resolved phylogeny
Michael H.J. Barfuss, Walter Till & Rosabelle Samuel
University of Vienna, Faculty of Life Sciences, Department of Systematic and Evolutionary
Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria
Author for correspondence: Michael H.J. Barfuss, michael.h.j.barfuss@univie.ac.at
Oral presentation: P. 356 in: Gradstein, R. & al. (eds.), Systematics 2008: 10th Annual Meeting
of the Gesellschaft für Biologische Systematik, 18th International Symposium "Biodiversity and
Evolutionary Biology" of the German Botanical Society (DBG) (abstracts). Göttingen, Germany:
University of Göttingen, 7–11 April 2008.
Abstract
Plastid Genomes have shown to be very uniform in recently diverged plant groups (like in
Bromeliaceae) mainly caused by rapid radiation (and subsequent quick speciation / isolation).
In addition to the less variable chloroplast regions which we analysed in our previous studies,
we sequenced another set of cpDNA markers, which are supposed to be phylogenetically much
more informative. Nevertheless, avoiding results of still insufficiently resolved phylogenies
derived from this additional chloroplast data we also established primers for nuclear DNA regions, especially with the target of getting well resolved backbones in our trees. Attempts of
several research groups to amplify the nrDNA region ITS (Internal Transcribed Spacer) in Bromeliaceae failed so far, because of very strong bounded secondary structures caused by repetitive GC-rich motives in the ITS 1 region. While we were able to successfully amplify and sequence over this difficult region for a set of species of Bromeliaceae by modifying PCR programs and sequencing chemistries, results of sequence analysis again showed very poor
amounts of phylogenetically informative characters among taxa, having a similar substitution
rate as it can be found in the investigated chloroplast regions (which is in contrast to many
other plant groups). All resulting trees (even from combined analyses) did not yet allow an
unambiguous interpretation of phylogenetic relationships of the whole subfamily Tillandsioideae; especially not at the backbone of very species-rich genera like Tillandsia and close relatives (Racinaea, Viridantha), and Vriesea and associates (Alcantarea, Werauhia). Therefore,
sequences (including exons as well as introns) of the assumed single-copy nDNA genes PRK
(Phosphoribulokinase), PHYC (Phytochrom C) and MS (Malate Synthase) have been analysed,
and results compared with phylogenies derived from ITS and more than 10 different plastid
regions; this nuclear markers seem to be much more promising in giving higher phylogenetic
signals per sequenced base pair than any of the other previously investigated DNA regions
does.
230
XVII IBC 2005
Phylogenetics of subfamily Tillandsioideae
(Bromeliaceae): A comparison of plastid and
nuclear DNA sequence with morphological data
Michael H.J. Barfuss, Walter Till & Rosabelle Samuel
University of Vienna, Faculty of Life Sciences, Department of Systematic and Evolutionary
Botany, Faculty Center of Biodiversity, Rennweg 14, 1030 Vienna, Austria
Author for correspondence: Michael H.J. Barfuss, michael.h.j.barfuss@univie.ac.at
Oral presentation: P. 150 (9.7.3.) in: XVII IBC 2005: XVII International Botanical Congress (abstracts). Vienna, Austria: Austria Center Vienna, 17–23 July 2005.
Abstract
Part of the low-copy nuclear gene phosphoribulokinase (PRK) and a multicopy nuclear rDNA
spacer region (ITS) were used to clarify relationships of subfamily Tillandsioideae (Bromeliaceae). These data are compared with the available cpDNA sequences of coding rbcL and matK
and noncoding trnL intron, trnL-F intergenic spacer, atpB-rbcL intergenic spacer, rps16 intron,
and partial 5' and 3' trnK intron. We added taxa of Tillandsia, Guzmania and Vriesea to get
better resolution within these genetically convergent genera. Molecular phylogenetic data is
compared with various morphological characters to test their usefulness for a new classification of Tillandsioideae. These characters are optimised onto a strict consensus tree of the
combined matrices of plastid and nuclear DNA sequences. Preference has been given to characters likely to be little affected by adaptive constraints, i.e., pollen, stigma, ovules, anther,
seed, and nectary morphology.
232
Conclusions
The current study demonstrates that the analyses of additional plastid and nuclear DNA markers are able to provide better resolution and support in phylogenetic trees of Bromeliaceae.
The inclusion of more taxa significantly improved the tree topologies, especially where more
distantly related taxa were sampled in order to break long branches, while sampling of lineages which were not included before tend to decrease the support for certain clades. However, in all cases several unknown relationships could be resolved.
Analyses of plastid DNA sequences including more markers and more species clarified
several uncertain relationships which existed among subfamilies of Bromeliaceae. The eight
subfamilies are confirmed, but uncertainty about a monophyletic origin of subfamily Puyoideae persists. Previously proposed hypotheses concerning biogeography, origin and age of Bromeliaceae could be re-analysed and refined with the enlarged data set. Rapid radiation and
relatively recent diversification within the last 8.7–4 My are most likely the explanation for the
low sequence divergence.
The investigation of the low-copy nuclear gene PRK has provided useful insights into phylogenetic relationships of Bromelioideae. The amplified region is at least 3-times more variable
than any plastid regions, thus making PRK suitable for phylogenetic reconstructions. However,
the amplified fragment alone did not provide sufficient information for resolving relationships
completely, only the combined analysis with plastid markers clarified several previously unresolved relationships at deeper nodes (“basal bromelioids”, “eu-bromelioids”). Genera within
core bromelioids remain problematic. Most parsimonious reconstructions for tank habit and
the sepal symmetry revealed strong evolutionary significance and indicated diagnostic utility
for future classifications.
The comparative studies of nuclear DNA sequences within Tillandsioideae showed that
some nuclear markers are able to provide more information and a higher degree of resolution
in phylogenetic trees than plastid markers. However, their utility cannot be measured only by
PICs, but depends also on methodological challenges. Nuclear ITS nrDNA is not recommended
for phylogenetic investigations of Bromeliaceae due to the presence of strong secondary structures, which create problems in amplification and sequencing, as well as few PICs to resolve
deeper nodes. Some other amplified fragments of genes were to short and showed few potentially PICs. The selected nuclear DNA regions of PRK and PHYC are able to provide reasonable
support in some parts of phylogenetic trees, but several relationships still remain unresolved.
So far, the search for “the ideal” phylogenetic marker for Tillandsioideae continues and perhaps it is impossible to find such a marker with standard PCR and Sanger-sequencing.
However, progress has been achieved in exploring phylogenetic relationships of Tillandsioideae by combining nuclear DNA sequence Data with already published plastid DNA sequence
data. DNA results together with re-evaluated morphological characters allowed the recircumscription of existing genera and description of three new genera and subgenera, respectively. However, taxonomic problems in certain groups persist due to poor sampling, limited
233
BARFUSS, M.H.J.
MOLECULAR STUDIES
resolution and variable morphology. To get a fully resolved picture of relationships within Tillandsioideae, more DNA data is needed as well as a careful revision of morphological characters which are already indicated to be useful to circumscribe taxonomic groups.
A major challenge that remains within Bromeliaceae is the great number of species, for
which it is sometimes impossible to get material for both DNA and morphological investigations. Many species are not cultivated in ex situ collections, because either they are simply not
available or require very special growing conditions. Several species were collected only once
in the wild or are known from a single locality. If plant material is available, it is rather simple
to generate additional DNA sequence data for phylogenetic studies, but collecting morphological characters is still challenging. Many useful characters are missing on herbarium specimens
and flowering material is sometimes hardly available for certain species. Without having flowering or fruiting specimens (ideally both) in good conditions, a careful taxonomic revision is
impossible. The future direction will be to establish improved international collaborations with
taxonomists in the countries of origin of Bromeliaceae to access important plant material more
easily.
The continuously improving next generation sequencing (NGS) technologies will play important roles in future phylogenetic investigations of Bromeliaceae. Sequencing and analysing
of plastoms has already been successfully implemented for monocots; this will definitely help
to clarify relationships and uncover the evolutionary history of the plastid genome in Bromeliaceae. The analysis of the entire 18S-5.8S-26S nrDNA repeat unit can also provide useful insights into the evolution of nrDNA in bromeliads and uncover the ecological significance of the
strong secondary structure. NGS of nrDNA might also be useful to differentiate species and
therefore could be a potential candidate marker for DNA barcoding. Furthermore, the possibility to gain huge amounts of allelic nuclear data will help to identify nuclear regions that are
useful for inferring relationships and uncover hybridisation events.
This work aimed to provide a starting point for a biological classification of Bromeliaceae
apart from the already well supported subfamilies. It intends to stimulate researchers interested in Bromeliaceae to focus on certain taxa, collect morphological information and apply
newest sequencing technologies to answer several, to date unresolved evolutionary questions
in Bromeliaceae.
234
Curriculum Vitae
Mag. Michael H.J. Barfuss
Name
Michael Harald Johannes Barfuss
Academic degree
Mag.rer.nat.
Date of birth, nationality
15.03.1977, Austria
Home address
Schillerstraße 12 Haus 12
2351 Wiener Neudorf, Austria
Corresponding address
University of Vienna, Faculty of Life Sciences,
Faculty Center of Biodiversity
Department of Systematic and Evolutionary Botany,
Rennweg 14, 1030, Vienna, Austria
Phone:
+43-(0)1-4277-54130 (office)
+43-(0)1-4277-54129 (lab)
Fax:
+43-(0)1-4277-9541
E-mails:
michael.h.j.barfuss@univie.ac.at
a9601611@unet.univie.ac.at
ariocarpus@gmx.at
Employment
since 2006
Technical laboratory assistant, molecular systematics laboratory (lab management and sequencing technology); research
associate, Department of Systematic and Evolutionary Botany,
University of Vienna
Academic career
2006–2012
Dr. student, Botany, University of Vienna, Austria
2006
Diplom (Mag.rer.nat), supervisors ao. Univ.-Prof. Dr. Rosabelle
Samuel, o. Univ.-Prof. Dr. Tod Stuessy, final exam
(Diplomprüfung) 25. August 2006, title of diploma thesis
Molecular studies in Bromeliaceae: Phylogenetic relationships
in subfamily Tillandsioideae based on evidence from plastid
sequences
1996–2006
Diploma student, Biology (Botany), University of Vienna, Austria
1996
Qualification for university entrance (Matura) 3. June 1996,
Höhere Bundeslehr- und Versuchsanstalt für Gartenbau, Vienna, Austria
235
BARFUSS, M.H.J.
MOLECULAR STUDIES
Project leadership
since 2011
Evolution of the Tillandsia capillaris complex (Bromeliaceae):
Adaptive radiation in a semi-arid environment?, project leaders Dr. Jorge Chiapella (Argentinia), Ass.-Prof. Dr. Walter Till,
Mag. Michael H.J. Barfuss (Austria) (BMWF, OeAD, WTZ)
since 2008
Molecular Phylogeny of Gymnocalycium (Cactaceae), project
leaders Mag. Michael. H.J. Barfuss, Ass.-Prof. Dr. Walter Till
(GÖK, AGG)
2007–2008
Evolution und Sekundärstruktur der nrDNS-Region ITS von
Tillandsia s. l. (Bromeliaceae): Zusammenhänge zwischen Sequenz-Alignments, Phylogenie und ökologisch-adaptiver Variabilität, project leaders Ass.-Prof. Dr. Walter Till,
Mag. Michael. H.J. Barfuss (ÖAW, KIÖS no. 2007-02)
Project collaboration
since 2012
Phylogeny and evolution of Tillandsia subgenus Tillandsia
(Bromeliaceae), project leaders Ass.-Prof. Dr. Walter Till, Mag.
Michael H.J. Barfuss; postdoc Juan Pablo Pinzón
since 2003
Phylogeny, adaptive radiation, and historical biogeography in
Bromeliaceae, The Bromeliad Phylogeny Group,
group leader Prof. Dr. Thomas J. Givnish
2003
Molecular phylogeny of Bromeliaceae subfamily Tillandsioideae: Insights from the nuclear and chloroplast genome,
project leader Ass.-Prof. Dr. Walter Till (ÖAW, KIÖS)
2002
Molekulare Analyse der Verwandtschaftsbeziehungen innerhalb der Gattung Tillandsia (Bromeliaceae, Unterfamilie
Tillandsioideae), project leader ao. Univ.-Prof. Dr. Rosabelle
Samuel (Hochschuljubiläumsstiftung der Stadt Wien)
Project assistance
since 2010
Evolution and Biodiversity of New Caledonian Diospyros (Ebenaceae), project leader ao. Univ.-Prof. Dr. Rosabelle Samuel
(FWF no. P22159)
2007–2011
Taxonomische Überarbeitung der Gattung Leontopodium,
project leader o. Univ.-Prof. Dr. Hermann Stuppner
(FWF no. P19480)
2006–2010
Species delineation and autecology of Spirogyra, project leader ao. Univ.-Prof. Mag. Dr. Michael Schagerl (FWF no. P18465)
2006–2010
Molecular Phylogeny, genome size and chromosomal evolution in genus Polystachya Hooker (Orchidaceae), project leader ao. Univ.-Prof. Dr. Rosabelle Samuel (FWF no. P19108)
2005–2006
Chromosomes and evolution of Melampodium (Asteraceae),
project leader o. Univ.-Prof. Dr. Tod F. Stuessy
(FWF no. P18201)
236
IN BROMELIACEAE
CURRICULUM VITAE
2004–2007
Phylogeny and Historical Biogeography of Ebenaceae, project
leader ao. Univ.-Prof. Dr. Rosabelle Samuel (FWF no. P17094)
2002
Phylogenie and historical biogeography of Phyllanthaceae,
project leader ao. Univ.-Prof. Dr. Rosabelle Samuel (FWF no.
P15333)
2001–2002
Intraspecific phylogeography of alpine plants, project leader
Univ.-Prof. Dr. Harald Niklfeld (FWF no. P13874)
2000–2002
Generic delimitations and molecular phylogeny of subfamily
Tillandsioideae (Bromeliaceae), project leader Ass.-Prof. Dr.
Walter Till (FWF no. P13690)
Grants (donator)
2011
XVIII IBC 2011, congress (University of Vienna)
2009
Jodrell Laboratory, Royal Botanic Gardens, Kew (Synthesys)
2008
Systematics 2008, conference (University of Vienna)
2008
Monocots IV, conference (ÖFB)
2003
Monocots III, conference (ÖFB)
Student tutorials
2008–2009
Project practical course in DNA markers and chromosomes in
plant systematics and evolutionary research
2005–2007
Practical course in population biology (AFLP, SSR)
2004–2010
Practical course in DNA sequencing analysis and molecular
phylogeny
2001–2002
Practical course in enzyme analysis in plant systematics
2001
Plant systematic-morphological introductory excursions
2000
Plant systematic-morphological practical course
Teaching experience
since 2012
Project practical course in molecular and karyological methods in evolution and ecology
since 2011
Project practical course in DNA barcoding - a new approach to
species identification in ecology and biodiversity research
since 2010
Practical course in DNA sequencing analysis and molecular
phylogeny
since 2010
Lectures in macromolecules and molecular phylogeny in plant
systematics and evolutionary research
2010–2011
Project practical course in DNA markers and chromosomes in
plant systematics and evolutionary research
237
BARFUSS, M.H.J.
MOLECULAR STUDIES
International field trips
2006
Ecuador
2006
USA (SW)
2002
Dominican Republic
2001
Borneo (Brunei Darussalam, Malaysia)
2000
USA (SW)
2000
Costa Rica
Invited talks
2009
Implications of five nuclear regions on the phylogenetic relationships of Tillandsioideae (Bromeliaceae);
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, UK
2006
Tillandsien und verwandte Gattungen: Kann uns die DNA in
der Systematik die fehlenden Antworten liefern? Ein Überblick
über unseren derzeitigen Wissenstand;
Biologiezentrum Linz der OÖ. Landesmuseen, Linz, Austria
International conferences
2011
XVIII IBC 2011, Melbourne, Australia; oral presentation, poster
2010
Biodiversity and Evolutionary Biology, Vienna, Austria
2008
Monocots IV, Copenhagen, Denmark; oral presentation
2008
Sytematics 2008, Göttingen, Germany; oral presentation
2007
9. Jahrestagung der GfBS, Vienna, Austria
2005
XVII IBC 2005, Vienna, Austria; oral presentation
2003
Monocots III, Claremont, California, USA; oral presentation
2001
Systematics 2001, London, UK; oral presentation
Scientific memberships
Since 2006
American Society of Plant Taxonomists, ASPT
Since 2006
International Association for Plant Taxonomy, IAPT
Since 2005
Botanical Society of America, BSA
Special interests
All topics concerning the angiosperm families Bromeliaceae and Cactaceae; succulent
plants; Asteraceae, Orchidaceae; special focus on molecular techniques (sequencing), (molecular) systematics, taxonomy, nomenclature, and field botany; plant cultivation and propagation, protection and preservation of endangered plant species (CITES).
238
IN BROMELIACEAE
CURRICULUM VITAE
Publications
00. Barfuss, M.H.J., W. Till & R. Samuel. In prep. A new classification of Bromeliaceae subfamily Tillandsioideae inferred from DNA sequences data of two genomes and morphology.
00. Barfuss, M.H.J., R. Samuel, M.W. Chase & F. Forest. In prep. Optimizing eight nuclear DNA
markers for phylogenetic studies in recently diverged angiosperms: A case study in Bromeliaceae subfamily Tillandsioideae.
00. Givnish, T.J., M.H.J. Barfuss, B. Van Ee, R. Riina, K. Schulte, R. Horres, P.A. Gonsiska, R.S.
Jabaily, D.M. Crayn, J.A.C. Smith, K. Winter, G.K. Brown, T.M. Evans, B.K. Holst, H.E. Luther, W. Till, G. Zizka, P.E. Berry & K.J. Sytsma. In prep. Adaptive radiation, correlated and
contingent evolution, and net species diversification in Bromeliaceae.
00. Sehr, E.M., M.H.J. Barfuss, G.E. Barboza, R. Samuel, E.A. Moscone & F. Ehrendorfer. In
prep. DNA sequences from the plastid genome and the single-copy nuclear gene waxy
suggest two dysploid karyotype changes in the phylogeny of Capsicum (Solanaceae).
17. Chen, C., M.H.J Barfuss, T. Pröschold & M. Schagerl. 2012. Hidden genetic diversity in the
green alga Spirogyra (Zygnematophyceae, Streptophyta). B.M.C. Evolutionary Biology
(12): 77, doi 1186/1471-2148-12-77.
16. Murillo-A., J., E. Ruiz-P., L.R. Landrum, T.F. Stuessy & M.H.J. Barfuss. 2012. Phylogenetic
relationships in Myrceugenia (Myrtaceae) based on plastid and nuclear DNA sequences.
Molecular Phylogenetics and Evolution, 62 (2): 764–776.
15. Demaio, P.H., M.H.J. Barfuss, R. Kiesling, W. Till & J. O. Chiapella. 2011. Molecular phylogeny of Gymnocalycium (Cactaceae): Assessment of alternative infrageneric systems, a
new subgenus, and trends in the evolution of the genus. American Journal of Botany 98
(11): 1841–1854.
14. Givnish, T.J., M.H.J. Barfuss, B. Van Ee, R. Riina, K. Schulte, R. Horres, P.A. Gonsiska, R.S.
Jabaily, D.M. Crayn, J.A.C. Smith, K. Winter, G.K. Brown, T.M. Evans, B.K. Holst, H.E. Luther, W. Till, G. Zizka, P.E. Berry & K.J. Sytsma. 2011. Phylogeny, adaptive radiation, and
historical biogeography in Bromeliaceae: Insights from an eight-locus plastid phylogeny.
American Journal of Botany 98 (5): 872–895.
13. Demaio, P.H., M.H.J. Barfuss, W. Till & J. O. Chiapella. 2010. Phylogenetic relationships
and infrageneric classification of the genus Gymnocalycium: Insights from molecular data.
Gymnocalycium (Sonderausgabe 2010): 925–946.
12. Russell, A., R. Samuel, V. Klejna, M.H.J. Barfuss, B. Rupp & M.W. Chase. 2010. Reticulate
evolution in diploid and tetraploid species of Polystachya (Orchidaceae) as shown by plastid DNA sequences and low-copy nuclear genes. Annals of Botany 106 (1): 37–56.
11. Russell, A., R. Samuel, B. Rupp, M.H.J. Barfuss, M. Šafran, V. Besendorfer & M.W. Chase.
2010. Phylogenetics and cytology of a pantropical orchid genus Polystachya (Polystachyinae, Vandeae, Orchidaceae): Evidence from plastid DNA sequence data. Taxon 59 (2):
389–404.
10. Blöch, C., H. Weiss-Schneeweiss, G.M. Schneeweiss, M.H.J. Barfuss, C.A. Rebernig, J.L.
Villaseñor & T.F. Stuessy. 2009. Molecular phylogenetic analyses of nuclear and plastid
DNA sequences support dysploid and polyploid chromosome number changes and reticulate evolution in the diversification of Melampodium (Millerieae, Asteraceae). Molecular
Phylogenetics and Evolution 53 (1): 220–233.
239
BARFUSS, M.H.J.
MOLECULAR STUDIES
9. Duangjai, S., R. Samuel, J. Munzinger, F. Forest, B. Wallnöfer, M.H.J. Barfuss, G. Fischer &
M.W. Chase. 2009. A multi-locus plastid phylogenetic analysis of the pantropical genus
Diospyros (Ebenaceae), with an emphasis on the radiation and biogeographic origins of
the New Caledonian endemic species. Molecular Phylogenetics and Evolution 52 (3): 602–
620.
8. Gruenstaeudl, M., E. Urtubey, R.K. Jansen, R. Samuel, M.H.J. Barfuss & T. F. Stuessy. 2009.
Phylogeny of Barnadesioideae (Asteraceae) inferred from DNA sequence data and morphology. Molecular Phylogenetics and Evolution 51 (3): 572–587.
7. Schulte, K., M.H.J. Barfuss & G. Zizka. 2009. Phylogeny of Bromelioideae (Bromeliaceae)
inferred from nuclear and plastid DNA loci reveals the evolution of the tank habit within
the subfamily. Molecular Phylogenetics and Evolution 51 (2): 327–339.
6. Möller, M., M. Pfosser, C.-G. Jang, V. Mayer, A. Clark, M.L. Hollingsworth, M.H.J. Barfuss,
Y.-Z. Wang, M. Kiehn & A. Weber. 2009. A preliminary phylogeny of the 'didymocarpoid
Gesneriaceae' based on three molecular data sets: Incongruence with available tribal classifications. American Journal of Botany 96 (5): 989–1010.
5. Till, W. & M.H.J. Barfuss. 2006. Progress towards a new classification of Tillandsioideae.
Journal of the Bromeliad Society 56 (6): 253–259.
4. Barfuss, M.H.J., R. Samuel, W. Till & T.F. Stuessy. 2005. Phylogenetic relationships in subfamily Tillandsioideae (Bromeliaceae) based on DNA sequence data from seven plastid regions. American Journal of Botany 92 (2): 337–351.
3. Samuel, R, H. Kathriarachchi, P. Hoffmann, M.H.J. Barfuss, K.J. Wurdack, C.C. Davis &
M.W. Chase. 2005. Molecular phylogenetics of Phyllanthaceae: Evidence from plastid
matK and nuclear PHYC sequences. American Journal of Botany 92 (1): 132–141.
2. Barfuss, M.H.J., R. Samuel & W. Till. 2004. Molecular phylogeny in subfamily Tillandsioideae
(Bromeliaceae) based on six cpDNA Markers: An update. Journal of the Bromeliad Society
54 (1): 9–17, 48.
1. Schönswetter, P., A. Tribsch, M.H.J. Barfuss & H. Niklfeld. 2002. Several Pleistocene refugia
detected in the high alpine plant Phyteuma globulariifolium Sternb. & Hoppe (Campanulaceae) in the European Alps. Molecular Ecology 11 (12): 2637–2647.
Abstracts (Presenter)
37. [Oral presentation] Turner, B., J. Munzinger, S. Duangjai, M.H.J. Barfuss, B. Wallnöfer, O.
Paun, M.W. Chase & R. Samuel. 2012. Diversification of endemic New Caledonian Diospyros (Ebenaceae). P. 53 in: Berichte des naturwissenschaftlich-medizinischen Vereins in
Innsbruck, Supplementum 2020, 15. Treffen der Österreichischen Botanikerinnen und
Botaniker (abstracts). Innsbruck, Austria: Universität Innsbruck, Institut für Botanik, 27–29
September.
36. [Oral presentation] Ehrendorfer, F., V. Vladimirov, J.-F. Manen & M.H.J. Barfuss. 2012.
Adaptive Radiation und weltweite Expansion der artenreichen Tribus Rubieae (Rubiaceae). P. 13 in: Berichte des naturwissenschaftlich-medizinischen Vereins in Innsbruck,
Supplementum 2020, 15. Treffen der Österreichischen Botanikerinnen und Botaniker (abstracts). Innsbruck, Austria: Universität Innsbruck, Institut für Botanik, 27–29 September.
240
IN BROMELIACEAE
CURRICULUM VITAE
35. [Oral presentation] Givnish, T.J., M.H.J. Barfuss, B. Van Ee, R. Riina, K. Schulte, R. Horres,
P.A. Gonsiska, R.S. Jabaily, D.M. Crayn, J.A.C. Smith, K. Winter, G.K. Brown, T.M. Evans,
B.K. Holst, H.E. Luther, W. Till, G. Zizka, P.E. Berry & K.J. Sytsma. 2012. Adaptive radiation, correlated evolution, and determinants of net diversification rates in Bromeliaceae:
Test of an a priori model. (638) in: Botany 2012: The Next Generation (abstracts). Columbus, Ohio: Greater Columbus Convention Center, 7–11 July.
34. [Oral presentation] Barfuss, M.H.J., W. Till & R. Samuel. 2011. Systematics, evolution, and
phylogeography of Tillandsia (Bromeliaceae) and related genera. P. 189 (SYM004) in: XVIII
IBC 2011: XVIII International Botanical Congress (abstracts). Melbourne, Australia: Melbourne Congress and Exhibition Centre, 23–30 July.
33. [Poster] Barfuss, M.H.J., W. Till & R. Samuel. 2011. New taxonomic implications in Tillandsioideae (Bromeliaceae) based on DNA data and morphology. Pp. 568–569 (P0730) in:
XVIII IBC 2011: XVIII International Botanical Congress (abstracts). Melbourne, Australia:
Melbourne Congress and Exhibition Centre, 23–30 July.
32. [Oral presentation] Givnish, T.J., M.H.J. Barfuss, B. Van Ee, R. Riina, K. Schulte, R. Horres,
P.A. Gonsiska, R.S. Jabaily, D.M. Crayn, J.A.C. Smith, K. Winter, B.K. Holst, H.E. Luther,
W. Till, G. Zizka, P.E. Berry, Ann Arbor & K.J. Sytsma. 2011. Origin, phylogeny, adaptive
radiation, and geographic diversification of Bromeliaceae. Pp. 186–187 (SYM004) in: XVIII
IBC 2011: XVIII International Botanical Congress (abstracts). Melbourne, Australia: Melbourne Congress and Exhibition Centre, 23–30 July.
31. [Oral presentation] Ehrendorfer, F., V. Vladimirov & M.H.J. Barfuss. 2011. Worldwide radiation, phylogeography and paraphyly of the speciose Rubiaceae-Rubieae. Pp. 253–254
(SYM094) in: XVIII IBC 2011: XVIII International Botanical Congress (abstracts). Melbourne,
Australia: Melbourne Congress and Exhibition Centre, 23–30 July.
30. [Poster] Russell, A., R. Samuel, M.H.J. Barfuss, B. Turner & M.W. Chase. 2011. Evolutionary inference from multiple incongruent DNA data matrices: Reticulate evolution of Polystachya (Orchidaceae). P. 665 (P1042) in: XVIII IBC 2011: XVIII International Botanical Congress (abstracts). Melbourne, Australia: Melbourne Congress and Exhibition Centre, 23–30
July.
29. [Poster] Samuel, R., B. Turner, S. Duangjai, J. Munzinger, B. Wallnöfer, M.W. Chase &
M.H.J. Barfuss. 2011. Origin and evolution of New Caledonian Diospyros (Ebenaceae): A
phylogenetic approach. P. 635 (P0947) in: XVIII IBC 2011: XVIII International Botanical
Congress (abstracts). Melbourne, Australia: Melbourne Congress and Exhibition Centre,
23–30 July.
28. [Poster] Michalak, I., D. Silvestro, D. Brie, M.H.J. Barfuss, K. Schulte & G. Zizka. 2011. Conflicting phylogenetic signal within the nuclear marker PRK highlights the importance of
hybridization events in the diversification of Bromeliaceae. Pp. 252–253 (II-74) in: BioSystematics Berlin 2011: 7th International Congress of Systematic and Evolutionary Biology
(ICSEB VII), 12th Annual Meeting of the Society of Biological Systematics (Gesellschaft für
Biologische Systematik, GfBS), 20th International Symposium “Biodiversity and Evolutionary Biology” of the German Botanical Society (DBG) (abstracts). Berlin, Germany: 21–27
February.
27. [?] Murillo-Aldana, J., E. Ruiz-P., L.R. Landrum, T.F. Stuessy & M.H.J. Barfuss. 2010. Relaciones filogeneticas en Myrceugenia(Myrtaceae). In: Latin American Botanical Congress.
La Serena, Chile: October.
241
BARFUSS, M.H.J.
MOLECULAR STUDIES
26. [Oral presentation] Russell, A., M.H.J. Barfuss, M.W. Chase & R. Samuel. 2010. Reticulate
evolution in diploid and tetraploid species of Polystachya (Orchidaceae) revealed by multiple nuclear and plastid loci. P. 55 in: Albach, D. & Greimler, J. (eds), 19th International
Symposium "Biodiversity and Evolutionary Biology" of the German Botanical Society (DBG)
(abstracts). Vienna, Austria: University of Vienna, Faculty Centre of Biodiversity, 16–19
September.
25. [Poster] Duangjai, S., R. Samuel, J. Munzinger, B. Wallnöfer, F. Forest, M.H.J. Barfuss &.
M.W. Chase. 2010. Biogeography origins and evolution of endemic species of Diospyros
(Ebenaceae) from New Caledonia: A molecular phylogenetic perspective. P. 85 in: Albach,
D. & Greimler, J. (eds), 19th International Symposium "Biodiversity and Evolutionary Biology" of the German Botanical Society (DBG) (abstracts). Vienna, Austria: University of Vienna, Faculty Centre of Biodiversity, 16–19 September.
24. [Poster] Stockenhuber, R.M., M.H.J. Barfuss, G. Cruz-Mazo & R. Samuel. 2010. Molecular
phylogeny of Scorzoneroides. P. 116 in: Albach, D. & Greimler, J. (eds), 19th International
Symposium "Biodiversity and Evolutionary Biology" of the German Botanical Society (DBG)
(abstracts). Vienna, Austria: University of Vienna, Faculty Centre of Biodiversity, 16–19
September.
23. [?] Russell, A., R. Samuel, V. Klejna, M.H.J. Barfuss, B. Rupp & M.W. Chase. 2010. Analysis
of multiple nuclear and plastid loci reveals reticulate evolution in diploid and tetraploid
species of genus Polystachya (Orchidaceae). In: New Frontiers in Plant Systematics and
Evolution (NFPSE 2010). Beijing, China: 7–9 July.
22. [Oral presentation] Schuetz, N., M.H.J. Barfuss, K. Weising & Georg Zizka. 2009. The genus
Deuterocohnia Mez (Bromeliaceae): Conflicting data in phylogenetic analysis. P. 118 in:
Systematics 2009, 7th Biennial Conference of the Systematics Association (abstracts). Leiden, The Netherlands: National Herbarium of The Netherlands and National Museum of
Natural History Naturalis, Leiden University Medical Centre, 10–14 August.
21. [Oral presentation] Brie, D., K. Schulte, M.H.J. Barfuss & G. Zizka. 2009. Phylogenetic utility of the nuclear marker PRK on a low taxonomic level: A case study in the genus Fosterella (Bromeliaceae). P. 33 in: Systematics 2009, 7th Biennial Conference of the Systematics Association (abstracts). Leiden, The Netherlands: National Herbarium of The Netherlands and National Museum of Natural History Naturalis, Leiden University Medical Centre, 10–14 August.
20. [Oral presentation] Russell, A., R. Samuel, B. Rupp, M.H.J. Barfuss, M. Safran, V. Klejna &
M.W. Chase. 2009. Phylogeny, cytology and biogeography of the pantropical orchid genus
Polystachya. P. 109 in: Systematics 2009, 7th Biennial Conference of the Systematics Association. Leiden, The Netherlands: National Herbarium of The Netherlands and National
Museum of Natural History Naturalis, Leiden University Medical Centre, 10–14 August.
19. [Oral presentation] Givnish, T.J., B. Van Ee, M.H.J. Barfuss, R. Riina, K. Schulte, R Horres,
P.A. Gonsiska, R.S. Jabaily, D.M. Crayn, J.A.C. Smith, K. Winter, G.K. Brown, T.M. Evans,
B.K. Holst, H.E. Luther, W. Till, G. Zizka, P.E. Berry & K.J. Sytsma. 2009. Classification,
adaptive radiation, and geographic diversification in Bromeliaceae: Insights from a new
multi-locus phylogeny. (753) In: Botany & Mycology 2009 (abstracts). Snowbird, Utah,
USA: Snowbird Center, 25–29 July.
242
IN BROMELIACEAE
CURRICULUM VITAE
18. [Poster] Russell, A., R. Samuel, M.H.J. Barfuss, B. Rupp, V. Klejna & M.W. Chase. 2009.
Low copy nuclear genes reveal hybrid speciation in Polystachya (Orchidaceae). In: International conference on polypoidy, hybridisation and biodiversity (abstracts). Saint-Malo,
France: 17–20 Mai.
17. [Poster] Russell, A., R. Samuel, B. Rupp, M.H.J. Barfuss & M.W. Chase. 2008. Phylogenetics, African biodiversity and intercontinental dispersal in Polystachya (Orchidaceae). Pp.
13–14 in: 10th Young Systematists’ Forum (abstracts). London, UK: Flett Theatre, Natural
History Museum, 2 December.
16. [Oral presentation] Barfuss, M.H.J., R. Samuel & W. Till. 2008. Molecular phylogenetics of
Tillandsia (Bromeliaceae) and related genera. In: Monocots IV: The 4th international conference on the comparative biology of the monocotyledons & the 5th international symposium on grass systematics and evolution (abstracts). Copenhagen, Denmark: University of
Copenhagen, H.C. Ørsted Institute, 11–15 August.
15. [Oral presentation] Givnish, T.J., R. Riina, B. Van Ee, P.E. Berry, K.J. Sytsma, M.H.J.
Barfuss, W. Till, R. Horres, K. Schulte & G. Zizka. 2008. Adaptive radiation and diversification in Bromeliaceae: Inferences from a new multigene phylogeny. In: Monocots IV: The
4th international conference on the comparative biology of the monocotyledons & the 5th
international symposium on grass systematics and evolution (abstracts). Copenhagen,
Denmark: University of Copenhagen, H.C. Ørsted Institute, 11–15 August.
14. [Oral presentation] Russell, A., R. Samuel, M.H.J. Barfuss, B. Rupp & M.W. Chase. 2008.
Molecular systematics of Polystachya (Orchidaceae). In: Monocots IV: The 4th international
conference on the comparative biology of the monocotyledons & the 5th international
symposium on grass systematics and evolution (abstracts). Copenhagen, Denmark: University of Copenhagen, H.C. Ørsted Institute, 11–15 August.
13. [Poster] Russell, A., B. Rupp, M. Šafran, R. Samuel, M.H.J. Barfuss, V. Klejna, H. WeissSchneeweiss, V. Besendorfer, D. Reich & M.W. Chase. 2008. Molecular systematics of
Polystachya (Orchidaceae). In: Botany 2008: Botany without borders (abstracts). Vancouver, Canada: University of British Columbia, 26–30 July.
12. [Oral presentation] Schneeweiss, G. M., M.H.J. Barfuss & M. Thiv. 2008. Evolving towards
the tops: Phylogeny and evolution of the European endemic Phyteuma (Campanulaceae).
P. 16 (O 15) in: 10th Symposium of the International Organization of Plant Biosystematists
(abstracts). Štrbské Pleso, Vysoké Tatry, Slovak Republic: 2–4 July.
11. [Oral presentation] Barfuss, M.H.J., W. Till & R. Samuel. 2008. The Backbone Problem:
Hunting for informative characters in DNA sequences of Tillandsioideae (Bromeliaceae)
for reconstructing a well-resolved phylogeny. P. 356 in: Gradstein, R. & al. (eds.), Systematics 2008, 10th Annual Meeting of the Gesellschaft für Biologische Systematik, 18th International Symposium "Biodiversity and Evolutionary Biology" of the German Botanical Society (DBG) (abstracts). Göttingen, Germany: University of Göttingen, 7–11 April.
10. [Oral presentation] Blöch, C., H. Weiss-Schneeweiss, M.H.J. Barfuss, C.A. Rebernig, J.L.
Villaseñor & T.F. Stuessy. 2008. Phylogeny of the genus Melampodium and the development of the x = 10 chromosomal line. P. 41 in: Gradstein, R. & al. (eds.), Systematics 2008,
10th Annual Meeting of the Gesellschaft für Biologische Systematik, 18th International
Symposium "Biodiversity and Evolutionary Biology" of the German Botanical Society (DBG)
(abstracts). Göttingen, Germany: University of Göttingen, 7–11 April.
243
9. [Oral presentation] Thiv, M., M.H.J. Barfuss & G. M. Schneeweiss. 2008. Evolving to the
peaks: Phylogeny and habitat evolution in the European endemic Phyteuma (Campanulaceae). P. 141 in: Gradstein, R. & al. (eds.), Systematics 2008, 10th Annual Meeting of the
Gesellschaft für Biologische Systematik, 18th International Symposium "Biodiversity and
Evolutionary Biology" of the German Botanical Society (DBG) (abstracts). Göttingen, Germany: University of Göttingen, 7–11 April.
8. [Poster] Russell, A., B. Rupp, M. Šafran, R. Samuel, M.H.J. Barfuss, H. Weiss-Schneeweiss,
V. Besendorfer, D. Reich & M.W. Chase. 2007. Molecular systematics of Polystachya (Orchidaceae). In: Orchid evolutionary biology and conservation: From Linnaeus to the 21st
century (abstracts). Richmond, UK: Royal Botanic Gardens, Kew, 31 October–2 November.
7. [Oral presentation] Blöch, C., H. Weiss-Schneeweiss, M.H.J. Barfuss, T.F. Stuessy, C.A.
Rebernig & J.L. Vil-laseñor. 2007. Molecular phylogeny and chromosomal changes in
Melampodium. P. 45 (96) in: Systematics 2007: The 6th Biennial Conference of the Systematics Association (abstracts). UK, Edinburgh, Royal Botanic Garden Edinburgh: 28–31 August.
6. [Oral presentation] Till, W. & M.H.J. Barfuss. 2006. Progress towards a new classification of
Tillandsioideae: Mapping promising morphological characters on a phylogenetic tree. In:
Bromeliads on the Border: 17th World Bromeliad Conference (BSI WBC 2006) (abstracts).
San Diego, California, USA: The Bromeliad Society International, The San Diego Bromeliad
Society, Town and Country Resort & Convention Center, 6–11 July.
5. [?] Blöch, C., M.H.J. Barfuss, T.F. Stuessy, C.A. Rebernig, H. Weiss-Schneeweiss & J.L. Villaseñor. 2006. Molecular phylogeny and chromosome numbers in Melampodium (Asteraceae, Heliantheae). P. 92 in: Systematics and Evolution of the Compositae: The International Compositae Alliance (TICA) Symposium (abstracts). Barcelona, Spain: 3–10 July.
4. [Oral presentation] Barfuss, M.H.J., W. Till & R. Samuel. 2005. Phylogenetics of subfamily
Tillandsioideae (Bromeliaceae): A comparison of plastid and nuclear DNA sequence with
morphological data. P. 150 (9.7.3.) in: XVII International Botanical Congress (XVII IBC 2005)
(abstracts). Vienna, Austria: Austria Center Vienna, 17–23 July.
3. [?] Kathriarachchi, H.S., R. Samuel, P. Hoffmann, M.H.J. Barfuss & M.W. Chase. 2003. Molecular phylogenetics of Phyllanthaceae based on plastid and nuclear sequence data. P. 26
in: Systematics 2003, 4th Biennial Conference of the Systematics Association (abstracts).
Dublin, Ireland: Trinity College, 18–22 August.
2. [Oral presentation] Barfuss, M.H.J., R. Samuel & W. Till. 2003. Phylogenetic relationships in
subfamily Tillandsioideae (Bromeliaceae) based on evidence from plastid trnL intron, trnLtrnF intergenic spacer, atpB-rbcL intergenic spacer, rps16 intron, partial 5’ and 3’ trnK intron, matK, and rbcL sequences. P. 4 in: Monocots III, The 3rd international conference on
the comparative biology of the monocotyledons & the 4th international symposium on
grass systematics and evolution (abstracts). Ontario, California, USA: Ontario Convention
Centre, 31 March–4 April.
1. [Oral presentation] Barfuss, M.H.J., R. Samuel & W. Till. 2001. Generic delimitations and
molecular phylogeny in subfamily Tillandsioideae (Bromeliaceae) based on cpDNA sequences of atpB-rbcL intergenic spacer, trnL (UAA) intron and trnL-trnF (GAA) spacer,
rps16 intron, matK and rbcL gene. P. 47 in: Systematics 2001, 3rd biennial conference of
the Systematics Association (abstracts). London, UK: Imperial College, 3–7 September.
244