investigating evolution of plant development in …
TRANSCRIPT
The Pennsylvania State University
The Graduate School
Department of Biology
INVESTIGATING EVOLUTION OF PLANT DEVELOPMENT
IN BASAL ANGIOSPERMS
A Dissertation in
Biology
by
Barbara Joanne Bliss
2008 Barbara Joanne Bliss
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
December 2008
The dissertation of Barbara Bliss was reviewed and approved* by the following:
Hong Ma
Professor of Biology
Dissertation Co-Advisor
Co-chair of Committee
Claude dePamphilis
Professor of Biology
Dissertation Co-Advisor
Co-chair of Committee
Paula McSteen
Assistant Professor
Siela Maximova
Research Associate
Doug Cavener
Professor of Biology
Head of the Department of Biology
*Signatures are on file in the Graduate School
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ABSTRACT
Understanding the evolution of modern plants requires integrating findings from
several disciplines, including plant physiology and development, molecular genetics, and
genomics. Observations from model and non-model plants are brought together in a
phylogenetic framework to derive hypotheses about how plant development evolved to
generate the abundant diversity we see today. Testing those hypotheses requires a plant
model system with the appropriate phylogenetic perspective: that of a basal lineage. The
greatest diversity of plants today is among the angiosperms (flowering plants), a lineage
which arose only about 160 million years ago. The most successful of these are the
monocot and core eudicot flowering plant lineages, from which current plant model
experimental systems are derived. For questions about the evolution of angiosperm
development, a plant model from among the basal lineages is required.
In addition to phylogenetic perspective, model systems possess features and
degrees of availability, representation, and utility not found in other members of the taxa
to which they belong. For all organisms, culturing requirements are central determinants
of utility, but for studying the evolution of plant development, amenability to studies
employing methods of genomics, genetics, molecular and developmental biology are also
required. This dissertation describes the search for and selection of a proposed basal
angiosperm experimental model, Aristolochia fimbriata, along with the development of
initial technologies required for testing hypotheses about the evolution of plant
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development. Culturing, hand pollination, genetic transformation, and in vitro
micropropagation and regeneration methods are described herein.
Genes involved in flower form and architecture have been particularly important
in the evolution of angiosperm diversity. The TCP gene family, so named for its founding
members (TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTOR)
has been shown to play important roles in evolution of form in both monocots and
eudicots. Prior functional and phylogenetic analyses of this gene family revealed clades
of TCP genes with two different kinds of gene function. Since then, additional sequence
data from basal lineages and new studies providing insight into TCP gene function have
become available. Together, these warrant an updated phylogeny and review of this
important gene family. Preliminary phylogenetic analyses of the TCP gene family is
described as a foundation for conducting future expression and functional analyses. A.
fimbriata has floral and vegetative features that will facilitate evaluating the role of TCP
genes in evolution of angiosperm form, and advance the use of this species as a basal
angiosperm model system.
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TABLE OF CONTENTS
LIST OF FIGURES ..................................................................................................... ix
LIST OF TABLES ....................................................................................................... xii
LIST OF ABBREVIATIONS ...................................................................................... xiv
ACKNOWLEDGEMENTS ......................................................................................... xvii
Chapter 1 Introduction ................................................................................................ 1
Innovations in land plants ..................................................................................... 2
Bryophytes and tracheophytes: Controlling water and gravity ..................... 2 Spermatophytes: Efficient reproduction ........................................................ 6
Angiosperms: “Flowering” plants ................................................................. 8 Angiosperm phylogeny ......................................................................................... 11
Phylogenetic analysis .................................................................................... 11
Selection of sequence data ............................................................................. 12 Basal angiosperms ................................................................................................ 14
Tracheary elements ........................................................................................ 16 Double fertilization ........................................................................................ 17
Flower ............................................................................................................ 18 Floral developmental genetics .............................................................................. 23
Floral organ identity model ........................................................................... 23 Genes involved in floral organ identity ......................................................... 25 Broader applications ...................................................................................... 29
Basal angiosperm model ....................................................................................... 30 Transformation systems ................................................................................. 31
Regeneration systems .................................................................................... 33 Overview of dissertation ....................................................................................... 34
Chapter 2 Aristolochia fimbriata: A proposed experimental model for basal
angiosperms .......................................................................................................... 37
Abstract ................................................................................................................. 41 Introduction ........................................................................................................... 42 Results................................................................................................................... 47
Evaluation of potential models in basal angiosperm orders and families ..... 47 Aristolochiaceae candidates considered ........................................................ 50
Physical and life cycle features of Aristolochia fimbriata ............................ 52 A phylogenetic perspective of genome sizes ................................................. 55 Methods for genetics ..................................................................................... 57
Discussion ............................................................................................................. 60
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Aristolochia fimbriata has many characteristics of a valuable
experimental model ................................................................................ 60
Aristolochia fimbriata is well positioned for studies of the evolution of
development ........................................................................................... 61 Aristolochia contains highly developed biochemical pathways offering
insight into evolution of biochemical synthesis and coevolution with
insects ..................................................................................................... 64
Aristolochia can provide insight into development of woodiness ................. 66 Aristolochia can reveal features of the ancestor common to monocots
and eudicots ............................................................................................ 67
Growing genomic resources in Aristolochiaceae support further
development of model system ................................................................ 68 Conclusion ............................................................................................................ 71
Materials & Methods ............................................................................................ 72 Cultivation ..................................................................................................... 72
Genome sizing ............................................................................................... 73 Phylogenetic analysis .................................................................................... 73 Pollination experiments ................................................................................. 74
Genetic transformation .................................................................................. 75 Genomic PCR analysis .................................................................................. 76
Genetic transformation .................................................................................. 77 Supplemental Material ................................................................................... 77
Acknowledgements ....................................................................................... 78 Cultivation Supplement ........................................................................................ 82
Background .................................................................................................... 82 Perianth maturation ....................................................................................... 83 Hand pollination methods .............................................................................. 84
Pollination is prevented with pollination bags .............................................. 86 Day two autogamous pollinations are most successful ................................. 87
Self-compatibility in Aristolochia elegans .................................................... 88 Seed germination methods ............................................................................ 88
Effect of seed age on germination ................................................................. 89
Chapter 3 In vitro propagation, shoot regeneration and rooting protocols for
Aristolochia fimbriata, potential model plant for basal angiosperms ................... 95
Abstract ................................................................................................................. 97 Introduction ........................................................................................................... 99 Materials and Methods ......................................................................................... 102
Plant material ................................................................................................. 102
Micropropagation initiation and multiplication ............................................. 103 In vitro rooting ............................................................................................... 106 Shoot regeneration from whole leaf explants ................................................ 107 Shoot regeneration from petiole and stem sections ....................................... 108
Results................................................................................................................... 110
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Micropropagation and in vitro rooting .......................................................... 110 Shoot regeneration ......................................................................................... 113
Discussion ............................................................................................................. 118 Conclusions........................................................................................................... 119 Acknowledgements ............................................................................................... 120
Chapter 4 TCP gene family evolution ........................................................................ 122
Founding members: TB1, CYC, and PCF ............................................................. 123
TCP gene functions ............................................................................................... 125 Class I TCP proteins ...................................................................................... 125
Class II TCP proteins ..................................................................................... 126 Class III TCP proteins ................................................................................... 130
Evolution by changing gene interactions .............................................................. 131 Regulation by methylation .................................................................................... 132
Materials and Methods ......................................................................................... 135 Automated pipeline ....................................................................................... 135
Verification of sequences .............................................................................. 135 Results................................................................................................................... 137
Sequence collection ....................................................................................... 137
Alignments .................................................................................................... 139 Phylogenetic analysis .................................................................................... 139
Discussion ............................................................................................................. 144
Chapter 5 Retrospective and future direction for studies of evolution of
development in basal angiosperms ....................................................................... 151
Improving the Aristolochia fimbriata transformation system .............................. 153
Evolution of gene function in basal angiosperms ................................................. 159
Appendix A Whole genome comparisons reveal intergenomic transfers ................... 161
Abstract ................................................................................................................. 162 Introduction ........................................................................................................... 163 Methods ................................................................................................................ 164
Sequence selection, alignment, and phylogenetic analysis ........................... 164 Comparing distributions of frequencies of similarity scores ......................... 166 Amplification and sequencing ....................................................................... 167
Results................................................................................................................... 168
Discussion ............................................................................................................. 179 Acknowlegements ................................................................................................. 180
Appendix B Cultivation Supplement .......................................................................... 181
Seed germination experiments .............................................................................. 182 Germinating in toweling ....................................................................................... 189
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Seed storage experiments ..................................................................................... 191 Inducing flowering ................................................................................................ 194
Hand pollination ................................................................................................... 198 Recording pedigree ............................................................................................... 205
Example 1: ..................................................................................................... 206 Example 2: ..................................................................................................... 207
Possible mutants ................................................................................................... 208
Literature cited ............................................................................................................. 210
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LIST OF FIGURES
Figure 1-1: Evolution of major innovations in embryophytes. ................................... 4
Figure 1-2: Angiosperm flowers. ................................................................................ 10
Figure 1-3: A consensus phylogeny of angiosperms. ................................................. 16
Figure 1-4: Floral specializations................................................................................ 20
Figure 2-1: Angiosperm phylogeny based on Stevens (2007)
(http://www.mobot.org/MOBOT/research/APweb/welcome.html). .................... 44
Figure 2-2: Overall approach for selecting a basal angiosperm model system. .......... 49
Figure 2-3: Genome sizes in basal angiosperm families updated with Cui et al.
(2006) and Bennett and Leitch (2005), shown on logarithmic scale.. .................. 50
Figure 2-4: Diversity of flower and growth forms in Aristolochiaceae.. .................... 53
Figure 2-5: Aristolochia fimbriata ............................................................................... 54
Figure 2-6: Phylogenetic relationships among sampled Aristolochiaceae, with
Tasmannia lanceolata (Cannelleales) as outgroup. .............................................. 56
Figure 2-7: Green fluorescent protein expression in Aristolochiaceae. ...................... 60
Figure 2S-1: Aristolochia fimbriata flower. ............................................................... 84
Figure 2S-2: Hand pollination, A. fimbriata ............................................................... 85
Figure 2S-3: Aristolochia fimbriata genotype and perianth detail. ............................ 93
Figure 2S-4: Phylogram of Aristolochiaceae relationships, showing minimal
variation in branch lengths within Aristolochiaceae. ........................................... 94
Figure 3-1: Aristolochia fimbriata regeneration. ........................................................ 105
Figure 3-2: Schematic representation of micropropagation and regeneration
protocols of A. fimbriata. ...................................................................................... 111
Figure 4-1: Supertribe 777, MUSCLE alignment of amino acids. ............................. 140
Figure 4-2: Supertribe 1148, MUSCLE alignment of amino acids. ........................... 141
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Figure 4-3: Supertribe 2066, MUSCLE alignment of amino acids. ........................... 142
Figure 4-4: RaxML phylogenetic analysis of TCP domain-containing sequences
from automated pipeline ....................................................................................... 143
Figure 4-5: Neighbor joining tree and expression pattern of TCP genes in rice and
Arabidopsis from Yao et al. (2007). ..................................................................... 145
Figure 4-6: Unrooted ML tree of 126 TCP proteins from land plants from
(Navaud et al., 2007). ........................................................................................... 147
Figure 4-7: Classes 1, 2, 3 TCP sequences ................................................................. 148
Figure 4-8: Class 1 (PCF-like) TCP sequences.. ........................................................ 149
Figure 4-9: HMM logo PFAM03634. ......................................................................... 150
Figure 5-1: Evaluating Kanamycin (Kan) as selection agent.. ................................... 156
Figure 5-2: GFP expression in A. fimbriata roots. ...................................................... 158
Figure A-1: MultiPipMaker plots of organelle genome sequences aligned to
chloroplast target genome sequences. ................................................................... 169
Figure A-2: Frequency of high identity regions in alignments of real and
simulated mitochondrial to chloroplast genome sequences. ................................. 171
Figure A-3: Distribution of similarity scores for 100 bp windows from 17
pairwise alignments of mt genome sequence or simulated mt genome
sequence to cp genome sequence ......................................................................... 173
Figure A-4: Strict consensus trees from NJ analysis with PAUP* (500 bootstrap
replicates) using aligned coding sequences from cp and mt genomes. ................ 176
Figure A-5: Successful amplification of psbA (A, C), rbcL (B, D), and ycf1 (E, F),
from the Arabidopsis thaliana (A, B, E) chloroplast and (C, D, F)
mitochondrial genomes. ........................................................................................ 177
Figure A-6: Strict consensus trees from PAUP* (500 bootstrap replicates)
analysis of aligned coding sequences from cp and mt genomes. ......................... 178
Figure B-1: Seeds planted in varying conditions on 9/28/2006. ................................. 183
Figure B-2: Comparing effects of light on germination of seeds planted on ½ MS
media.. ................................................................................................................... 184
Figure B-3: Comparing seed germination on wet filter paper or medium. ................. 185
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Figure B-4: Comparing effect of light on germination of seeds planted in wet
toweling.. .............................................................................................................. 186
Figure B-5: Effects of light on germination of seeds planted in wet toweling. .......... 187
Figure B-6: Comparing effect of light on germination of seeds planted on soft
media. .................................................................................................................... 188
Figure B-7: Toweling germination method.. .............................................................. 189
Figure B-8: Short term effects of seed age on germination. ....................................... 192
Figure B-9: Longer-term effects of seed age on germination.. ................................... 193
Figure B-10: Inducing blooms .................................................................................... 195
Figure B-11: A. fimbriata produces successive axillary blooms along the shoot. ...... 196
Figure B-12: A. fimbriata in bloom.. .......................................................................... 197
Figure B-13: Pollination bag. ...................................................................................... 199
Figure B-14: Pollination bag attachment detail. ......................................................... 200
Figure B-15: Day of anthesis. ..................................................................................... 201
Figure B-16: Gynostemium ........................................................................................ 202
Figure B-17: Self-pollination in a day two flower ...................................................... 203
Figure B-18: Inferior ovary. ........................................................................................ 204
Figure B-19: Abnormal seedlings.. ............................................................................. 209
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LIST OF TABLES
Table 2-1: Homologs of genes involved in development, cell wall biosynthesis,
and response to biotic and abiotic stress revealed in preliminary sequencing
of two cDNA libraries constructed from combined A. fimbriata tissues. ............ 69
Table 2S-1: A summary of relevant basal angiosperm characteristics. ...................... 79
Table 2S-2: Cultivation features of 25 Aristolochiaceae taxa evaluated, with
findings. ................................................................................................................ 81
Table 2S-3: Evaluating self-compatibility in A. fimbriata. ........................................ 86
Table 2S-4: Percent fruit set (sample size) in A. elegans resulting from hand
pollinations of flowers on days 1-4 with autogamous, geitonogamous and
xenogamous pollen from flowers on days 1-3. ..................................................... 88
Table 2S-5: Genome sizes, vouchers, sources and accessions for sequence data
used.. ..................................................................................................................... 90
Table 3-1: Media formulations for micropropagation and shoot organogenesis. ....... 105
Table 3-2: The effect of plant growth regulators on axillary shoot proliferation at
21 days after culture initiation. ............................................................................. 112
Table 3-3: Effect of indole-3-butyric acid (IBA) on rooting of shoots multiplied
in vitro. .................................................................................................................. 113
Table 3-4: Direct organogenesis of Aristolochia fimbriata from whole leaf
explants.. ............................................................................................................... 114
Table 3-5: Shoot regeneration of petiole and stem explants. ...................................... 116
Table 3-6: Shoot regeneration, rooting and acclimation of plants regenerated
from petiole and stem explants from plants maintained in REN2 medium. ......... 117
Table 4-1: Arabidopsis thaliana (At) and Oryza sativa ssp. japonica (Os) TCP
genes from Yao et al. (2007) and Navaud et al. (2007) ........................................ 136
Table 4-2: Three supertribes of genomic sequences comprise Arabidopsis and
rice genes of interest. ............................................................................................ 137
Table 4-3: Genomic sequences containing TCP domain (PFAM domain 03634). .... 138
Table A-1: List of species and GenBank accessions for 25 organelle genome
sequences.. ............................................................................................................ 165
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Table A-2: Primers to amplify chloroplast genes from Arabidopsis cp and mt
genomes. ............................................................................................................... 167
Table A-3: Locations of chloroplast-specific sequences in pairwise alignments of
mt genome sequences with cp genome sequences. .............................................. 175
Table B-1: Pedigree record, example 1. ...................................................................... 206
Table B-2: Pedigree record, example 2. ...................................................................... 207
Table B-3: Pedigree and incidence of “tricots.” ......................................................... 208
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LIST OF ABBREVIATIONS
6BA: 6-benzoamino-purine
AAGP: Ancestral Angiosperm Genome Project (NSF DEB 0638595)
AG: AGAMOUS
AGL6: AGAMOUS-Like 6
AP1: APETAL1
bp: base pairs
CaMV: cauliflower mosaic virus
cp: chloroplast
CUC: CUP-SHAPED COTYLEDON
cup: cupuliformis
CYC: CYCLOIDEA
DICH: DICHOTOMA
DIV: DIVARICATA
EGFP: enhanced green fluorescent protein
ERF: ethylene responsive factor
EST: expressed sequence tags
FGP: Floral Genome Project (NSF DBI-0115684)
GFP: green fluorescent protein
HGT: Horizontal gene transfer
IBA: indole-3-butyric acid
Kan: kanamycin
MADS: MCM, AGAMOUS, DEFICIENS, SRFC
MAX: MORE AXILLARY GROWTH
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MI: micropropagation initiation medium
MP: micropropagation medium
MS: Murashige and Skoog (basal medium)
mt: mitochondrial
mya: million years ago
NAA: -naphthalene acetic acid
nptII: neomycin phosphotransferase
orf: open reading frame
PCF: proliferating cell factor
PCNA: proliferating cell nuclear antigen
PCR: polymerase chain reaction
petG: cytochrome b6/f complex subunit 5
PGR: plant growth regulator
pip: percent identity plot
PGR: plant growth regulator
psaA: photosystem I P700 apoprotein A1
psbA: photosystem II reaction center polypeptide D1
psbD: photosystem II reaction center protein D2
QTL: quantitative trait loci
rbcL: ribulose 1, 5–bisphosphate carboxylase/oxygenease, large subunit
RAD: RADIALIS
REN1, REN2: root elongation media one and two
RI: root initiation medium
SI: shoot induction medium
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SR: shoot regeneration medium
TB1: TEOSINTE BRANCHED1
TCP: TB1, CYC, PCF
TDZ: thidiazuron
ycf1, ycf2: large orf common in angiosperm chloroplast genomes
xvii
ACKNOWLEDGEMENTS
I would like to thank my committee, for their individual support and collective
service on my behalf. In the lab of Dr. Claude dePamphilis, I conceived of my project
and brought it to bear, a feat I certainly could have done nowhere else. The hand of Dr.
Hong Ma kept me guided firmly toward my goal; providing me with technical expertise
and supporting my interest in classical and molecular genetics. Dr. Siela Maximova has
been an unfailing inspiration, even when all else failed, and Dr. Paula McSteen has
served to moderate and tell me plainly what the outcome should look like. My work at
Penn State was supported in 2002-03 by a University Graduate Fellowship from the
graduate school of Penn State University and a Braddock Fellowship from the Eberly
College of Science. During 2003-08, the work was supported by NSF grants DBI0115684
(Floral Genome Project) and DEB 0120705 (Ancestral Angiosperm Genome Project) to
Claude dePamphilis, Teaching Assistantships from the Department of Biology, and travel
grants from the Floral Genome Project. I received another Braddock Fellowship from the
Eberly College of Science in 2008.
I deeply appreciate my colleagues and mentors. My lab mates Jill R. Duarte, Yan
Zhang, and most especially Joel McNeal shared their expertise. Jim Leebens-Mack, Kerr
Wall, Paula Ralph, and Ali Barakat provided the eagle‟s perspective, and Laura Zahn
contributed the outsider‟s viewpoint, encouragement, and friendship. My training has
also been shaped by the excellent habits of Sharon Pishak and the focus and
determination of Stefan Wanke.
xviii
I am grateful to my family and friends who recklessly urged me to embark on this
endeavor, knowing I would not rest until I had done so. They have not ceased to remind
me that despite circumstances encountered along the way, this was - and is - the right
path for me. I especially want to acknowledge the sacrifices of my two children, who
gave up so much of Mom just so that she could go to school to get a job that she liked. I
hope they, too, will not hesitate to go to school to get a job that they like, and that if they
are also parents when they do, that their children give them just as hard a time as they
have given me. They have been the wind beneath my wings; which has been, indeed, the
wind of a firestorm threatening me with certain immolation if I do not rise high, quickly
enough
Chapter 1
Introduction
Research into the evolution of plant development aims to understand the
mechanisms which have given rise to the wide variety of plants we observe today (Raff,
2000). The most speciose extant plant lineage is that of the angiosperms, with over
250,000 species. In the angiosperms, the innovations of flower, fruit, and double
fertilization supported such a rapid radiation of diversity that Darwin described it as an
“abominable mystery” (1903). Although the origins of the angiosperms have seemed
enigmatic, as the disciplines of developmental biology (from early embryology),
genetics, molecular biology, molecular evolution, and modern genomics are brought to
bear, Darwin‟s “abominable mystery” is giving way to a rudimentary understanding of
processes involved in the evolution of angiosperm development (Friedman et al., 2004).
2
Innovations in land plants
"Ontogeny (embryology or the development of the individual) is a concise and
compressed recapitulation of phylogeny (the paleontological or genealogical
series) conditioned by laws of heredity and adaptation." (Haeckel, 1909)
The evolution of development that led to the flowering plant lineage was directed
and limited by development that had occurred previously, and had proved to be
advantageous. Development in an individual flowering plant does not strictly recapitulate
the evolutionary history of angiosperms, but major changes in form and organization can
be identified in the lineage leading to land plants (embryophytes) since they last shared a
common ancestor with charophycean algae (Endress, 2006; Qiu et al., 2007). Thus, it is
necessary to study the predecessors of flowering plants in order to recognize and
construct hypotheses about what features evolved to give rise to the flowering plants.
What follows is a brief review of the major vegetative and reproductive innovations in
land plants leading to the success of the angiosperm lineage.
Bryophytes and tracheophytes: Controlling water and gravity
Early adaptive changes required for successful colonization of land arose around
480 million years ago (mya) in the Ordovician period, in an aquatic common ancestor the
embryophytes shared with Characeae (Karol et al., 2001; Judd et al., 2002; Simpson,
2006). These early adaptations included apical development and phragmoplastic cell
division, necessary for producing land plant architecture. Cellulosic cell walls and
specialized gametangia (antheridia and archegonia) with encased egg cells (cortication)
3
contributed to the ability to survive drying conditions of land, particularly for
reproductive structures (Figure 1-1).
Land plants appeared in the lower Silurian (approx 430 mya) and shared further
adaptations, including a cuticle and jacketed gametangia to further protect the gametes
and embryo from desiccation (Figure 1-1) (Qiu, 2008). The basal nonvascular
embryophytes are known as the bryophytes and include the mosses, hornworts, and
liverworts (Figure 1-1) (Nickrent et al., 2000). The genome sequence of the moss
Physcomitrella patens (Physcomitrella) (Qiu et al., 2007), reveals a loss of genes
required for success in an aquatic environment, and acquisition of new genes required for
long-term success on land (Qiu et al., 2006; Rensing et al., 2008). The Physcomitrella
genome sequence contains evidence of a single large-scale genome duplication event,
which would have supported the birth of new genes by providing the raw material for
innovation (Ohno, 1970; Howarth and Donoghue, 2005; Rensing et al., 2008). Sequence
similarity with the modern angiosperms Arabidopsis thaliana (Arabidopsis) or Oryza
sativa (Oryza) (Rensing et al., 2008) reveals acquisition or expansion of genes
functioning in desiccation and UV tolerance, in hormonal pathways coordinating
development (auxins, abscisic acid, and cytokinin signaling pathways), in control of
morphogenesis (phytohormones and light receptors), and in vesicle trafficking (adenosine
triphosphate binding cassette proteins) in the Physcomitrella genome, indicating further
adaptations to the atmosphere and gravity.
Vascular tissue (tracheids) arose, approximately ten million years after plants
colonized land, potentiating an adaptive radiation of vascular plants (tracheophytes)
including vascular non-seed plants (club mosses, whisk ferns, horsetails, ferns), and
4
Figure 1-1: Evolution of major innovations in embryophytes. Nonvascular, basal
embryophytes (blue). Tracheophytes include vascular non-seed plants (orange) and seed
plants. Gymnosperms (green) and angiosperms (pink) are monophyletic .
5
vascular seed plants (cycads, gnetophytes, ginkgos, conifers, angiosperms). These
tracheophytes attained much greater size and independence from water in the
environment than had been possible for nonvascular plants (Judd et al., 2002; Qiu et al.,
2007).The gametophyte became reduced and specialized for reproduction, eventually
entirely dependent on and retained by the sporophyte (endosporic development). The
sporophytes became the dominant generation in the tracheophytes, superseding the
dominant gametophyte generation of nonvascular plants (Figure 1-1) (Judd et al., 2002;
Simpson, 2006). Expressed sequence tags (ESTs) from the basal tracheophyte and
lycopod Selaginella moellendorffii (Selaginella) include genes for several functions
required for vascularization and adaptation to land not found in Physcomitrella, including
genes involved in lignification, cell division control, intracellular transport, responses to
sulfur starvation, dehydration, and viral infection (Weng et al., 2005).
The basalmost lineage of tracheophytes, the lycophytes (club mosses), diverged
from the lineage which later led to the euphyllophytes (whisk ferns, horsetails, ferns, and
seed plants) around 400 mya (Qiu et al., 2007). The lycophytes lack lateral leaf traces and
lateral root branches, and have microphylls instead of euphylls (Kenrick and Crane,
1997) mya (Figure 1-1). Later diverging tracheophytes, including the pteridophytes
(ferns, fern allies) and spermatophytes (seed plants) share euphylls (true leaves) as well
as a 30 kB inversion in the chloroplast genome (Raubeson and Jansen, 1992).
6
Spermatophytes: Efficient reproduction
The spermatophytes, in which the seed serves as the unit of dispersal, first
appeared near the end of the Devonian period, about 360 mya (Judd et al., 2002;
Simpson, 2006). The seed consists of an embryonic sporophyte enclosed with food stores
in a protective coating (seed integument) derived from sterile, parental, sporophyte
tissues. The seed allows the dormant sporophyte generation to disperse over long
distances and survive harsh conditions of drought and cold. Sperm, which is delivered to
eggs by water droplets in non-seed plants, is carried to eggs by pollination in
spermatophytes. The spermatophyte megasporangium typically, and ancestrally retains
just one megaspore after the abortion of three of the four haploid products of meiosis
(Friedman and Williams, 2004). The female gametophyte contained therein is enveloped
by sterile sporophytic tissue forming the integument, which has a single opening at one
end (the micropyle).
Microspores, immature male gametophytes in the form of pollen grains, develop
in microsporangia on the same plant (monoecy) or on another plant (dioecy). The seed
plant microspore germinates, giving rise to a haustorial pollen tube which grows and
extracts nutrition from the sporophytic tissue surrounding the megasporangium on its
way to deliver sperm to the ovule (siphonogamy). In addition to the shared seed
characteristic of spermatophytes, seed plants also produce wood via secondary growth of
xylem from a bifacial cambium (Figure 1-1) (Judd et al., 2002).
Seed plants comprise the most diverse vascular plant lineage, with about 270,000
extant species. The major extant spermatophytes lineages include cycads (Cycadales),
7
ginkgos (Ginkgoales), conifers (Coniferales), gnetophytes (Gnetales), and flowering
plants (angiosperms) (Figure 1-1) (Judd et al., 2002; Simpson, 2006). In the first four
seed plant lineages, megasporangia are exposed except for the coverage provided by
megasporophylls, hence they are collectively known as the “gymno-“ (naked) “-sperms”
(seeds). In the gymnosperms, pollen grains are delivered (presumably originally by wind
pollination) near the micropyle, where the microspore germinates and slowly grows
toward the egg.
Relative to the angiosperms, the four gymnosperm lineages contain greater
diversity than the angiosperms, but only a fraction of the species (Raven et al., 1999;
Judd et al., 2002; Simpson, 2006). The cycad lineage is limited to about 130 species,
which feature limited secondary xylem, dioecy, and compound leaves. They have lost the
axillary branching that appeared earlier in land plants, and have derived coralloid roots
which host nitrogen-fixing cyanobacteria. Cycads have derived a specialized pollination
system employing irregular thermogenesis in the cone, and mutualistic relationships with
its pollinators . Cycad seeds are usually colorful and fleshy, attracting animal dispersal
agents. Although the basalmost cycad has several ovules borne on leaf-like
megasporophylls, more derived cycads bear two ovules on peltate megasporophylls in
strobili.
Ginkgos form a monospecific taxon, with secondary growth and fan-shaped
leaves. Ginkgos are also dioecious, bear ovules in pairs on axillary stalks, and produce
seeds with a fleshy, smelly outer integument and hard inner integument. Both cycads and
ginkgos have giant swimming sperm, which are interpreted as an ancestral feature in seed
plants (Raven et al., 1999; Judd et al., 2002; Simpson, 2006; Roemer et al., 2008).
8
Conifers, gnetophytes and angiosperms have nonmotile sperm, thus the pollen tube must
grow farther to deliver them directly to the female gametophyte.
Conifers comprise approximately 600 living species with well-developed wood
and, often, needle-like leaves adapted for drought. Conifers release pollen from strobili
bearing microsporangia on microsporophylls (Raven et al., 1999; Judd et al., 2002;
Simpson, 2006). Seed-producing strobili have megasporophylls bearing receptive ovules,
in which one remaining haploid cell gives rise to the female gametophyte, which then
produces one or more eggs. Polyembryony is common in conifers, due either to separate
fertilization events or by subdivision of a single embryo in early development.
Gnetophytes contain three genera and about 80 species, including Ephedra,
Gnetum, and Welwitschia mirabilis. Gnetophytes share unusual features including vessels
with pore-like perforation plates, a form of double fertilization that does not produce
endosperm, both compound microsporangiate and compound macrosporangiate strobili,
unique pollen shape and markings, and opposite leaves (Friedman, 1992; Carmichael and
Friedman, 1996; Raven et al., 1999; Judd et al., 2002; Simpson, 2006). These relatively
few species in the four basal seed plant lineages contain advantageous innovations
(Figure 1-1) which can be seen as precursors or initial debuts of features later optimized
and exploited in the angiosperms.
Angiosperms: “Flowering” plants
Angiosperms arose in the seed plant lineage about 160 mya (Bell et al., 2005;
Leebens-Mack et al., 2005). Although they are known as the “flowering plants,” the
9
specific innovation that changed the condition of gymnospermy to angiospermy was the
innovation of the carpel. The carpel has a lower portion (ovary) enclosing the ovules and
an upper portion (stigma), often elongated (style), which is receptive to pollen (Figure 1-
2 A). Angiosperm pollen is produced in stamens, which are microsporophyll-like organs
bearing two thecae (anther), each containing two sporangia (Figure 1-2 A, C). The
angiosperm flower is comprised of the staminate and carpellate reproductive structures,
together with sterile tissues, including those of the perianth (Figure 1-2 A), but the sterile
tissues can be highly variable. Wind-pollinated flowers may lack a perianth entirely
(Figure 1-2 B, C, D). Lineage-specific flower and inflorescence modifications vary
widely. In particular, the grass spikelet (Poaceae) (Figure 1-2 C), and spathe/spadix
structures of Araceae (Figure 1-2 E), bear little resemblance to the typical angiosperm
flower .
Pollen grains (microspores) delivered to the stigmatic surfaces of the angiosperm
carpel germinate there, and the pollen tube grows toward the ovule to deliver two sperm
cells through the micropyle (Raven et al., 1999; Simpson, 2006). Unlike consequences of
double fertilization in the gymnosperms, double fertilization in angiosperms involves one
sperm fertilizing the egg cell to form a diploid zygote, and the other fusing with polar
nuclei to form endosperm. Angiosperm endosperm provides essential nutrition to the
embryo, replacing that role of the female gametophyte in earlier spermatophyte lineages
(Friedman and Williams, 2003; Friedman, 2008). Maturing embryos in the angiosperm
seed are enclosed in two integuments, as are also found in gnetophyte seeds. The
angiosperm seed is enclosed by additional sterile ovary and carpel tissue, forming the
fruit, which is the angiosperm dispersal unit.
10
Angiosperms consist of two major lineages, and several less speciose ones. The
eudicot lineage contain about 75% of all angiosperm species, and diverged about 125
Figure 1-2: Angiosperm flowers. A. Diagram showing the main parts of a “typical”
flower. B. Ragweed inflorescence (Ambrosia trifida); highly reduced perianth facilitates
wind pollination (Robert H. Mohlenbrock) C. Bouteloua sp. spikelet (Poaceae)
featherlike stigmas and stamens extended; two thecae visible on red anther. D. Tag alder
(Alnus serrulata, Betulaceae) inflorescences with unisexual flowers, highly reduced for
wind pollination. Male catkins on right, mature female catkins left E. Jack-in-the-pulpit
(Arisaema triphyllum, Araceae) (Robert H. Mohlenbrock)
11
mya, while the monocots contain about 22% of all angiosperm species, and diverged
about 120 mya (Magallon et al., 1999; Leebens-Mack et al., 2005). Angiosperms
coevolved with insect pollinators and animal dispersal agents, and early angiosperm
fossils show evidence of herbivory and insect pollination (Labandeira et al., 1994; Hu et
al., 2008). However, the rapid radiation of 97% of all extant angiosperm species is not
attributable to a few key innovations, but to several potentially interacting factors,
including specialized pollination and dispersal modes, life history, habit, and
geographical distribution (Davies et al., 2004; Sargent, 2004; Friedman and Barrett,
2008). The capacity of the angiosperm lineage to diversify in response to these factors
was a function of the potential for evolution of development contained in the basal taxa.
Angiosperm phylogeny
A clear and accurate understanding of the evolutionary relationships among taxa
informs comparisons of species and gene sequences, providing insight into relationships
and lineage-specific phenotypes. Such an understanding is essential for constructing
hypotheses about the evolution of development (Soltis and Soltis, 2003).
Phylogenetic analysis
A phylogenetic tree represents a hypothesis about the evolutionary relationships
among taxa, inferred from the data used, and according to the model of evolution applied
(Page and Holmes, 1998). Data used for phylogenetic analyses are derived from
12
observations of conserved features, including fossil evidence, morphological (e.g.,
embryological, vasculature, reproductive organs), palynological, karyological, and
chemical characters. Character evolution is analyzed (originally, with parsimony
methods) to make inferences about organism evolution. As technology has advanced,
more and more molecular sequence data have become available (Soltis et al., 2005).
Today, sequence data from the three plant genomes (chloroplast, mitochondrion, or
nucleus) has largely replaced the use of physical and chemical features as the basis for
phylogenetic analysis, and has brought resolution to long mettlesome questions, including
the phylogenetic position of the monocots (Qiu et al., 1999; Doyle and Endress, 2000;
Cai et al., 2006; Jansen et al., 2007; Doyle, 2008). The resulting hypothesis of organismal
evolution provides a framework for analyzing the evolution of angiosperm development
(Doyle and Endress, 2000; Soltis and Soltis, 2003).
Selection of sequence data
Sequences used for phylogenetic analysis must be conserved enough among the
taxa of interest so that the sequences can be aligned, but they must have enough
differences to be phylogenetically informative, distinguishing the taxa (Soltis and Soltis,
1998). For plants, chloroplast genome sequences are frequently used for phylogenetic
analysis due to the small size of the genome, conserved function of the genes in the
genome, and presence of most of these genes in single copy, in contrast with the
multigene families found in the diploid nuclear genome (Soltis and Soltis, 1998).
Chloroplast genes (protein-coding sequences as well as introns) vary in number of
13
nucleotides and rate of evolution, so different sequences can be chosen to provide
resolution at different taxonomic levels (Soltis and Soltis, 1998).
If the sequence data are appropriate for the level of discrimination required, and
the models of molecular evolution are appropriate for the data, the evolutionary
relationships described from the analysis of the sequence data may be expected to
accurately reconstruct organismal relationships. However, phylogenetic analysis of any
given sequence will reflect the history of that sequence. Events other than vertical
transmission will generate a topology that differs from that of the organismal history
(Wendel and Doyle, 1998). Hybridization, introgression, and lineage-sorting obscure
species-level phylogenies (Doyle and Davis, 1998; Rieseberg and Welch, 2002).
Horizontal transfer has a similar effect, across even greater phylogenetic distances
(Wendel and Doyle, 1998). Genes which have been transferred between the chloroplast
and mitochondrial genomes include the photosystem II reaction center polypeptide D1
(psbA); the large subunit of ribulose bisphosphate. carboxylase (rbcL); the cytochrome
b6/f complex subunit 5 (petG), the photosystem II reaction center protein D2 (psbD), two
conserved, large open reading frames frequently found in angiosperms (ycf1, ycf2), the
photosystem I P700 apoprotein A1 (psaA), and an approximately 500 bp open reading
frame (orf) (Appendix A).
Combining several genes for phylogenetic analysis limits the distortion any single
gene can produce, and using a taxon-rich dataset reduces the potential for long-branch
distortions of the resulting phylogeny (Leebens-Mack et al., 2005). The most current,
well-supported hypothesis of evolutionary relationships among the angiosperms is based
on analysis of 81 sequences from the chloroplast genomes of 64 taxa (Jansen et al.,
14
2007). That phylogeny (Figure 1-3), resolves ambiguities regarding the basalmost
angiosperm lineages, and provides an exceptionally robust context in which to analyze
evolution of features which historically may have been used as characters for
phylogenetic analysis.
Basal angiosperms
Findings from basal angiosperms inform and polarize the interpretation of
flowering plant features, and are necessary for inferring ancestral states and considering
evolution of development in land plants (Endress, 2001a; Soltis et al., 2002; Soltis et al.,
2005). The diverse habits, vegetative and floral forms, and specialized adaptations found
in basal angiosperms display a level of diversity not found in more recently diverged
lineages (Endress and Igersheim, 2000; Floyd and Friedman, 2001; Williams and
Friedman, 2002; Friedman and Williams, 2004; Endress, 2006). Over one hundred
macro- and microscopic morphological characters vary among the basal angiosperms and
have been evaluated in a phylogenetic context (Doyle and Endress, 2000). In the context
of well-resolved phylogenetic relationships, the diversity in these basal lineages can
provide insight into the evolution of development of key innovations associated with
angiosperm success. In particular, highly efficient tracheary elements, endosperm, and
carpels with variably attached sterile tissues (including perianth and ovary tissues) are
angiosperm innovations that became genetically fixed in later diverging lineages, after
selective advantage of these features had been proved (Endress, 2001b).
15
SolanumLycopersiconNicotianaPetunia*
Antirrhinum*Mimulus
HelianthusGerbera*
MedicagoGlycinePisumLotus*
Populus (tree)
Arabidopsis*Brassica
Gossypium
BetaMesembryanthemum*
Aquilegia*Eschscholzia*
OryzaZea*Brachypodium
Asparagus
Persea (tree)
Liriodendron (tree)
Aristolochia
Acorales
Alismatales
Petrosaviales
Pandanales
Dioscoreales
Liliales
Asparagales
Arecales
Poales
Commelinales
Zingiberales
Dipsacales
Asterales
Apiales
Aquifoliales
Garryales
Solanales
Gentianales
Lamiales
Ericales
Cornales
Caryophyllales
Dilleniales
Malvales
Brassicales
Huerteales
Sapindales
Geraniales
Myrtales
Fabales
Rosales
Fagales
Cucurbitales
Zygophyllales
Oxalidales
Malpighiales
Celastrales
Crossosomatales
Vitales
Saxifragales
Santalales
Berberidopsidales
Gunnerales
Buxales
Trochodendrales
Proteales
Sabiales
Ranunculales
Ceratophyllales
Chloranthales
Magnoliales
Laurales
Piperales
Cannellales
Austrobaileyales
Nymphaeales
Amborellales
16
Tracheary elements
Vessel elements are a unique tracheary element that have perforation plates at
each end of the cell, providing greater efficiency of solute transport, (Raven et al., 1999).
A kind of vessel element, with pore-like perforation plates, first appeared in the
gymnosperm gnetophyte lineage, but has not been found in the conifers or other
gymnosperms. Vessel elements with simple, scalariform, or reticulate perforation plates
have been reported in the basal angiosperm families Hydatellaceae, Canellaceae,
Piperaceae, and Saururaceae, but in many basal angiosperms, which are typically woody,
only tracheids have been reported (Stevens, 2008).
However, closer examination using scanning electron microscopy revealed that
vessel elements are not found in a bimodal state of presence or absence in the basal
angiosperms (Carlquist and Schneider, 2002). A series of criteria can be used to describe
the extent to which tracheary elements should be described as vessels or tracheids, based
on cell dimensions, perforations, and associations with other cells (Carlquist and
Schneider, 2002). Using these criteria, incipient vessel elements (in transition from
tracheids) have been identified in all basal angiosperm families previously reported to be
lacking them (Carlquist and Schneider, 2002; Stevens, 2008).
Figure 1-3: A consensus phylogeny of angiosperms. Adapted from Stevens (2008),
incorporating added resolution based on whole plastid genome phylogenies of Jansen et
al. (2007) and Moore et al. (2007). Species indicated are floral models. Blue shading
designates basal angiosperm lineages, beige designates basal eudicot lineages, pink
designates the rosid core eudicot lineages, yellow designates the asterid core eudicot
lineages, green designates monocot lineages.
17
Double fertilization
Too sperm cells are delivered from the gymnosperm pollen tube, but typically
only one sperm cell is functional. When double fertilization does occur, additional
embryos are produced, not sterile, nutritive tissue (Carmichael and Friedman, 1996). In
gymnosperms, the embryo obtains nutrition from the large female gametophyte which
becomes enriched to nourish the embryo regardless of whether fertilization occurs
(Friedman, 2008). Angiosperm double fertilization involves the fusion of one sperm
nucleus with the egg nucleus and one sperm nucleus with a central cell nucleus. The
associated production of endosperm in angiosperms provides them the advantage of
deferring production of tissue for embryonic nutrition until after fertilization has taken
place ff(Friedman et al., 2008).
Over 70% of extant angiosperms produce a seven-celled, eight-nucleate
Polygonum-type female gametophyte from a single megaspore, and form triploid
endosperm when a second sperm cell fuses with two haploid nuclei in the central cell (or
one diploid nucleus, if the two haploid nuclei have already fused) (Friedman et al., 2008).
Six other female gametophyte genetic constructs have been described for the
angiosperms, each of which can be found among the basal lineages, but are rare among
the core eudicots. The specific ontogeny of the female gametophyte determines the
number and meiotic descent of the central cell nuclei, affecting endosperm ploidy,
heterozygosity, maternal to paternal genome ratio, and potential for genetic conflict.
Studies in basal angiosperms reveal that the female gametophyte can be derived from
one, two, or four megaspores, and produce diploid (2N) (Nymphaeles, Austrobaileyales),
18
triploid (3N) (Amborella, Polygonum), 5N (Penea, Plumbago), 9N or 15N (Peperomia)
endosperm ff(Williams and Friedman, 2002; Friedman et al., 2003; Williams and
Friedman, 2004; Friedman, 2006; Arias and Williams, 2008).
The basal angiosperm family Hydatellaceae (Nymphaeales), a lineage with
diploid endosperm, offers a missing link to the gymnosperms (Friedman, 2008). In
Hydatella and its sister genus, Trithuria, megasporangia make a precocious investment to
seed nutrition, building up starch in the female gametophyte (perisperm) prior to
fertilization. After fertilization, nutrients cease to accumulate in the perisperm, and
accumulate, to some extent, in the endosperm. The presence of both endosperm and
perisperm nutritive tissues in the seed occurs frequently in basal angiosperms and may
represent an intermediate step from gymnosperm lineages, which lacked endosperm
(Friedman, 2008; Rudall et al., 2008). In addition, the frequent observation of double
megagametophytes in the same ovule in Trithuria and other basal angiosperms
underscores the developmental lability of basal angiosperm female gametophytes.
Organismal phylogenies can be used to interpret the evolution of female gametophyte
development in angiosperms, but still lacking are the developmental genetics to support
or test hypotheses of homology between basal and later diverging angiosperms, and
between angiosperms and gymnosperms.
Flower
Stamens and carpels in the angiosperm flower derive from the leaf-like
microsporophylls and megasporophylls found in gymnosperms, and the flower itself may
19
have originated by union of those reproductive organs to form modules, as is suggested
by the close association of the reproductive organs in Gnetales (Endress, 2001b). Stamens
in basal angiosperms may have thecae extending the length of the anther and open
longitudinally (laminar stamens), as in Amborellaceae, Nymphaeaceae, Magnoliaceae
(Judd et al., 2002). In other basal angiosperms, stamens have distinct anthers and a broad,
laminar filament, with valvate anther dehiscence (e.g., Laurales). Overall, basal
angiosperm stamens do not display pollination mode-specific canalizations of
development observed in later diverging lineages. For example, in Poaceae, the anthers
dangle from the filament to disperse pollen into the wind (Figure 1-2 C), and in the core
eudicot Kalmia (Ericaceae), stamens are released from a retracted position when the
pollinator lands, slapping the pollinator and releasing pollen onto its body (Figure 1-4
A).
Carpels in basal angiosperms are generally superior and apocarpous, often
assembled on an elongated axis, as in Annonaceae and Magnoliaceae (Figure 1-4 B).
However, carpels are half-inferior to inferior in Aristolochiaceae (Figure 1-4 C), and
fully inferior in Monimiaceae (Figure 1-4 D). In Aristolochia, the inferior ovary is
associated with a specialized structure, the gynostemium, which is composed of the
thecae fused to the outer tissues of the style. Development of reproductive organs in
Aristolochia and in less enigmatic genera in the family (e.g., Saruma) is well
documented, and suggests transitions in form that may have led to the gynostemium
(Gonzalez and Stevenson, 2000b, a; Simpson, 2006). The placentation in basal
angiosperm carpels varies, thus the ovaries mature to produce a variety of fruits,
including berries (Nymphaeaceae),
20
Figure 1-4: Floral specializations. A. Core eudicot adaptation: stamens release (black
arrows) from retracted position to hit pollinator when disturbed (Kalmia latifolia;
Ericaceae: Joel McNeal) B. Basal angiosperm flower with superior, apocarpous carpels
on elongated axis, laminar stamens (Magnolia tripetala, Magnoliaceae: Kurt Stueber) C.
Basal angiosperm flower with inferior ovary (Aristolochia arborea, Aristolochiaceae: T.
Voelker) D. Basal angiosperm with hypanthium (black arrow) (male Tambourissa
21
follicles (Magnoliaceae), capsules (Aristolochiaceae), samaras (Hernandiaceae), drupes
(Lauraceae), and aggregate fruits (Annonaceae).
In Tambourissa (Monimiaceae; Laurales), carpels and stamens are attached to a
thickened hypanthium (Figure 1-4 D) (Endress and Lorence, 1983). The evolutionary
trend from monoecy to dioecy has occurred repeatedly in this genus (Endress, 1992). The
female Tambourissa flower tends toward forming a cup- or urn-like structure, rimmed by
very small sterile organs (tepals), so that the reproductive organs are enclosed deep
within. The hypanthium of the male Tambourissa flower tears as it opens to expose its
many stamens. In one species of Tambourissa, a hyperstigma on the female flower blocks
access to the carpels entirely, so that pollen germinates on the hyperstigma, and feeding
pollinators (beetles) cannot devour the styles of the flower (Endress and Lorence, 1983).
Sepals and petals also first appear in the basal angiosperms, although organ
identity and merosity are often indeterminate or variable (Endress, 1994). There may
appear to be only one type of perianth organ, occurring in several spirally arranged
whorls (tepals), as in Amborella, Nymphaeaceae, Austrobaileya, and several Magnoliales
(e.g., Calycanthus, Magnolia) (Figure 1-4 B) (Endress, 2001b). Perianth organs may
grade into laminar stamens, and include a whorl or two of organs displaying features of
both tepals and stamens (intergrading floral organs). Perianth whorls that do display
regular merosity are typically trimerous (e.g., Cabomba, Annonaceae, Lauraceae, and
Asaroideae), as is found in the monocots.
elliptica, Monimiaceae: B. Navez)
22
Basal angiosperm perianth parts, and sometimes carpels, are colored for pollinator
attraction, with pigments limited to reddish browns, white, yellow, cream, and green
(Figure 1-4 B-D). Although the perianth organs in basal angiosperms usually lack fusion
and are radially symmetric, Aristolochia (Aristolochiaceae, Piperales) has a
monosymmetric unipartite perianth (Figure 1-4 C). The monosymmetric perianth of
Aristolochia is associated with extensive speciation (Wanke et al., 2006b), as is floral
monosymmetry in later diverging lineages (Sargent, 2004).
Some basal angiosperms lack perianth structures altogether, a feature which has
evolved repeatedly in Hydatellaceae, Chloranthaceae, Piperaceae, and Eupomatiaceae
(Endress, 2001b). These families are animal-pollinated nonetheless, as they use scent and
heat to effect pollinator attraction . Many Magnoliales produce generally sweet or musky
fragrances, and ethereal oil cells are found in some basal angiosperms. Thermogenesis in
Nymphaeaceae, Annonaceae, and Magnoliaceae volatilizes fragrances produced by the
plant and encouraged pollinators to remain active or breed (Thien et al., 2000; Seymour
et al., 2003). Pollinators attracted to the basal angiosperm flower may be rewarded in a
variety of ways, with stigmatic secretions, nectar, pollen (typically produced in copious
amounts in basal angiosperms), breeding habitat, or additional sterile tissues presented
for pollinator consumption, such as the staminodes of Eupomatia (Bergstrom et al., 1991)
Dipteran pollinators attracted to fermenting, fruity, wine-like scents, and rotting meat or
dead fish odors (Austrobaileya, Illicium, Aristolochia, Asarum), are succumbing to
pollination by deceit, or may be responding to pheromones in the fragrance (Rulik et al.,
2008).
23
Floral developmental genetics
Functional studies in current flowering plant models have shaped our
understanding of floral developmental genetics and led to the discovery of broadly
homologous traits, including conservation of floral organ identity genes among core
eudicots and monocots (Pnueli et al., 1994; Samach et al., 1997; Kramer and Irish, 1999;
Ambrose et al., 2000). These homologies, and others observed in current plant models
(Figure 1-3), have suggested hypotheses about the common ancestor of monocots and
eudicots. However, current eudicot models are in the highly diverged rosid (Arabidopsis)
and asterid (e.g., Antirrhinum, Petunia) “core” eudicot lineages, which comprise over
three quarters of all extant angiosperms (Magallon et al., 1999) and display lineage-
specific, highly canalized floral forms (Endress, 1994; Endress, 2001b). Similarly, the
monocot models Zea and Oryza are in Poaceae, a highly derived, species-rich family with
a unique inflorescence and floral form. Genetic models such as the ABC model of floral
organ identity developed for these later diverging lineages may not apply to basal
angiosperms. Expression studies and genomics data from basal species provide insight
into the genetics of flower development, showing that floral developmental genetics in
basal angiosperms is far more labile than in later diverging lineages.
Floral organ identity model
The original ABC model proposed functional and evolutionary homology
between genes in Arabidopsis and in Antirrhinum based on similarity of homeotic
mutants, and, when data was available, sequence similarity (Coen and Meyerowitz, 1991;
24
Ma, 1994). Mutant analysis, overexpression studies, and in situ hybridizations suggested
a set of homeotic genes, most belonging to the type II MADS family of transcription
factors (Reichmann and Meyerowitz, 1997), were required to establish organ identity in
sepal, petal, stamen, and carpel whorls. As originally conceived, A-function alone
specified sepal identity, B- and A-functions acting together specified petal identity, B-
and C- functions acting together specified specified stamen identity, C-function alone
specified carpel identity, and A- and C-functions negatively regulated each other. Similar
findings in the highly modified floral organs of Zea and Oryza demonstrated B function
is required to establish lodicules, which are sterile organs positionally equivalent to
petals, and C function is required for stamen organ identity (Mena et al., 1996; Ambrose
et al., 2000).
In Petunia, another type II MADS family gene was discovered with the capacity
to produce ectopic ovules on perianth organs, suggesting an ovule organ identity, or D-
function (Colombo et al., 1995), but D function has not been discovered in any other
species (Theissen, 2001). Three additional members of the type II MADS family, the
SEPALLATA (SEP) genes, were found to physically interact with A,B and C whorl genes
to transform vegetative tissues to floral ones in Arabidopsis. These were designated E-
function genes (Honma and Goto, 2001). Phylogenetic analyses of plant and animal
MADS family genes have indicated the type II A-E genes, or a progenitor, were present
in the common ancestor of angiosperms and gymnosperms, and that some type II MADS
genes were even present in mosses (Nam et al., 2003).
25
Genes involved in floral organ identity
The A function genes, APETALA1 (AP1) and APETALA2 (AP2) in Arabidopsis,
are both involved in transition from an inflorescence meristem to a floral meristem, as
well as in sepal and petal floral organ identity. However, as originally conceived, A-
function, has only been clearly demonstrated in Arabidopsis. The AP1 lineage of type II
MADS-box genes has duplicated numerous times, including prior to the divergence of
the core eudicots (Becker and Theissen, 2003; Nam et al., 2003), and, in the monocots
prior to the divergence of the grass family (Preston and Kellogg, 2006). AP2 is a member
of an ethylene response factor (ERF) required for transition to a floral meristem in
Arabidopsis. The euAP2 clade of these genes provides a cadastral function of restricting
AG expression to the inner two whorls by translational repression attributable to
miRNA172 (Chen, 2004; Kim et al., 2006). This cadastral function is not performed by
identified A-function genes in Antirrhinum. Orthologues of both AP1 and AP2 genes
have been found in basal angiosperms, but they are expressed broadly across the sterile
and reproductive whorls in a manner inconsistent with organ identity genes (Litt and
Irish, 2003; Kim et al., 2004; Kim et al., 2005a; Kim et al., 2005b; Shan et al., 2007).
Furthermore, floral organ identity can be difficult to interpret in basal angiosperms. In
Amborella, the sister to all other angiosperms, bracts grade to tepals, then to sporophylls,
which are structurally homologous in the unisexual flowers (Buzgo et al., 2004b). There
have been many duplications in the AP1 and AP2 lineages, and the ancestral role of A-
function genes remains difficult to determine (Becker and Theissen, 2003; Nam et al.,
2003).
26
Expression of the B function (petal identity) requires members of a different type
II MADS-box gene lineage. These are APETALA3 (AP3) and PISTILLATA (PI) in
Arabidopsis; DEFICIENS (DEF) and GLOBULOSA (GLO) in Antirrhinum. These B
function genes arose after a duplication event occurring after the divergence of
angiosperms from gymnosperms (Kramer and Irish, 2000; Kim et al., 2004; Zahn et al.,
2005b). Additional duplication events, particularly just prior to the divergence of the
eudicots and another prior to the divergence of the core eudicots, have provided
opportunities for neofunctionalization and subfunctionalization (Force et al., 1999). The
expression of B-function paralogs in core eudicots suggest the DEFICIENS (DEF) and
GLOBULOSA (GLO) lineage has acquired diverse functions in roots, vasculature, leaves,
and young seedlings (Zahn et al., 2005b). Expression of a GLO ortholog in the basal
eudicot Eschscholzia californica, is consistent with ancestral function of DEF/GLO in
petal and stamen organ identity (Zahn et al., 2005b), but in the basal eudicot Sanguinaria
and the monocot Sagittaria, expression has been reported to be more broad, sometimes
expanding into sepals and carpels (Kramer and Irish, 2000). Expression patterns of B-
function homologs are more broad in many basal angiosperms, including Amborella,
giving rise to the “fading borders” model of floral organ identity (Buzgo et al., 2004b;
Kim et al., 2005b; Chanderbali et al., 2006).
The Petunia ovule identity (D function) genes are a subset of the C function
subfamily, which includes AGAMOUS (AG) and SEEDSTICK (STK, formerly AGL11)
from Arabidopsis (Zahn et al., 2006). The C (AG) and D (AGL11) clades are derived
from a duplication in the type II MADS box lineage prior to the angiosperm radiation
(Kramer et al., 2004), which would have allowed conservation of ancestral C function
27
while allowing neofunctionalization within the AGL11 lineage. The AGL11 lineage
includes ovule-specific genes expressed in basal eudicots and basal angiosperms and
would have afforded the ovule greater independence from the megasporophyte during
development (Zahn et al., 2006). The ancestral role of AG lineage genes, which are
expressed in male and female gymnosperm cones, was likely carpel and stamen identity
(Rutledge et al., 1998; Becker et al., 2003; Zhang et al., 2004). Although C-function
genes are usually expressed only in the reproductive tissues of basal angiosperms, the
presence of C-function genes in Illicium and Persea suggest the tepals of these taxa may
originate from staminate primordia (Kim et al., 2005b; Chanderbali et al., 2006).
The effects of duplication are also evident in the E function (SEPALLATA) genes,
which were formerly named AGL2, -3, -4, and -9, and which are not readily detected due
to redundancy . Phylogenetic analysis suggests a duplication prior to the divergence of
extant gymnosperms and angiosperms gave rise to the SEPALLATA (SEP) genes, which
have been found in basal angiosperms and basal eudicots, and are most closely related to
the AGL6 lineage, which has been recovered in gymnosperms (Zahn et al., 2005a).
Phylogenetic analysis reveal several duplications within the angiosperm SEP lineage,
with two copies of SEP genes recovered in basal angiosperms, four in the rosids, and up
to six in the asterids (Zahn et al., 2005a; Chanderbali et al., 2006). SEP expression and
function in plant tissues have evolved repeatedly, including roles in fruit maturation and
plant architecture, as well as floral development (Malcomber and Kellogg, 2005). SEP
genes appear to have conserved expression in the floral meristem and ovules, although
the conserved floral organ identity function has sometimes been lost, perhaps due to
overlapping function with other genes. SEP genes display lineage-specific differences in
28
sequence evolution, expression, and function, particularly within the monocots
(Malcomber and Kellogg, 2004), demonstrating the opportunities for neo- and
subfunctionalization provided by relaxed constraint on paralogs (Force et al., 1999;
Lynch and Conery, 2000).
Rooting the developmental genetics of the floral organ identity model with
information from basal angiosperms has revealed elements of the floral organ identity
model which are not more broadly applicable. Unlike the four whorled flower of core
eudicots, floral form varies widely in basal angiosperms, and is often characterized by
gradual shifts from one type of floral organ to the next (Endress, 2001b). Gene expression
studies in basal angiosperms have revealed broad patterns of conserved B and C function
across the floral meristem, with “fading borders” of influence at the outer and inner edges
of their area of expression (Buzgo et al., 2004a; Kim et al., 2004; Kim et al., 2005a). The
core eudicot ABC regulatory pattern appears to be a relatively recent innovation,
accompanying canalization of floral form and arising subsequent to duplications of
ancestral floral organ identity genes (Kramer and Irish, 2000; Kramer and Zimmer,
2006). An earlier model of floral organ identity (“B, C model”), in which only petals and
carpels are specified, might better apply to basal angiosperms (Schwarz-Sommer et al.,
1990).
29
Broader applications
"The rapid development as far as we can judge of all the higher plants within
recent geological times is an abominable mystery." (Darwin, 1903)
Beyond floral organ identity, existing studies in model systems have generated
many hypotheses about ancestral features of flowering plants and the developmental
changes that led to early-diverging lineages, as well as to the more canalized eudicots and
monocots (Endress, 2001a; Floyd and Friedman, 2001; Soltis et al., 2002; Soltis et al.,
2005). Several genome-scale duplications within the angiosperms have provided the raw
material for conservative experimentation with new gene functions (Cui et al., 2006). The
history of those genes and their roles in evolution of development will provide – at least
in part - the solution to Darwin‟s abominable mystery.
Floral symmetry, for example, is ancestrally radial, but important lineages in both
the monocots and core eudicots display monosymmetry. The first gene to be associated
with floral symmetry was CYCLOIDEA (CYC), discovered in the asterid, Antirrhinum
(Lamiales) (Luo et al., 1996). Duplication and evolution of CYC genes is implicated in
floral form evolution in a later diverging asterid lineage, Dipsacales , and is also required
for monosymmetry in the rosids Lotus, Pisum, and Lupinus (Fabales) (Ree et al., 2004;
Howarth and Donoghue, 2005; Feng et al., 2006; Wang et al., 2008). CYC genes have
also been implicated in the bisymmetric flowers of the basal eudicots (Papaveraceae;
Ranunculales) (Damerval et al., 2007; Damerval and Nadot, 2007). The extent to which
CYC genes play a role in the monosymmetry of the basal angiosperm, Aristolochia
fimbriata, has yet to be determined.
30
Evolution of development in basal angiosperms can be studied by comparative
studies of gene function between systems, as well as by studies of gene family evolution.
Functional analyses employ classical and molecular genetics, including mutant analysis,
complementation experiments, and over- and under-expression studies to derive an
understanding of gene function. Reverse genetics, employing transformation methods, are
effective ways to test hypotheses about gene function. In order to test hypotheses about
the roles of B and C function genes in basal angiosperms, or hypotheses about the role of
CYC genes in basal angiosperms, it will be necessary to have a basal angiosperm
experimental model system.
Basal angiosperm model
The purposes of a model system are, primarily, to represent the taxa to which it
belongs, to provide an easier to use system in which to conduct experimentation, and to
facilitate inductive reasoning so conclusions drawn from the model system can be applied
more generally (Bolker, 1995). To support those purposes, a model system must be in a
phylogenetically informative position, ideally derived from phylogenetically nested taxa
so comparisons can be made to closely related species (Baum et al., 2002). To be useful,
a “good” model system should have features that support its current and continued use. In
biology, this means the model organism must be easily obtained, demanding of few
resources, fast growing, productive of ample tissues as needed, and amenable to widely-
used technologies.
31
The highly successful plant model Arabidopsis thaliana meets all of these criteria
for the core eudicots, particularly the highly successful rosids (Arabidopsis, 2000).
Arabidopsis has structures broadly representative of closely related species, and can be
readily compared with Brassica, a closely related genus in the economically important
family, Brassicaceae. Arabidopsis is readily obtained, has a short generation time, easy to
follow and well-developed protocols for commonly used technologies, and produces
ample tissue and seed in a small amount of space. As a result, Arabidopsis can be used in
forward genetics, in which mutants are analysed to gain information about genetic
processes, and it can be exceptionally easily transformed and used in reverse genetics, for
functional analyses. It also has a very small genome, which has facilitated genome
mapping, sequencing, and studies of gene function (Arabidopsis, 2000). The large
community of researchers using Arabidopsis has contributed crucial value-added
resources (e.g., libraries, genome sequence and expression databases, detailed
developmental studies, mutant collections, seed banks), supporting shared problem-
solving and leading to rapid advances in understanding. A careful review of basal
angiosperms was conducted to identify the best possible candidate for model system
development, keeping the Arabidopsis thaliana model system in mind as the “gold
standard” to be met.
Transformation systems
A genetic transformation system is essential for generating transgenic plants in
which hypotheses about the evolution of development in basal angiosperms can be tested.
32
Agrobacterium-mediated methods of transformation use the machinery found in the soil
bacterium, Agrobacterium, to transfer DNA to plant cells and integrate it into the plant
genome (Gelvin, 2000)f. In the commonly used binary vector method, the tumor-
inducing (Ti) plasmid in the Agrobacterium is disarmed by removing the section of
endogenous DNA that is transferred (T-DNA) to the plant cell to induce tumor formation.
The endogenous virulence (vir) genes, which are essential for effecting the transfer and
susbsequent integration of the T-DNA, remain on the disarmed plasmid. The genetically
modified Agrobacterium are induced to take up a binary plasmid containing origins of
replication for E. coli and Agrobacterium, the endogenous right and left border sequences
from the Ti plasmid flanking a T-DNA, as well as a selectable marker and multiple
cloning sites. The T-DNA typically includes at least one marker for selection in plant
cells, a selectable reporter gene, the exogenous gene of interest to be evaluated in the
plant system, the appropriate promoter sequences, and other sequences to improve
transformation (e.g., matrix attachment regions) (Maximova et al., 2003).
When plant tissues are infected with A. tumefaciens, the vir genes are activated to
transfer the TDNA in the genetically modified plasmid to the plant chromosome (Gelvin,
2000; Gelvin, 2003). After a period of co-cultivation with A. tumefaciens, the plant
material is subjected to chemical selection to eliminate non-transformed cells, and
counter-selection to kill the Agrobacterium. Aminoglycoside antibiotics are toxic to plant
cells to some extent, so the neomycin phosphotransferase (NPTII) selective marker gene
can be constitutively expressed in transformed cells to confer resistance to
aminoglycoside antibiotics (Norelli and Aldwinckle, 1993; Petri et al., 2005). The
transformed tissue is cleared of A. tumefaciens with an antibiotic that is effective against
33
the Gram-negative cells.of Agrobacterium but is not toxic to plant cells. The transformed
plant cells are regenerated under ongoing conditions of selection to prevent regrowth of
bacteria or non-transformed cells.
Regeneration systems
Transformation of Arabidopsis was originally implemented in vitro (An et al.,
1986). In planta floral dip and spray methods were developed later (Clough and Bent,
1998; Chung et al., 2000). Other plant species still rely on tissue culture-based
transformation systems (Ishida et al., 1996; Negrotto et al., 2000), which permit greater
control of variables which influence plant growth, including light, water, temperature,
nutrients, pH, container, carbohydrate source, and condition of source material
(Mohamed et al., 1992; Razdan, 2003; Vega et al., 2008). Plant tissues or callus in culture
can often be induced to regenerate in response to treatment with plant growth regulators
(PGRs).
PGRs influence development both absolutely and in proportion to one another
(Razdan, 2003). Auxins were identified in 1926 by the ability of indole acetic acid, a
naturally occurring plant hormone, to promote growth in oat coleoptiles (Went, 1926).
Cytokinins were identified in 1948 by their ability to stimulate cell division (Caplin and
Steward, 1948). Skoog and Miller (1957) showed that organ formation depended on the
balance between cytokinin and auxin. High concentrations of auxin promoted root
formation, and high concentrations of cytokinin promoted shoot formation. Theoretically,
a 1:1 ratio of cytokinin to auxin could beexpected to produce callus (Razdan, 2003).
34
Plant tissues differ in response to PGRs, which also differ in potency with respect
to their ability to induce growth or cell division. The synthetic plant growth regulator
thidiazuron (TDZ) has properties of both auxin and cytokinin and is particularly effective
at inducing shoot primordia in the first phase of shoot organogenesis (Murthy et al., 1998;
Chen and Chang, 2001; Wu et al., 2004). In comparison, somatic embryogenesis
protocols begin with a very high auxin level to induce formation of pro-embryos, which
are then removed to a more balanced ratio of PGRs for regeneration (Juretic and Jelaska,
1991). The synthetic auxin (2, 4, -D) is particularly effective for inducing somatic
embryos (Li et al., 1998; Maximova et al., 2003). The presence of antibiotic compounds
in growth media typically alters the response of plant tissue to plant growth regulators, so
regeneration protocols must be optimized for transformed cells in the presence of
selection agents (de Mayolo et al., 2003).
Overview of dissertation
There currently is a need for a basal angiosperm model system amenable to
genomics studies and experimentation using methods of developmental biology,
molecular biology and classical genetics. Towards this end, I undertook a study to select
the best available basal angiosperm with a small genome, self-compatibility, closely
related family members with which it could readily be compared, and the ability to be
transformed with a genetic marker. Fourteen species were cultivated and evaluated. I
developed hand pollination methods and demonstrated self-compatibility for Aristolochia
elegans and Aristolochia fimbriata (A. fimbriata), two species with small genome sizes. I
35
then used those methods to develop in-bred lines to serve as a source of tissue and seed
for the NSF supported (DEB 0638595) Ancestral Angiosperm Genome Project (AAGP).
I developed and implemented standardized tissue collection methods for A. fimbriata
greenhouse and seedling tissues from which AAGP generated expressed sequence tags
(ESTs). I used a disarmed strain of Agrobacterium tumefaciens to transform Saruma
henryi and two genotypes of A. fimbriata with a plasmid conferring constitutive
expression of an NPTII antibiotic resistance gene for selection of transformed tissues, and
an enhanced green fluorescent protein (EGFP) marker gene for identifying transformed
cells. Chapter 2 includes the manuscript covering that study, including the survey of basal
angiosperms and experimental results that led to the selection of A. fimbriata, as well as
hand pollination methods and preliminary EST sequencing results.
In vitro micropropagation and shoot organogenesis methods were developed to
support the transformation protocol, as well as to provide in vitro tissue for biochemical
analysis of the secondary metabolites produced by A. fimbriata. In addition to
micropropagation methods, I developed a direct shoot organogenesis method using small
pieces of petiole explants, on which shoot primordia were induced in 14-21 days and
regenerated within three months. Chapter 3 includes the manuscript of a paper describing
the in vitro propagation and shoot organogenesis methods for A. fimbriata.
Chapter 4 includes preliminary findings from a study of TCP gene family
evolution using the available sequences from fully sequenced angiosperm genomes, as
well as ESTs from basal angiosperms. This study will be incorporated into a manuscript
after further analysis is complete. Chapter 5 reports the conclusions of my work and
discusses what should be done next to advance the development of the A. fimbriata
36
model system, including recommendations for early experiments to conduct in a basal
angiosperm experimental model. Two appendices follow: Appendix A reviews my work
on organelle genome evolution, documenting horizontal transfers from angiosperm
chloroplasts to mitochondria. Appendix B is a cultivation supplement, presenting results
of seed germination data from several experiments, illustrations of hand pollination
methods and descriptions of cultivation methods in greater detail than that provided with
the manuscript included in Chapter 2, instructions for using the seed pedigree system I
devised, and documentation of two mutant phenotypes observed during the AAGP
seedling tissue collection efforts.
Chapter 2
Aristolochia fimbriata: A proposed experimental model for basal angiosperms
This chapter contains a manuscript of an article that will be submitted for
publication by Barbara Joanne Bliss, Stefan Wanke, Abdelali Barakat, Patrick Kerr Wall,
Lena Landherr Sheaffer, Yi Hu, Hong Ma, Christoph Neinhuis, Jim Leebens-Mack, K
Arumuganathan, Sandra Clifton, Siela N. Maximova, Claude W. dePamphilis* to Plant
Physiology.
This work resulted from collaboration among the labs of Drs. Claude
dePamphilis, Hong Ma, and Siela Maximova. Dr. Stefan Wanke produced the phylogeny
of the taxa sampled and contributed to polymerase chain reaction (PCR) methods
development to verify the green fluorescent protein (GFP) marker gene. Dr. Abdelali
Barakat contributed miRNA genomics resources for A. fimbriata. Lena Landherr
constructed A. fimbriata cDNA libraries, conducted transformation experiments and
optimized PCR methods to verify transgenic tissues. Dr. Jim Leebens-Mack and Sandra
Clifton sequenced libraries and managed EST data. Dr. Kerr Wall conducted
bioinformatic analyses of EST data and resulting unigenes. Yi Hu assisted with
maintenance of in vitro and greenhouse cultivated plants, tissue collection, and RNA
extraction. Dr. K Arumuganathan conducted genome size analysis. All other results were
generated by Barbara Bliss.
38
Running head: Aristolochia fimbriata as a basal angiosperm model
Corresponding author: Claude W. dePamphilis, Department of Biology, Institute of
Molecular Evolutionary Genetics, and the Huck Institutes of the Life Sciences, 201 Life
Sciences Building, Pennsylvania State University, University Park, PA 16802, US (814)
863-6412 [email protected]
Journal research area: Genetics, Genomics, and Molecular Evolution
39
Title: Aristolochia fimbriata: A proposed experimental model for basal angiosperms
Author full names: Barbara J. Bliss, Stefan Wanke, Abdelali Barakat, P. Kerr Wall, Lena
Landherr, Yi Hu, Hong Ma, Christoph Neinhuis, Jim Leebens-Mack, K Arumuganathan,
Sandra Clifton, Siela N. Maximova, Claude W. dePamphilis*
Institution addresses: Department of Biology, Institute of Molecular Evolutionary
Genetics, and the Huck Institutes of the Life Sciences, 201 Life Sciences Building,
Pennsylvania State University, University Park, PA 16802, USA (B.J.B., P.K.W., L.L.S.,
Y.H., H.M., C.W.D.); Technische Universität Dresden, Institut für Botanik, Plant
Phylogenetics and Phylogenomics Group, D-01062 Dresden, Germany (S.W., C.N.);
Benaroya Research Institute at Virginia Mason, Flow Cytometry and Imaging Core
Laboratory, 1201 Ninth Avenue, Seattle, WA 98101, USA (K.A.); Department of Plant
Sciences, University of Georgia, Athens, GA 30602 (J. L-M.); Washington University
School of Medicine, 4444 Forest Park Boulevard, St. Louis, MO 63108, USA (S.C.);
Department of Horticulture, 421 Life Sciences Building, Pennsylvania State University,
University Park, PA 16802, USA (S.N.M.)
40
Footnotes:
Financial sources: This work was supported in part by NSF Plant Genome Research
Program, awards DBI-0115684 (The Floral Genome Project) and DEB 0638595 (The
Ancestral Angiosperm Genome Project), by the Department of Biology and Huck
Institute of Life Sciences of the Pennsylvania State University, and by a postdoctoral
grant to SW from the German Academic Exchange Service (DAAD).
Corresponding author; email Claude W. dePamphilis, [email protected]
41
Abstract
“Basal angiosperms” are still lacking a model system, which is crucial for
understanding flowering plant evolution via comparative approaches. An evolutionary
perspective is essential for designing genetic, developmental, and comparative genomic
studies of angiosperms. Using a broad approach among basal angiosperm families, we
first reviewed general characteristics critical for model systems. These included
availability to a broad scientific community, growth habit, and suitability to represent
basal angiosperms displaying a wide spectrum of phenotypic diversity. Most basal
angiosperms, predominantly woody or aquatic, were excluded using these criteria.
Aristolochiaceae were investigated further for ease of culture, life cycle, genome size,
and chromosome number. We demonstrated self-compatibility for Aristolochia elegans
and A. fimbriata, and transformation with a GFP reporting construct for Saruma henryi
and A. fimbriata. Consequently, we recommend Aristolochia fimbriata as a basal
angiosperm model system. Native to Brazil, this rapidly growing perennial is widely
cultivated with a seed-to-seed life cycle of just 3 months. Tissue culture regeneration
methods are available for this species to support a high-throughput transformation
system. Emerging genetic and genomic tools will aid the investigation of floral biology,
developmental genetics, biochemical pathways important in plant-insect interactions and
human health, and various other features present in early angiosperms.
42
Introduction
Our present understanding of genetics, genomics, development, evolution, and
physiology of living organisms is largely based on work done in model systems. Model
systems provide more favorable conditions for observing phenomena and testing
hypotheses than other systems afford. Models support inductive reasoning, in which one
builds on the understanding of living organisms in general, based on observations made
in a specific model organism (Bolker, 1995). Model organisms also provide a focus for
researchers to work on a common system, resulting in collaborative and complementary
efforts which can yield rapid progress and development of further resources. Models are
crucial for understanding basic biological processes.
Models should have several key attributes. At the very least, model organisms
should be good representatives of the taxon or process they are chosen to represent
(Kellogg and Shaffer, 1993; Bolker, 1995) and they must be accessible (Kellogg and
Shaffer, 1993) so that a broad community of scientists can utilize and develop them.
Models used for developmental and genetic studies must also offer rapid development,
short generation time, be amenable to large scale cultivation, and provide ample tissue for
experimentation (Bolker, 1995). Models should also support forward and reverse
genetics, as is required for hypothesis testing (Soltis et al., 2002), and have a small
genome size, to facilitate molecular genetics and genomics work as well as genome
sequencing (Pryer et al., 2002). Finally, for studying the evolution of development,
models should support studies among closely related species, so that comparative studies
can be interpreted at the molecular level. To the extent that model systems exemplify the
43
taxa to which they belong, deep studies of the models, along with comparative studies
between them and closely related species, can inform and advance theory. The lack of
appropriate experimental models limits development of theoretical ones, and such is the
case in plant biology today.
Our current understanding of angiosperm evolution has been shaped by multiple
phylogenetic studies (Jansen et al., 2007; Moore et al., 2007; Stevens, 2008) which
provide the context in which the evolution of any aspect of flowering plants is studied. Of
particular interest to both basic and applied plant biology are changes leading to the
success and diversification of angiosperm lineages, beginning with the early, mostly
species poor lineages of angiosperms (Table 2S-1) previously known as the ANITA
grade, followed by the magnoliids (Figure 2-1). The magnoliids contain four major
branches and several thousand species (Table 2S-1). Among the species in this lineage
are the tree genome model, Liriodendron tulipfera (Liang et al., 2007) and the tree fruit
model, Persea americana (Awad and Young, 1979), as well as the most economically
important spice, black pepper (Piper nigrum) (Wanke et al., 2007). Taxa included in the
magnoliids exhibit many features first appearing in basal angiosperms or angiosperms in
general e.g., vessel elements, perianth monosymmetry, alkaloid chemistry, specialized
pollination systems, diverse forms of female gametophyte development (Soltis et al.,
2002; Soltis et al., 2005; Madrid and Friedman, 2008). The magnoliids are the sister clade
and therewith the closest outgroup to the highly diverged monocot and eudicot lineages
(Jansen et al., 2007).
44
Figure 2-1: Angiosperm phylogeny based on Stevens (2007)
(http://www.mobot.org/MOBOT/research/APweb/welcome.html). Important model
systems and the proposed model, Aristolochia fimbriata, are shown along the
corresponding clade. Liriodendron, Persea, Populus, and Carica are tree species. Species
that have been used as flower development models are indicated with an asterisk. .
45
The rich diversity of basal angiosperms provides glimpses into early successful
experiments in angiosperm adaptation (Endress and Igersheim, 2000; Floyd and
Friedman, 2001; Soltis et al., 2002; Williams and Friedman, 2002; Friedman and
Williams, 2004; Endress, 2006). Novel features which are otherwise constrained by
function may have evolved more than once, in parallel, such as the structural and
developmental similarities of the inflorescence of the monocot Acorus with that found in
some Piperales (Piperaceae, Saururaceae; members of Magnoliids) (Buzgo and Endress,
2000). Basal lineages retain evidence of “trials” of features that became genetically fixed
in later lineages (Endress, 2001b). For example, perianth parts are much more variable in
basal angiosperms, as well as basal eudicots and basal monocots, and only became fixed
in the core eudicots (Irish, 2006).
Current flowering plant genetic models are all derived from the highly diverged
monocot and eudicot lineages (Figure 2-1). Among them, the monocot models Zea and
Oryza occur in Poaceae, which has specialized floral organs and an inflorescence found
only in that family. Similarly, the current eudicot models are derived from the highly
diverged rosid (Arabidopsis) and asterid (e.g., Antirrhinum, tomato) “core eudicot”
lineages, each of which displays lineage-specific floral forms (Endress, 1994; Endress,
2001b). Studies in current plant models have led to the discovery of broadly homologous
traits, including conservation of floral organ identity genes (ABC/quartet models) (Coen
and Meyerowitz, 1991; Ma and dePamphilis, 2000; Theissen, 2001). These homologies,
and others observed in current plant models (Figure 2-1), have suggested hypotheses
about the common ancestor of monocots and eudicots.
46
An understanding of the evolution of floral development or any fundamental
process in flowering plants will require a basal angiosperm experimental model in which
to test these hypotheses (Baum et al., 2002; Soltis et al., 2002; Buzgo et al., 2004a; Soltis
et al., 2006; Endress, 2007). Although the current model systems represent well the
highly successful and derived lineages in which they occur, they do not represent the
overall diversity of angiosperms. Information from basal lineages is necessary both to
better describe that diversity, to polarize the changes that occurred angiosperm evolution,
and to make functional inferences about the common ancestor of early angiosperms.
Current plant models have been selected to address particular questions, but very
few are available for use both as genomic and as genetic models. Genomic resources,
which emphasize plants with small genomes, have been developed for tree wood and fruit
species, including Populus (Jansson and Douglas, 2007), Liriodendron (Liang et al.,
2007), Persea (Albert et al., 2005) and Carica (Wei and Wing, 2008). However, woody
species are too large at maturity and do not have short enough life cycles for general use
in genetics experiments. Forward genetics requires a very small organism with a rapid
life cycle, and is facilitated by the ability to self-pollinate individuals with desired
characteristics. Reverse genetics requires manipulation of DNA or RNA in a targeted
manner. Both benefit from a small genome, and transformability is essential for testing
hypotheses about gene function. Therefore, we sought to identify a basal angiosperm
species having as many important features of a model system as possible to support
widespread use as an experimental model in genetics and genomics. We present these
essential features in Aristolochia fimbriata - small size at maturity, rapid life cycling,
47
self-compatibilty, small genome size, and transformability - along with relevant findings
for other taxa evaluated in our study.
Results
Evaluation of potential models in basal angiosperm orders and families
We followed a formal selection process to identify a suitable candidate for an
experimental model organism among basal angiosperms (Figure 2-2). Many basal
angiosperms are uncommon, with limited distribution, and often occur in families with
only one genus and few species (Table 2S-1). Taxa were considered readily accessible to
a broad scientific community if they could be obtained commercially at a low price and
could be readily propagated by seed. Those of limited availability were eliminated,
including Amborellales (Amborellaceae), Austrobaileyales (Illiciaceae,
Austrobaileyaceae, Trimeniaceae), and Chloranthales (Chloranthaceae) (Figure 2-2).
Woody plants requiring a year or more to attain maturity, as well as extensive space for
cultivation, were eliminated (Fig 2). These included most Magnoliales (Myristicaceae,
Magnoliaceae, Annonaceae, Himantandraceae, Degeneriaceae, Eupomatiaceae), Laurales
(Lauraceae, Hernandiaceae, Monimiaceae, Atherospermataceae, Gomortegaceae,
Siparunaceae, Calycanthaceae), and Cannellales (Cannellaceae, Winteraceae). The forest
tree model species, Liriodendron (Magnolicaceae) and the fruit tree model Persea
(Lauraceae), were among those eliminated due to woody habit and long generation time.
48
49
Many of the families listed above would also be eliminated due to limited commercial
availability, as e.g., Trimeniaceae, Himantandraceae, and Gomortegaceae (Table 2S-1).
Similarly, many of the families elimated for limited accessibility have a woody habit
(Table 2S-1); these would have been eliminated for that character if they had been more
accessible (e.g., Amborella trichopoda, the sister of all other flowering plants). Plants
with an aquatic habit, including water lilies (Nymphaeales: Nymphaceae, Cabombaceae)
and Ceratophyllum (Ceratophyllales: Ceratophyllaceae), were similarly eliminated due to
intensive cultivation requirements (space, maintainance) (Figure 2-2). The order
Piperales (Aristolochiaceae, Hydnoraceae, Piperaceae, Saururaceae, Lactoridaceae)
contains several herbaceous taxa (Table 2S-1). Parasitic plants (Hydnoraceae) and those
with highly reduced flowers (Saururaceae, Piperaceae) do not generally represent
angiosperms, and so were eliminated (Figure 2-2). Hydnoraceae and Lactoridaceae
would also be excluded due to limited availability (Table 2S-1). Among the basal
angiosperm families, only Aristolochiaceae contains highly accessible, easily cultivated
herbaceous plants with features broadly representative of angiosperms in general.
Figure 2-2: Overall approach for selecting a basal angiosperm model system. General
criteria are indicated; for full description refer to methods. Taxa generally eliminated
after initial application of each criterion are indicated. Some taxa may have been
eliminated for more than one reason. For example, Illicium, in family Illiciaceae, is
increasingly available in cultivation, unlike most Austrobaileyales, but was eliminated
due to its woody growth habit. Many taxa in order Piperales were generally accessible
and of amenable growth habit, yet family Lactoridaceae was eliminated due to
inaccessibility and woodiness. In genus Aristolochia, subgenus Pararistolochia was
eliminated due to large genome size. Basal angiosperm family characteristics and those
of Aristolochiaceae species cultivated are described in Tables 2S-1, 2S-2.
50
Aristolochiaceae candidates considered
We surveyed Aristolochiaceae, seeking species with rapid growth, no requirement
for vernalization in the life cycle, ease of large scale cultivation, and a small genome size
Figure 2-3: Genome sizes in basal angiosperm families updated with Cui et al. (2006) and
Bennett and Leitch (2005), shown on logarithmic scale. Filled symbols indicate taxa used
in Floral Genome Project (www.floralgenome.org) or Ancestral Angiosperm Genome
Project, and for which EST resources are available. Compare basal angiosperm genome
sizes to Arabidopsis at 125 Mb (Arabidopsis, 2000) and Oryza at 389 Mb (International-
Rice-Genome-Sequencing-Project, 2005). The symbol representing Aristolochia is the
proposed model Aristolochia fimbriata. Other species of Aristolochia have smaller
genome sizes (see Figure 2-6).
51
to facilitate gene cloning and eventual genome sequencing (Figure 2-2). Members of
genus Aristolochia have some of the smallest basal angiosperm genome sizes currently
known (Figure 2-3). Therefore, we evaluated each genus in this family to identify the
best species for model system development. Subfamily Asaroideae genera (Asarum,
Saruma) (Figure 2-4A,B) require cold treatment to induce flowering, resulting in
increased culturing efforts and extended time to flower, so they were eliminated
(Figure 2-2). Thottea, here represented with T. siliquosa (Figure 2-4C), was not possible
to obtain for a detailed culture survey (Table 2S-2), as it can only be cultivated with very
high maintenance, in a narrow range of conditions. Furthermore, it grows slowly,
produces little tissue, few flowers, and few seeds. Of the Aristolochia species available
for culturing, those requiring cold treatment (A. serpentaria, A. clematitis of subg.
Isotrema, Figure 2-4D) were eliminated from consideration. Members of subgenus
Pararistolochia were not available for culturing (Figure 2-4E). Those species that did
not bloom in six months (A. californica, A. anguicida, A. macrophylla, A. tomentosa),
produced very few flowers (A. trilobata, A. passiflorafolia; Figure 2-4F, G), or formed
very large vines (A. grandiflora, A. ringens, A. labiata, A. gigantea) (Table 2S-2,
Figure 2-2) were also eliminated. The remaining candidates that met our criteria were
two smaller members of subgenus Aristolochia (A. elegans and A. fimbriata), belonging
to a group of subtropical and tropical species from South and Middle America (Neinhuis
et al., 2005; Wanke et al., 2006a).
52
Physical and life cycle features of Aristolochia fimbriata
We characterized the physical and life cycle features of Aristolochia fimbriata
(Figure 2-4H) to further assess its potential as a model system. Seeds planted in potting
53
medium in the greenhouse germinated at rates up to 100%, and flowered in as few as 62
days after planting. Cultivated as a small pot crop in the greenhouse, A. fimbriata stock
plants occupy minimal bench space (Figure 2-5A), and were not particularly susceptible
to any pest or pathogen, though commercial pesticide treatments were applied
greenhouse-wide as needed. Vines are supported by a small trellis during periods of
flowering or fruit ripening (Figure 2-5B) to prevent mechanical damage and facilitate
fruit harvest. Plants flower copiously from axillary nodes on multiple indeterminate stems
that arise from the woody root. A new flower opens every two to three days along the
stem (Figure 2-5C) facilitating collection of staged tissues. Fruits from open-air
pollinations on one-year-old plants averaged over one hundred seeds per capsule.
Although seasonally green in its native habitat, A. fimbriata grows year round in
greenhouse culture. It forms a large perenniating root that can be divided to produce
clones. Stems can be pruned back to the root after fruit collection to support greenhouse
sanitation efforts, or “as needed” to stimulate new growth. New stems generated in this
way will flower in two weeks. Fruit size, leaf size, and number of seeds increase in older,
larger plants. The longevity of individual plants provides an ongoing source of seed from
a single experimental subject.
Figure 2-4: Diversity of flower and growth forms in Aristolochiaceae. Herbaceous
perennials with radially symmetric 3-merous flowers include A. Asarum chingchengense
B. Saruma henryii C. Thottea siliquosa, a small shrub D. A. arborea (subgenus Isotrema),
a tree-like shrub with flowers that mimic fungi E. A. triactina (subgenus
Pararistolochia) F. A. trilobata (subgenus Aristolochia) with three lobed, evergreen
leaves, grows as a vine with woody branches (liana) from which new growth emerges. G.
A. passiflorafolia (subgenus Aristolochia) (photo used with permission from Changbin
Chen) and H. A. fimbriata (subgenus Aristolochia).
54
We developed in vitro methods for germinating seeds to support collection of
seedling tissues and for comparisons of seed viability. Light during germination is
required for true leaves to emerge. Seeds germinated significantly better in wet toweling
(mean= 61%, n=11) compared to germination on plates containing solid, sucrose-free
media (mean=9%, n=3). Light and age of seed (up to 2.5 years) had no significant effect
on germination with 80% germinating by 70 days. More details are available in the
Cultivation Supplement.
Figure 2-5: Aristolochia fimbriata A. Twelve three-year old stock plants maintained in
pots (12 cm diameter) occupy 1 m x 1.5 m bench space in the greenhouse B. Plants in use
for genetic crosses, seed or tissue collection are trellised C. Closeup of vine showing
flowers and floral buds at successive developmental stages on one stem [with arrows]..
55
A phylogenetic perspective of genome sizes
Because a small genome size facilitates cloning genes of interest, detecting
regulatory regions, and sequencing the genome of a model organism, our analysis of
genome size evolution in Aristolochiaceae focused on species having small genomes,
particularly in subgenus Aristolochia, for which we report here the smallest genome size
to date from a basal angiosperm (A. lindneri) (Figure 2-6). In order to gain insight into
the evolution of genome structure as well as genome size in Aristolochiaceae,
chromosome numbers from previously published studies (Sugawara, 1987a; Ohi-Toma et
al., 2006) were plotted along with genome sizes on a strict consensus tree using
TreeGraph (Müller and Müller, 2004) (Figure 2-6).
A direct correlation between chromosome numbers and genome size for the
family of Aristolochiaceae was not observed. Aristolochiaceae is subdivided into two
subfamilies, Asaroideae and Aristolochioideae. Asaroideae, which has 2n= 26, 52
(Saruma henryi) and 2n=26 (Asarum caudatum) chromosomes, has about two to ten
times the genome size of genus Aristolochia (Figure 2-6). Thottea, the earliest diverging
branch in subfamily Aristolochioideae has the same number of chromosomes (2n=26) as
Asarum and Saruma, but has only 1/9 and 1/5 of the genome size of Asarum and Saruma,
respectively.
In contrast, within genus Aristolochia, species in subgenus Aristolochia exhibit
the smallest genome sizes, but have a wide range of chromosome numbers. Species in
subgenus Isotrema (Figure 2-4D) are generally characterized by 2n=32 chromosomes
56
and have small genome sizes (554-774 Mbp mean genome size, 1C). Within subgenus
Pararistolochia (Figure 2-4E) (1793-4321 Mbp mean genome size, 1C), which is sister
Figure 2-6: Phylogenetic relationships among sampled Aristolochiaceae, with Tasmannia
lanceolata (Cannelleales) as outgroup. Maximum parsimony strict consensus tree with
genome sizes (Mbp/1C) and chromosome counts indicated. If different genome sizes
were obtained from different plants belonging to the same species, the smallest size was
plotted on the tree. For range of genome sizes within one species and standard deviation
refer to Table 2S-5. Bootstrap values from 1000 replicates are indicated on the branches.
57
group to subgenus Aristolochia, and therefore nested within the clade having species with
small genomes, a lineage-specific increase in genome size can be seen. The increase is
not associated with an increase in chromosome number, so it can be inferred that there
has been an increase in chromosome size. The Australian species of Pararistolochia have
less than half of the genome size of the African species, but still have the same number of
chromosomes (Ohi-Toma et al., 2006). Within the subgenus Aristolochia (Figure 2-4F,
G, H), the different monophyletic groups recovered (Figure 2-6) are in accordance with
previous studies (Wanke et al., 2006a; Wanke et al., 2007). It is interesting to notice that
Asarum and subgenus Pararistolochia have large chromosomes similar to those of
monocots, whereas the remaining clades in Aristolochiaceae have small chromosomes
(Ohi-Toma et al., 2006). Aristolochia fimbriata is a member of a clade of Aristolochia
species with 2n=14 chromosomes and genomes roughly the size of Oryza sativa.
Methods for genetics
We further evaluated selected taxa for self-compatibility and potential for genetic
engineering, both of which are critical features of genetic model systems. Self-pollination
experiments were conducted with A. elegans and A. fimbriata because of their small
genomes, ease of culture, and prolific flowering (Figure 2-2, Table 2S-2). Both species
could be hand pollinated to accomplish cross- and self-pollination events using simple
methods (Tables 2S-3, 2S-4). Morphological changes in the perianth and gynostemium
associated with maturation of the anthers and stigmatic surfaces are described for A.
fimbriata from the day of anthesis (day 1) through day 3 (Figure 2S-1). Self-pollination
58
in A. fimbriata was most effective on day 2, both in terms of fruit production and seed
viability (Figure 2S-1, Table 2S-3). Using in vitro germination methods, 59% of seeds
produced in open-air pollinations of A. fimbriata germinated normally, compared to 50%
of seeds produced from self-pollination of day 2 flowers (Table 2S-3). Additional details
are provided in the Cultivation Supplement.
Agrobacterium tumefaciens-mediated genetic transformation experiments were
performed with two varieties of A. fimbriata (NV, VL) and with Saruma henryi. The
binary vector utilized (Maximova et al., 2003) contained a neomycin phosphotransferase
marker gene (NPTII) for antibiotic selection and an enhanced green fluorescent protein
reporter gene (EGFP); both genes were under the control of the E12-Ω CaMV-35S
promoter (Maximova et al., 2003). High frequency transient expression was observed in
the leaf explants at seven days after culture initiation (Figure 2-7A-D), followed by
production of transgenic calli and shoot primordia at 24 days after culture initiation
(Figure 2-7E, F). Stable transformation was observed in 40% of the S. henryi explants
(n=25), and in 58% of the A. fimbriata explants (n=59). Additionally, transgenic calli,
shoot, and root primordia (Figure 2-7G, H) were successfully regenerated from stem
explants of A. fimbriata tissue cultured plants. The integration of EGFP in the transgenic
calli was confirmed by genomic PCR analysis (Figure 2-7I). The PCR reactions
including DNA from green fluorescent calli (Figure 2-7I, lanes 3-5) resulted in the
amplification of one 426bp fragment similar to the control reaction including plasmid
pGH00.0131 DNA (Figure 2-7I, lane 7). Amplification was not detected in the control
reaction containing DNA from non-transgenic Aristolochia leaf tissue (Figure 2-7I, lane
59
2). and the control reaction without DNA (Figure 2-7I, lane 6). Additional experiments
have been initiated for further optimization of the transformation system.
60
Discussion
Aristolochia fimbriata has many characteristics of a valuable experimental model
A basal angiosperm experimental model is needed to explore the diversity of
basal angiosperms and to test hypotheses about the evolution of development in
flowering plants. Aristolochia fimbriata possesses many features desired in a model
system. A. fimbriata has the physical features for large-scale greenhouse cultivation. It
can be made to flower continuously, and provides the essential feature of self-
compatibility, which permits the production of large numbers of homozygous individuals
required for gene functional analysis in a single life cycle. We have begun to develop
inbred lines to facilitate genome mapping and large scale mutagenesis experiments. High-
efficiency transformation with a GFP construct allows rapid, nondestructive identification
of transformed tissues for subsequent processing, and our in vitro micropropagation and
regeneration methods (Figure 2-7J) can yield greenhouse acclimated plants in three
months (Bliss et al., submitted, December 2008). Individual A. fimbriata plants survive
indefinitely, providing ongoing access to mutant lines that can be cloned for distribution
Figure 2-7: Green fluorescent protein expression in Aristolochiaceae. A, C, E, G. Light
images. B, D, F, H. Fluorescent images. A-D. Leaf explants 10 days after Agrobacterium
tumefaciens infection. A, B. Saruma henryii. C, D. A. fimbriata. E, F. Regenerating A.
fimbriata stem explant. G, H. Regenerating A. fimbriata roots. I. Gel image of PCR
products; Lane L- 200bp ladder, bright band at 1K bp with corresponding bands at each
200bp; Lane 2-A. fimbriata (WT) DNA; Lane 3- In vitro transformed callus 1; Lane 4- In
vitro transformed callus 2; Lane 5- In vitro transformed callus; Lane 6-negative control;
Lane 7-plasmid PC (1ng/ul) J. A. fimbriata in vitro regeneration methods support
transformation system and generate sterile tissue..
61
to the research community. Currently available for analysis, the VL and NV genotypes
possess a number of readily discernible traits including leaf variegation (Figure 2S-3C)
and perianth details (Figure 2S-3B, D, E) amenable to investigation at the genetic level.
Further inbreeding and crossbreeding these genotypes can be used to dissect the genetic
basis for these or other differences, which may yield useful markers, identify linked
genes, and ultimately contribute to mapping and assembling the Aristolochia fimbriata
genome sequence.
Aristolochia fimbriata is well positioned for studies of the evolution of development
Aristolochia fimbriata is in a strong phylogenetic position to support its use as a
model system in evolutionary studies. Aristolochiaceae, with its approximately 550
species in four genera (Wanke et al., 2006a), is one of the most diverse and speciose
families among the basal angiosperms. The largest genus in the family, Aristolochia,
contains approximately 450 species, and has long been of interest to botanists due to its
monosymmetric, unipartite fly-trapping perianth (Figure 2S-3E) and unique
gynostemium, which is a structure formed by the fusion of the gynoecium with the
anthers. Aristolochia fimbriata is a typical member of the family, such that its flower was
modelled in glass by Leopold and Rudolf Blaschka in the latter half of the nineteenth
century (Glass flowers from the Ware Collection in the Botanical Museum of Harvard
University, 1940). A. fimbriata differs from closely related Aristolochia species in several
late stage modifications (e.g., fimbriae, papillae, pubescence), as well as in perianth and
gynostemium development (Gonzalez and Stevenson, 2000b, a); these features are
62
potentially suitable for molecular analysis and studies of gene function using an A.
fimbriata experimental system.
Comparisons between closely related taxa in Aristolochiaceae could support
discovery of differences responsible for speciation and may resolve long-standing
questions about the origins of floral structures. Unlike Aristolochia, the three other genera
in the family (Asarum (Figure 2-4A), Saruma (Figure 2-4B), and Thottea (Figure 2-
4C)) have radially symmetric perianths, and account for approximately 100 species
(Table 2S-1). The bilaterally symmetric perianth of Aristolochia represents the most
“basal” occurrence of this important floral adaptation in angiosperms. Aristolochia
fimbriata presents the opportunity to investigate the genetic basis of bilateral symmetry in
magnoliids and compare it with that found in the eudicot models Antirrhinum (Cubas et
al., 2001) and Lotus (Feng et al., 2006). Existing microscopic studies of anatomy and
development in Aristolochiaceae will facilitate comparative and functional studies, and
provide insight into the evolution of development in this family. For Saruma the anatomy
of stem, leaf, flower, and pollen have been described (Dickison, 1992, 1996), and across
the family, ovule and seed development (Gonzales and Rudall, 2003), microsporogenesis
(Gonzalez et al., 2001), and inflorescence morphology (Gonzalez, 1999) have been
described in detail.
Morphological and gene expression studies argue that Aristolochiaceae offers an
excellent system in which to study the role of homologs of B-class MADS-box genes
which are required for the organ identities of petal and stamen in higher eudicots and
putative homologous organs in grasses (Ma and dePamphilis, 2000; Irish, 2003). The
flower of the monotypic genus Saruma (Figure 2-4B) resembles that of the typical
63
magnoliid flower more than any other species in the family, having apparent sepals,
petals, stamens, and carpels. A thorough examination of development in the petaloid
perianth whorl of Saruma indicates it is of staminoid origin (de Craene et al., 2003). In
Aristolochia, the utricle, tube, and limb (Figure 2S-3E) of the single-whorled perianth
originate from the outermost whorl and are interpreted as a calyx (Gonzalez and
Stevenson, 2000b). However, Jaramillo and Kramer (2004) reported that putative
orthologs of B-class genes (AP3, PI) are expressed in the stamens and staminoid “petals”
of Saruma, as well as in the anthers and inner layer of utricle cells of the Aristolochia
sepalloid perianth. In Asarum and Thottea, the appendages opposite the sepals have been
interpreted to be petaloid (Leins and Erbar, 1985; Renuka and Swarupanandan, 1986;
Sugawara, 1987b; Leins et al., 1988; Gonzalez and Stevenson, 2000a, b), and putative
AP3 orthologs have been found in Asarum (Kramer and Irish, 2000; Zhao et al., 2006).
Variations in the expression of putative orthologs of the B-class genes in Saruma
and Aristolochia suggest they might not regulate perianth floral organ identity in the
same way as they do in eudicots and grasses, even though expression is found in
pollinator-attracting structures. For example, homologs of B-class genes might regulate
the identity of the perianth in Aristolochiaceae, even though the perianth does not have
the same structure (i.e., petals) found in other taxa. Alternatively, these genes might not
regulate the perianth identity in Aristolochiaceae. Experimental evidence from
Aristolochiaceae is needed to determine if B-class homologs play a role in organ identity
in basal angiosperms, particularly since the expression patterns of these genes in the first
perianth whorl of other basal taxa is variable (Kramer and Irish, 1999, 2000; Zahn et al.,
2005b). \
64
Aristolochia contains highly developed biochemical pathways offering insight into
evolution of biochemical synthesis and coevolution with insects
Aristolochiaceae produce a complex mixture of secondary metabolites, as is
common in basal angiosperms,. In particular, aristolochic acids and aristolactams are
produced in Aristolochiaceae (reviewed in Kumar et al. 2003) found throughout Piperales
and the basal eudicots. Compounds produced by alkaloid biosynthesis pathways in the
poppy family (Papaveraceae, Ranunculales) are of great pharmacological importance.
Compounds from a parallel pathway in Aristolochiaceae (aristolochic acids and
aristolactams) are associated with the use of some Aristolochia species in traditional
medicines (Reddy et al. 1995). The common name “birthwort” attributed to Aristolochia
refers to traditional use of some species, particularly extracts from root tissues, as
abortifacients, emmenagogues, or post-coital antifertility agents (Pakrashi and Pakrasi,
1979). More recently, constituents of primarily root extracts from Aristolochia species
have been isolated and evaluated for biological activity as antibiotics, antivenoms and
tumor-inhibiting agents (Gupta et al., 1996; Broussalis et al., 1999; Otero et al., 2000;
Qiu et al., 2000; Gadhi et al., 2001a; Shafi et al., 2002; Elizabeth and Raju, 2006; Meinl
et al., 2006), although aristolochic acids become bioactivated and carcinogenic when
consumed in the diet (Hwang et al., 2006; Grollman et al., 2007).
In addition to its pharmacological properties, Aristolochia provides an opportunity
to explore coevolution of secondary metabolites with insects. Dipterans commonly serve
as pollinators in Aristolochia (Petch, 1924; Lindner, 1928; Lu, 1982; Wolda and
Sabrosky, 1986; Hall and Brown, 1993), sometimes having specialized, mutualistic
relationships involving egg deposition in the flowers (Vogel, 1978; Disney and Sakai,
65
2001; Sakai, 2002; Burgess et al., 2004). Dipteran pollinators are thought to be attracted
to secondary metabolites mimicking the aroma of a food source (Petch, 1924; Burgess et
al., 2004), or acting as pheromones to attract species-specific, sex-specific pollinators
(Rulik et al.), or stimulate oviposition (Sakai, 2002).
Aristolochia species are also important host plants for the larval stages of
swallowtail butterflies (Papilionidae, Lepidoptera) (Rausher, 1981; Nishida and Fukami,
1989; De Morais and Brown, 1991; Nishida, 1994; Klitzke and Brown, 2000; Fordyce et
al., 2005). Secondary metabolites found in particular Aristolochia species are critical for
the defense and survival of associated species of swallowtail butterfly species during their
feeding stage, to the extent that the decline of butterfly populations is attributed to
decreased distributions of particular Aristolochia species (Sands et al., 1997). Finally,
secondary metabolites of Aristolochia and related species are of interest for their
repellent, insecticidal, and antifeedant activities (Lajide et al., 1993; Alexenizer and
Dorn, 2007; Palacios et al., 2007). Biochemistry in Aristolochia can be evaluated in
greenhouse grown or micropropagated plants, using chemical analyses (e.g., mass
spectroscopy, gas chromatography) to further characterize constituents of specific plant
tissues in A. fimbriata. Biochemical pathways can be further investigated using
transformed roots (Figure 2-7G, H) or callus currently available in the transformation-
regeneration system, without further optimization.
66
Aristolochia can provide insight into development of woodiness
Woodiness is an important seed plant feature, both for commercial and ecological
purposes (Stevens, 2008). Growth forms within the Aristolochiaceae vary widely,
presenting an opportunity to investigate growth form traits including flexibility, stiffness,
and woodiness of closely related species (Speck et al., 2003). Arisolochiaceae are most
commonly perennial, self-supporting herbs (Figure 2-4A, B), procumbent or trailing,
non-self-supporting vines (A. passiflorafolia, A. fimbriata) (Figure 2-4G, H), and woody
lianas (Figure 2-4F). Rarely, they are small woody shrubs (Figure 2-4C); and even more
rarely trees (or tree-like forms) (Figure 2-4D). Ancestral members of the family (Asarum
and Saruma) are characterized as perennial rhizomatous herbs, and the sister group to
Aristolochia is comprised of the woody sub-shrub Thottea (Figure 2-4C). Perennial
herbs appeared iteratively in the topology (Figure 2-6) of the family phylogeny and are
nested within groups of woody vines. Most eudicots and monocots are modular
organisms with indeterminate body plans. Shifts in the ontogenetic trajectory may be
expected to have a profound effect on the overall size and potential life history of the
descendant. This effect might have played a key role in growth form evolution and the
development of flexibility, stiffness, and woodiness in Aristolochiaceae (Lahaye et al.,
2005; Rowe and Speck, 2005). Differences in these growth form traits can be
investigated in Aristolochia species of interest, including A. fimbriata, beginning with
cellular level observations and descriptions of development of secondary growth
(“wood”). Molecular and cell biological methods can be used to locate and describe in
Aristolochia homologs of genes involved in growth form traits in other species (e.g.,
67
Arabidopsis, Populus, Liriodendron), to further characterize the role of interesting gene
products in Aristolochiaceae.
Aristolochia can reveal features of the ancestor common to monocots and eudicots
Aristolochiaceae occurs in Piperales, in the magnoliid clade, which is sister to the
very large and diverse monocot plus eudicot clades (Jansen et al., 2007). As such,
Aristolochia can provide a close outgroup for analysis of ancestral traits in the major
groups of angiosperms. The ancient features of different lineages in Piperales have long
been recognized, earning them classification as “paleoherbs” in early taxonomic works
(Taylor and Hickey, 1990; Taylor and Hickey, 1992). Aristolochiaceae and close
relatives show a mixture of features of monocots and eudicots. The more ancestral clade,
Asaroideae, is comprised of rhizomatous perennials similar to basal monocots and
eudicots. Also found in Piperales are aquatics (e.g., Saururaceae) a common life form in
both basal eudicots and monocots (e.g. Acorales, Alismatales). Piperales, and
Aristolochiaceae in particular displays other features more commonly associated with
monocots, including floral organs in multiples of three, median prophylls (which are
shared with nearly all monocots and only few dicot clades), and subtype PII sieve-tube
plastids (Huber, 1993; Behnke, 2003). Piperales also share with early diverging monocots
(Alismatales, Arales) distichous placement of leaves and palmate leaf venation. Indeed,
in some phylogenies, Piperales appear as the closest relative to Acorus, the sister of all
other monocots (Duvall, 2000). Consequently, many features in monocots and eudicots,
both genetic and phenotypic, can be expected to have a homolog in Aristolochiaceae and
68
its relatives, and would help to characterize the extinct ancestors of the eudicot, monocot
and magnoliid clades.
Growing genomic resources in Aristolochiaceae support further development of
model system
Genomic resources are growing rapidly for Aristolochiaceae, which will facilitate
the identification and study of genes, gene families, and functional studies in basal
angiosperms. The Ancestral Angiosperm Genome Project
(www.ancestralangiosperm.org) has selected Aristolochia fimbriata for deep EST
sequencing using a combination of traditional capillary (Albert et al., 2005) and extensive
next generation (454, Solexa Illumina) sequencing of libraries constructed from multiple
vegetative and reproductive tissues and stages. There are currently 16,451 Sanger EST
sequences from A. fimbriata in the NCBI EST database
(http://www.ncbi.nlm.nih.gov/sites/entrez). In this initial set of A. fimbriata ESTs, we
used BLAST analyses as described in Albert et al 2005 to identify putative homologs of
many interesting regulatory and signaling genes (see Table 2-1 for examples).
Surprisingly, Aristolochia cDNA sequences had greater sequence similarity with other
monocot or eudicot species than with Arabidopsis, highlighting the important role A.
fimbriata will play in rooting phylogenetic analyses of gene function [results not shown].
Consistent with the composition of tissues included in the libraries, we found putative
orthologs of many genes important for development such as auxin efflux carrier PIN1,
phytochrome signaling protein GIGANTEA, as well as floral development genes AP2,
AP3, AINTEGUMENTA, and SEP3 (Table 2-1). We anticipate these cDNA sequences,
69
and many more being currently generated, will contribute to new and ongoing studies of
evolution of development in our lab and in others.
Table 2-1: Homologs of genes involved in development, cell wall biosynthesis, and
response to biotic and abiotic stress revealed in preliminary sequencing of two cDNA
libraries constructed from combined A. fimbriata tissues. All genes searched (blastx)
against the sequenced plant genomes of Arabidopsis thaliana, Oryza sativa, Populus
trichocarpa, Vitis vinifera, Carica papaya, Medicago truncatula, and Sorghum bicolor
(http://www.floralgenome.org/tribedb/index.pl). Values reported are the number of ESTs
in each unigene, the unigene length (bp), percent identity of the unigene to its best blastx
hit, evalue, Arabidopsis AGI, and the annotation of the best Arabidopsis hit as well as the
species with the best overall hit following (in parentheses).
ESTs Length Identity Evalue AGI Annotation
Dev
elo
pm
ent
1 330 bp 82% 6e-49 AT4G36920 AP2/EREBP floral homeotic protein (Vitis vinifera)
9 755 bp 71% 1e-18 AT1G22770 GIGANTEA (Sorghum bicolor)
3 1254 bp 84% 2e-73 AT1G73590 PIN1 auxin efflux carrier (Medicago truncatula)
2 613 bp 88% 3e-44 AT4G37750 AINTEGUMENTA (Carica papaya)
7 1420 bp 50% 1e-79 AT2G37630 Homologous to Antirrhinum PHANTASTICA (PHAN), maize
ROUGH SHEATH2 (RS2) (Vitis vinifera)
4 747 bp 61% 2e-41 AT1G24260 SEP3 MADs box transcription factor (Vitis vinifera)
1 736 bp 87% 1e-112 AT1G48410 RNA Slicer that selectively recruits microRNAs and siRNAs
(Populus trichocarpa)
2 956 bp 81% 1e-151 AT2G34710 PHABULOSA contains hd-leucine zipper domains and domain similar to a mammalian sterol binding domain (Vitis vinifera)
2 835 bp 33% 6e-40 AT3G54340 AP3/APETALA 3 DNA binding / transcription (Vitis vinifera)
Cel
l w
all
bio
syn
thes
is 1 495 bp 56% 6e-14 AT5G42100
Plasmodesmal (Pd)-associated membrane protein involved in Pd
callose degradation and gating (Vitis vinifera)
8 787 bp 55% 4e-28 AT2G38540 Cell wall lipid transfer protein binds calmodulin in a Ca2+-
independent manner (Medicago truncatula)
5 932 bp 70% 2e-65 AT5G08380 ATAGAL1 alpha-galactosidase similar to ATAGAL2 (Vitis vinifera)
1 737 bp 53% 4e-39 AT4G03210 Xyloglucan endotransglucosylase/hydrolase (XTH) (Vitis
vinifera)
16 986 bp 81% 6e-80 AT1G26770 EXPA10: expansin involved in the formation of nematode-
induced syncytia in roots (Carica papaya)
Str
ess
resp
on
se
3 1725 bp 65% 1e-41 AT3G12500 BASIC CHITINASE in ethylene/jasmonic acid mediated signalling pathway during SAR (Oryza sativa)
2 972 bp 82% 1e-145 AT5G67030 Zeaxanthin epoxidase gene (Populus trichocarpa)
3 1372 bp 79% 1e-164 AT3G45640 MAP KINASE 3 (MPK3) upregulated in response to touch, cold, salinity, chitin (Vitis vinifera)
2 572 bp 71% 1e-59 AT1G05850 POM-POM1; Chitinase-like protein essential for tolerance to
heat, salt, drought stresses (Vitis vinifera)
1 685 bp 55% 2e-45 AT1G59870
ATP binding cassette transporter contributes to SA independent
nonhost resistance and heavy metal resistance (Populus
trichocarpa)
6 904 bp 52% 4e-46 AT1G06040 Salt tolerance protein (STO) (Populus trichocarpa)
1 763 bp 61% 2e-54 AT5G51700 RAR1 disease resistance protein ; Required for R protein
accumulation (Carica papaya)
70
Comparative analyses of cDNA sequences within Aristolochiaceae will be
facilitated by 10,274 EST sequences from Saruma henryi (Figure 2-4B). Saruma was
selected by the Floral Genome Project (FGP) to represent Aristolochiaceae for floral
transcriptome sequencing (http://www.floralgenome.org/taxa/) because it appears to
display ancestral morphological characters in the family (Dickison, 1992; Leins and
Erbar, 1995; Kelly, 1998) and because its flower includes all four (sepal, petal, stamen,
and carpel) floral whorls. The ESTs from the Saruma premeiotic flower bud cDNA
library (Albert et al., 2005) and from A. fimbriata form the foundation of the genomic
resources available today from this important basal angiosperm family.
In addition to EST sequencing, our group has isolated miRNAs from A. fimbriata
using cloning and capillary sequencing as well as ultrahigh throughput pyrosequencing
(Ali Barakat, P. Kerr Wall, and Claude dePamphilis, in prep.). This study allowed the
isolation of hundreds of different miRNA sequences belonging to 32 conserved families
as well as several non-conserved families. MicroRNAs are small RNAs (21 nt) that play
a major role as regulator of gene expression in various physiological, cellular, but, mainly
developmental processes (Jones-Rhoades et al., 2006). Several potential miRNA targets
were found in cDNA sequences of A. fimbriata. Predicted target genes include
transcription factors but also genes implicated in various metabolic processes and in
stress defense. The isolation of miRNA from Aristolochia presents a good opportunity for
analyzing the function of miRNAs in basal angiosperms as well as understanding how
miRNA-mediated regulation of gene expression has evolved in land plants by comparing
miRNAs in basal angiosperms to those in basal eudicots and lower land plants (Axtell et
71
al., 2007; Barakat et al., 2007). This core of genetic, genomic, and methodological
resources is presently available as a foundation for further development of Aristolochia
fimbriata as a basal angiosperm model system as well as for immediate use by the
scientific community working on various areas of research including evolution of
development, plant resistance to biotic and abiotic stresses, gene functional analysis, and
comparative genomics.
Conclusion
We have used a rigorous process to select and develop resources for the basal
angiosperm, A. fimbriata. Culturing and hand pollination methods required for rapid
generation of homozygous lines needed for genetic experiments are described. The small
genome size and immediate availability of sequence data supports ongoing studies of
molecular genetics and evolution. Hypotheses about gene evolution and gene function
can be tested using a reverse genetic approach, i.e., over and under expression studies in a
transformable species suitable for large-scale cultivation. Optimizing the selection phase
for transformed A. fimbriata explants will yield a high throughput transformation system
for investigating evolution of gene function in a basal angiosperm. Along with continued
development of genomic resources, A. fimbriata promises to be an excellent experimental
system to provide further insight into the diversity found among basal angiosperms and to
test hypotheses about the evolution of angiosperm genomes, development and
biochemistry.
72
Materials & Methods
Cultivation
We evaluated 24 species of Aristolochiaceae. These were selected to encompass
the phenotypic plasticity of the four-whorled, actinomorphic, Saruma, the actinomorphic,
single-whorled perianths of Asarum and Thottea, and the monosymmetric, highly
modified and diverse flowers of Aristolochia. The sampled taxa also reflected the genetic
diversity of the whole family recovered by phylogenetic analysis. Plant material was
obtained from commercial nurseries, private donations, and academic sources. Vouchers
of specimens included in the phylogenetic analysis and sampled for genome sizes have
been entered into herbaria as described in Table 2S-5. For these species, we evaluated
evolution of genome size and chromosome number in a phylogenetic context. For 14
species of Aristolochia we evaluated life cycle and cultivation characteristics. Plants were
maintained in the greenhouse at The Pennsylvania State University, University Park, PA.
All seeds were germinated in soil-free potting medium (Pro-Mix BX, Premier
Horticulture Inc., Quakertown , PA) in shallow germination trays with drainage holes, in
the greenhouse at 18-27C (varying from night to day) and 40-70% humidity. The trays
were incubated on heating mats operating at approximately 27C, as needed. Natural day
length was supplemented with high-pressure sodium lamps (1000 watt) October through
April to provide twelve-hour days. Plants received regular watering as needed.
Depending on the stage of growth, regular fertilizer applications were provided, as a
drench, alternating Peter's Professional 15-16-17 Peat Lite Special at 200 PPM nitrogen
73
(once to twice weekly) with Peters Professional 21-7-7 Acid Special (Scotts Horticulture,
Marysville, OH) at 200 PPM nitrogen (approximately every six weeks). The plants were
drenched once a month with 100 ppm chelated iron (Sprint 330 10% iron, RoseCare.com,
Santa Barbara, CA).
Genome sizing
Nuclear genome size estimation were obtained by flow cell cytometry following
the protocol described by Arumuganathan and Earle (1991). The mean nuclear DNA
content of each plant sample (expressed as pg) was based on 1000 scanned nuclei from
sample tissue, compared to a preparation of tissue from the internal standard. Each
nuclear preparation was sampled 4 times.
Phylogenetic analysis
To clarify the phylogenetic positions of the taxa surveyed and to evaluate genome
size in an evolutionary context we constructed a phylogenetic tree based on the plastid
trnK/matK region. Total DNA was extracted using the CTAB method of (McNeal et al.,
2006). Vouchers, DNA, and tissue samples are stored at Dresden, PAC or DD.
Amplification and sequencing was performed following methods described in detail by
Wanke et al. (2007) using published primers (Wanke et al., 2006a; Wanke et al., 2006b;
Wanke et al., 2007). Sequences were manually aligned using PhyDE® (Müller et al.,
2007). The alignment is available from TreeBASE (www.treebase.org). Phylogenetic
74
analysis was performed under maximum parsimony in PAUP* 4.0b10 (Swofford, 2002)
using PAUP scripts written by PRAP (Müller, 2004). PRAP was used to implement the
Parsimony Ratchet (Nixon, 1999) following procedures described in Wanke et al. (2007)
but with 1000 ratchet replicates. Bootstrap values were additionally calculated to infer
branch support with 1000 replicates. For an independent evaluation of relationships, a
likelihood approach was chosen using the likelihood ratchet described by Morrison
(2007) as implemented in PRAP v. 2.0 (Müller, 2004) with default settings.
Pollination experiments
Several species of Aristolochia had been reported to be self-compatible (A.
fimbriata, A. elegans, A. ridicula, A. ringens) and generally protogynous , having a
receptive stigma before the anthers dehisce. To determine if autogamous pollination
could be successful, we attempted hand pollinations of A. elegans and A. fimbriata on
day one (the day of anthesis), day two, day three, day four and day five. Unopened flower
buds were covered with pollination bags prior to anthesis and observed daily. Pollinations
were accomplished by severing the perianth midway across the utricle, just above the
gynostemium (Figure 2S-1 (A)). Pollen was transferred with a toothpick (Figure 2S-1
(B)), and the remnant of the perianth was taped closed to prevent additional pollinator
entry. Mature fruits were collected and seeds germinated on wet toweling at 27°C
exposed to 16/8 hour day/night cycles. Hand pollination methods are detailed in the
Pollination Protocol.
75
Genetic transformation
Protocol for genetic transformation of A. fimbriata was developed based on in
vitro shoot regeneration from leaf and stem explants coupled with Agrobacterium-
mediated transformation. Leaf and Internodal stem segments (2-3 cm long) from rooted
tissue cultured A. fimbriata plants were excised and immediately immersed in induction
media (Petch, 1924; Maximova et al., 2003; Bliss et al., submitted, December 2008) to
keep moist. The explants were inoculated with Agrobacterium strain AGL containing
plasmid pGH00.0131 as previously described for Theobroma cacao L. (Maximova et al.,
2003). After the inoculation the explants were blotted on sterile paper towels and co-
cultivated on callus initiation medium (SI) (0.5mg/L 6BA, 1mg/L NAA and 1mg/L
TDZ) for 64 hours in the dark at 27C. Following co-cultivation, the explants were
transferred to CI supplemented with 50 mg/L Geneticin (G418) (Cellgro, Herndon, VA)
and 200 mg/L Claforan (Aventis, New Jersey) and incubated at 27C in the dark for the
remainder of the 14 days. After culture on CI medium, the explants were transferred on
shoot regeneration medium (1.75 mg/L 6BA and 1.0 mg/L NAA) with 25 mg/L G418 and
200mg/L Claforan and incubated in the dark for an additional 14 days. After the 28 days,
the cultures were incubated under dim light until the development of shoot primordia.
Individual glowing shoot primordia were then excised and transferred and maintained for
further shoot elongation and rooting on REN2 medium (hormone free) in culture vessels
(Sweetheart DSD8X and LDS58) under dim light conditions at 27C (Bliss et al.,
submitted, December 2008). EGFP fluorescence was observed and recorded as
previously described (Maximova et al., 2003).
76
Genomic PCR analysis
The incorporation of the transgenes was confirmed by genomic PCR. A sets of
EGFP specific PCR primers were used for the analysis. The primers amplify a 426bp
EGFP fragment (5 ‟-CCA GGA GCG CAC CAT CTT CT-3‟ and 5‟-CTC GTC CAT
GCC GAG AGT GA-3‟ (Maximova et al., 2003). Genomic DNA was isolated from non-
transgenic Aristolochia leaf tissue and from transgenic calli from independent lines by
using a CTAB method of (McNeal et al., 2006). Each PCR reaction (final volume of 20
µl )contained: 5ng/ul DNA (Qiagen purified DNA kit #69104), 10ul JumpStart™
REDTaq® ReadyMix (Sigma #0982), 5uls water, forward and reverse primers at final
concentration 0.5 µM. Reactions were prepared on ice. Control PCR reactions were also
performed with 1ng/ul plasmid DNA from vector pGH00.0131 and PCR reaction mix
without DNA. This represents an equal molar amount of plasmid DNA compared to the
GFP DNA contained in 5ng total Aristolochia genomic DNA present in the leaf extract,
assuming single copy/insertion of the GFP gene. For the plasmid reactions DNA was
isolated using QIAGEN plasmid midi purification kit (QIAGEN Inc., Valencia, CA).
PCR conditions for all reactions were: 94ºC for 2 min, then 35 cycles of 94ºC for 45 sec.,
62ºC for 45 sec, 72ºC for 1 min. The final cycle was followed by incubation at 72ºC for 7
min. 5 µl of each PCR reaction were loaded onto 1.5% high-resolution agarose gel
(Sigma-Aldrich Co., St. Louis, MO, #A-4718) for electrophoresis.
77
Genetic transformation
Protocol for genetic transformation of A. fimbriata was developed based on in
vitro shoot regeneration from leaf and stem explants coupled with Agrobacterium-
mediated transformation. Leaf and Internodal stem segments (2-3 cm long) from rooted
tissue cultured A. fimbriata plants were excised and immediately immersed in induction
media (Maximova et al., 2003; Bliss et al., submitted, December 2008) to keep moist.
The explants were inoculated with Agrobacterium strain AGL containing plasmid
pGH00.0131 as previously described for Theobroma cacao L. (Maximova et al., 2003).
After the inoculation the explants were blotted on sterile paper towels and co-cultivated
on callus initiation medium (CI)
Supplemental Material
Table 2S-1: Basal angiosperm characteristics
Table 2S-2: Cultivation features of 25 species of Aristolochiaceae
Cultivation Supplement: Descriptions of perianth maturation, hand pollination methods,
and self-compatibility in A. fimbriata and A. elegans. Includes: Table 2S-3, Table 2S-4,
Figure 2S-1, Figure 2S-2
Table 2S-5: Genome sizes, vouchers, sources, and accessions
Figure 2S-3: Aristolochia fimbriata genotype and perianth detail
Figure 2S-4: Phylogram of Aristolochiaceae relationships
78
Acknowledgements
We thank A. Omeis for plant care, Jason Stetson and Yunjiao Joy Wang for DNA
isolations; Paula Ralph for plant care and observations, pollination, seed germination, lab
assistance and tissue collection; Yi Hu for tissue collection, RNA isolations, and lab
assistance; Joel McNeal, Mario Blanco, Al Hill, Larry Rosen, Russ Strover, Victor
Wong, the New York Botanical Garden, the Botanical Gardens at the University of Ulm,
Dresden, Universidade de Coimbra, and Dawe‟s Arboretum for seed and plant material.
This research was supported by The Floral Genome Project [], The Ancestral
Angiosperm Genome Project [NSF DBI-0638595], the Biology Department of Penn State
University, and a postdoctoral fellowship by DAAD for Stefan Wanke is gratefully
acknowledged.
79
Table 2S-1: A summary of relevant basal angiosperm characteristics. Familiar genera
include representatives from the family and are not intended to comprise a
comprehensive listing. Taxa are considered “commercially available” if one hundred
plants or more can be purchased, inexpensively, and can be readily propagated from seed.
1(Stevens, 2008; Bliss et al., submitted, December 2008)
2(Mabberley, 2008)
3Number of species shown for Aristolochiaceae genera from (Wanke et al., 2006a)
Order Family1,2 # Genera
/species3 Familiar genera
3 Growth form
1,2 Commercially
available
AMBORELLALES Amborellaceae 1/1 Amborella shrub, tree no
NYMPHAEALES
Nymphaeaceae 5/95
Nymphaea
Nuphar
Barclaya
Victoria
Euryale aquatic herb yes
Cabombaceae 2/6
Cabomba
Brassenia aquatic herb yes
Hydatellaceae 2/10
Hydatella
Trithuria aquatic herb no
AUSTROBAILEYALES
Austrobaileyaceae 1/2 Austrobaileya liana no
Illiciaceae 3/92
Illicium
Schisandra
Kadsura shrub, tree yes
Trimeniaceae 1/8 Trimenia
shrub, tree,
liana no
CERATOPHYLLALES Ceratophyllaceae 1/2 Ceratophyllum aquatic herb yes
CHLORANTHALES Chloranthaceae 4/75
Chloranthus
Ascarina
Hedyosmum
Sarcandra
herb, shrub,
tree no
MAGNOLIALES
Annonaceae 123/2100
Annona
Guatteris
Xylopia
Uvaria
Polyalthia
Rollinia
Artabotrys
Asimina
shrub, tree,
liana yes
Eupomatiaceae 1/3 Eupomatia shrub, tree yes
Magnoliaceae 2/221
Magnolia
Lioriodendron shrub, tree yes
Degeneriaceae 1/2 Degeneria tree no
Himantandraceae 1/2 Galbulimima tree no
Myristicaceae 19/520
Myristica
Horsfieldia
Virola
Knema shrub, tree yes
LAURALES
Calycanthaceae 3/10
Chimonanthus
Calycanthus
Idiospermum shrub, tree yes
Hernandiaceae 4/60
Hernandia
Illigera
shrub, tree,
liana no
Lauraceae 52/2550
Laurus
Litsea
shrub, tree,
parasitic vine yes
80
Ocotea
Cinnamomum
Cryptocarya
Persea
Lindera
Cassytha
Nectandra
Phoebe
Apollonias
Beilschmiedia
Umbellularia
Monimiaceae 24/200
Doryphora
Peumus
Xymalos
Mollinedia
Tambourissa
Kibara shrub, liana no
Siparunaceae 2/57
Siparuna
Glossocalyx shrub, tree no
Gomortegaceae 1/1 Gomortega shrub, tree no
Atherosperma-taceae 7/25
Atherosperma
Daphnandra
Doryphora
Dryadodaphne
Laurelia
Nemuaron shrub, tree no
CANELLALES
Canellaceae 5/13
Canella
Cinnamodendron
Cinnamosma shrub, tree yes
Winteraceae 4/65
Drimys
Zygogynum
Pseudowintera
Takhtajania shrub, tree yes
PIPERALES
Hydnoraceae 2/15
Prosopanche
Hydnora parasitic herb no
Piperaceae 6/2750 Peperomia herb yes
Piper herb yes
Zippelia herb no
Manekia liana no
Verhuellia herb no
Saururaceae 4/6 Anemopsis herb yes
Houttuynia herb yes
Saururus herb yes
Gymnotheca herb no
Lactoridaceae 1/1 Lactoris shrub no
Aristolochiaceae 4/550 Saruma (1 sp.) herb yes
Asarum (ca. 86
spp.) herb yes
Thottea (ca. 29
spp.) shrub no
Aristolochia (ca.
450 spp.)
herb, shrub,
liana yes
81
Table 2S-2: Cultivation features of 25 Aristolochiaceae taxa evaluated, with findings.
Observations based on greenhouse cultivation (at Penn State and/or Botanical
Garden, Dresden) except where indicated.
Taxon Floral productivity Maintenance Vernalization
required
Asaroideae
Saruma henryii Oliv. moderate1 low
2, 7 required
Asarum canadense L. moderate1 low
2, 7 required
Aristolochioideae genus Aristolochia L
subgenus Isotrema A. serpentaria L. ample low
2-4, 7 required
A. macrophylla Lam. sparse1 low
4, 7 required
A. californica Torr. sparse1 low
4, 7 no
A. tomentosa Sims sparse1 low
4, 7 required
A. holostylis (Duchartre) F. Gonzalez 9 sparse low no
subgenus Pararistolochia A. goldieana (Hook.f.) Hutch. & Dalz
9 sparse moderate
no
A. prevenosa F.Muell. 9
sparse moderate no A. promissa (Mast.) Keay
9 sparse moderate no
A. triactina (Hook. f.) Hutch & Dalz sparse moderate no
subgenus Aristolochia A. acuminata Lam. sparse
1 high5
no A. anguicida Jacq. sparse
1 high4, 8 no
A. clematitis L. sparse1 high
2-4, 7 no
A. elegans Mast. ample moderate4, 8 no
A. fimbriata Cham. ample low2-4 no
A. gigantea Mart. & Zucc. sparse moderate5, 8 no
A. grandiflora Sw. sparse high5, 8 no
A. passiflorafolia Rich., A. sparse1 low
3 no A. ringens Vahl. sparse
1 high5, 8 no
A. trilobata L. sparse1 high
5 no
A. lindneri Berg. 9
sparse moderate no
A. maxima Jacq9 sparse moderate no
Aristolochia. sp. 9
sparse moderate no
genus Thottea
T. siliquosa (Lam.) Hou9 sparse moderate
6, 7, no
1few or no flowers in first season
2herbaceous perennial
3diminutive vine
4vine
5very large vine
6low tree/shrub
7slow growing
8aggressive
9Botanical Garden, Dresden
10Botanical Garden, Bonn
82
Cultivation Supplement
Background
Several species of Aristolochia have been reported to be protogynous, and self-
compatible (A. fimbriata, A. elegans, A. ridicula, A. ringens ; A. bracteata (Petch, 1924;
Razzak et al., 1992); A. inflata (Sakai, 2002). Autonomous self-pollination has been
reported in A. paucinervis, (Berjano et al., 2006) and cleistogamy has been suspected in
A. serpentaria . Control of pollination events, including preventing unintended pollen
delivery, is essential for reproducible genetic experiments. Therefore, we set out to 1)
determine if pollination could be prevented, 2) document post-anthesis changes in the
perianth and gynoecium useful for evaluating the receptiveness of the stigma and 3)
identify the day on which hand pollinations would be most successful. We evaluated fruit
production from autogamous pollination, in which both the pollen and stigmatic surfaces
were from the same flower (Faegri and Van Der Pijl, 1979). We also evaluated fruit
production from geitogamous pollination, in which pollen was applied to stigmatic
surfaces of a different flower on the same plant, selecting older flowers as pollen donor to
better represent the presumed protogynous strategy used by Aristolochia. Results were
compared with “outcrossings,” (xenogamous pollinations) and fruit set on bagged flowers
(Faegri and Van Der Pijl, 1979). We subsequently evaluated seed viability from pods
produced by self-pollination events, and compared it with the viability of bulk seed
collected from open air pollinations. Open air pollinated fruit was collected from the
general population of plants in greenhouse rooms housing both VL and NV cultivars, as
well as large community of pollinators, which were mostly common greenhouse “fungus
83
gnats.” We confirmed reports of self-compatibility in A. elegans and A. fimbriata, and
regularly germinated seeds (up to three years old) produced from self-pollinations and
open-air pollinations.
Perianth maturation
The perianth of A. fimbriata opens early on the day of anthesis (Figure 2S-1 A),
hence referred to as day one (D1). The lobes of the gynostemium of the D1 flower appear
smooth, clean, and curved slightly inward (Figure 2S-1 D). The anthers, located on the
outside of the gynostemium are closed, but a portion of an anther locule can be pried
loose and placed onto the stigmatic surfaces to effect self-pollination on D1. The fimbriae
of the limb of day two (D2) flowers are curved inward, covering the tube and preventing
the exit of any trapped pollinators (Figure 2S-1 B). The lobes of the gynostemium are no
longer curved inward and pollen is visibly dehiscing from the locules on the outer
surfaces of the gynostemium lobes (Figure 2S-1 E). The loose pollen in D2 flowers (and
later) can be collected on the tip of the toothpick from the dehiscing anthers on the
outside of the gynostemium. The perianth of the day three (D3) flower is beginning to
visibly decline (Figure 2S-1 C). The D3 flower releases pollen abundantly, the stigma has
lost turgor, and the lobes of the gynostemium are beginning to recurve outward (Figure
2S-1 F).
84
Hand pollination methods
Selected flowers were enclosed in a pollination bag at an early stage of
development to prevent pollinator access to the flower after anthesis. Each pollination
bag was constructed of an approximately 12 cm x 30 cm piece of tulle (bridal veil) with
an approx. 1.5 mm diameter pore size. The piece of tulle was folded in half, to create the
closed end of the bag. The two long sides were sewn shut with a strong, durable,
upholstery thread using 2 cm long stitches, leaving two drawstrings at the corners of the
Figure 2S-1: Aristolochia fimbriata flower A. Day one perianth at anthesis B. Day two
perianth the day after anthesis C. Day three perianth D. Day one gynostemium; anthers
have not dehisced E. Day two gynostemium; anthers have dehisced F. Day three
gynostemium, pollen abundant.
85
open, shorter side. Pollination bags were wrapped gently two to three times around a stem
containing one or more flower buds, then the drawstring on the bag was tied around the
stem below the petiole of the most proximal flower. To conduct hand pollination, the
pollination bag was removed from a newly opened flower, and the perianth was removed
about halfway through the utricle (Figure 2S-2 A). A toothpick was used to transfer
pollen from the anther locules on the outside of the gynostemium of the pollen donor and
place it on the stigmatic tissue on the inner surfaces of the gynostemium of the pollen
recipient (Figure 2S-2 B). In the self pollinations conducted in these experiments, a single
flower was used both as the pollen donor and recipient (autogamous pollination). The
mature fruit was harvested approximately 4 weeks after hand pollination, when the
capsule dehisced along the six carpel valves, revealing dry seed (Figure 2S-2 C), which
was stored in paper envelopes at ambient room conditions. Seeds collected from pods
harvested before fully mature germinated poorly, if at all (data not shown).
Figure 2S-2: Hand pollination, A. fimbriata A. Sever the perianth midway through the
utricle B. Apply pollen to stigmatic surfaces. (Seal to prevent further pollen delivery) C.
Mature fruit dehiscing.
86
Pollination is prevented with pollination bags
Pollination bags prevented pollination in A. fimbriata except on one occasion,
representing less than one percent of all occurrences (Table 2S-3). On that occasion, the
perianth of the “not pollinated” flower producing fruit was lost, so it could not be
inspected for the presence of a pollinator, which was likely responsible for the pollination
event. Throughout the experiments, holes in the pollination bag and poorly tied bags were
occasionally noted. Observation of an insect in the perianth at any time invalidated hand
pollination efforts for that flower.
Table 2S-3: Evaluating self-compatibility in A. fimbriata. Day two autogamous
pollination yielded greatest percent fruit set and germination success (bold).
1Percent fruit set as a fraction of hand pollinations attempted, reported as sample size
2Percent germination success as a fraction of seeds collected, reported as sample size
Self-
pollination
(autogamous)
Percent
fruit set
(sample
size) 1
Percent
germination
success
(sample
size) 2
D1 19.3 (57) 28.0 (164)
D2 29.2 (154) 49.6 (733)
D3 7.9 (76) 29.9 (128)
D4 0 (12) 0
not pollinated 0.8 (127)3
not bagged 59.0 (144)
87
Day two autogamous pollinations are most successful
Hand pollination in A. fimbriata was most successful on day two, both in terms of
the numbers of seed pods produced from attempted pollinations and the viability of seed
produced in those fruits (Table 2S-3). Pods were produced from 29% of the hand
pollinations attempted on day two, from 19% of the pollinations attempted on day one,
and only 8% of hand pollination attempted on day three. The gynostemia of day four
flowers were fragile and often broke, constraining efforts to attempt hand pollinations in
flowers more than two days beyond the day of anthesis. Fruits produced by hand
pollination contained a far more variable number of seeds than fruit produced by open
air, insect pollinator-mediated pollination, which typically contained 10/15 seeds per
carpel, six carpels per fruit. The reduced seed production in fruits from self (hand-)
pollination events may be due to limited pollen transfer with hand pollination, or to
mechanical damage.
88
Self-compatibility in Aristolochia elegans
Autogamous, geitonogamous, and xenogamous pollinations were evaluated using
the hand pollination methods described for A. fimbriata, using very small sample sizes.
Self pollinations of day one and day two flowers were very successful (Table 2S-4).
Viability of seed produced by hand-pollinations was not evaluated for A. elegans.
Seed germination methods
We developed in vitro methods for germinating seeds to support collection of
seedling tissues and for comparisons of seed viability. Seeds collected from fruits
Table 2S-4: Percent fruit set (sample size) in A. elegans resulting from hand pollinations
of flowers on days 1-4 with autogamous, geitonogamous and xenogamous pollen from
flowers on days 1-3. Compare to 0% fruit set on 19 flowers, bagged before anthesis to
prevent pollinator entry. Day 1 and 2 autogamous pollinations worked well, as did
xenogamous pollinations (bold)..
Age of
stigmatic
surfaces ↓
A g e o f p o l l e n s o u r c e
P o l l i n a t i o n t y p e
Autogamous Geitonogamous Xenogamous
D1 D2 D3 D2 D3 D2
D1 90 (10) 33 (3) 67 (24)
D2 80 (5)
D3 20 (5)
D4 0 (1)
89
produced by open-air pollination of mixed VL and NV greenhouse stocks germinated
significantly better in wet toweling packets in a growth chamber providing 16h days, 8h
nights, 30°C days, 16°C nights than in sucrose-free media. Seeds incubated in 16h light/8
h did not germinate significantly better than seeds incubated in total darkness, but the
first set of true leaves never developed in seedlings emerging in total darkness, thus the
etiolated seedlings died (data not shown). However, seeds germinated most quickly in
potting medium, with bottom heat, in the greenhouse (up to 95% in two weeks) than in
wet toweling in a growth chamber. Furthermore, seedlings transferred from wet toweling
to potting medium in the greenhouse after the first set of true leaves appeared typically
bloomed two to three weeks later (at an older age) than did seeds germinated in potting
medium in the greenhouse. This delay reflects the additional resources available to seeds
germinating in potting medium, evident as a larger rooted seedling at four weeks than
observed on seedlings germinated in toweling (data not shown).
Effect of seed age on germination
Using in vitro (in wet toweling, 25 seeds per replicate) germination, we evaluated
the effect of age on seed viability and found no significant difference in total percent
germination of seeds planted at seven day intervals, from 7 days after collection through
147 days after collection, and mean germination was 90% (n=15) by day 63 (data not
shown). Furthermore, germination in 2.5 year old seeds was not significantly lower than
in 2 year old or 1.5 year old seeds, and mean germination was 80% (n=12) by day 77.
90
Table 2S-5: Genome sizes, vouchers, sources and accessions for sequence data used.
Abbreviations: R, Rice (Oryza sativa ssp. japonica cv. „Nipponbare‟ 0.9 pg/2C); S,
Soybean (Glycine max cv. „Dunbar‟ 2.35 pg/2C), T, Tobacco (Nicotiana tabacum
cv.‟SR-1‟ 9.15 pg/2C); BG, Botanical Garden; PAC, Pennsylvania State University; DR,
Dresden. Mbp DNA for plant species is based on the assumption 1pg=980 Mbp
according to (Pfeifer, 1966; Wang et al., 2005). (BJB=collection number of the first
author). XXX Genbank accession numbers -- need to submit them when reviews come
back).
Taxon genome size (pg/2C)
+/-std. dev. (n=4) S
tandar
d
DNA content of
sample species (Mbp/1C)
Voucher, herbarium
Source GenBank
Accession
Aristolochia acuminata Lam.
1.15 +/-0.007 S 564 BJB06.06A, PAC Victor Wong (private coll.)
Wanke & Neinhuis 146, DR BG Dresden XXX
Aristolochia
anguicida Jacq.
0.81 +/-0.005 S
397
BJB06.03A, PAC Mario Blanco (private coll.)
0.89 +/-0.011 436
Wanke & Neinhuis s.n., DR BG Bonn XXX
Aristolochia
californica Torr.
1.58 +/-0.006 S 774
BJB05.02A, PAC Albert J. Hill (private coll.)
1.59 +/-0.011 R 779
Wanke & Neinhuis 143, DR BG Dresden XXX
Aristolochia
clematitis L.
0.96 +/-0.001 S
470
BJB03.07A, PAC
Seneca Hill Perennials, New
York
0.99 +/-0.001 485
0.90 +/-0.012 S
441
BJB03.03A, PAC BG University Ulm
0.98 +/-0.008 480
W. Stahmüller, KL Croatia, Is. Ilovik/Asinello XXX
Aristolochia elegans Mast.
0.81 +/-0.007 S
397
BJB03.02A, PAC Park Seed Company, cat. #0179-7 XXX 0.81 +/-0.004 397
Aristolochia
fimbriata Cham.
0.91 +/-0.004 S
446
BJB03.04A, PAC
Larry D. Rosen (“VL”)
(private coll.) XXX 0.97 +/-0.004 475
0.96 +/-0.001 S
470 BJB03.05A, PAC
Russ Strover (“VL”)
(private coll.)
0.84 +/-0.009
S
412
BJB04.08A, PAC
Jardim Botanico, Departamento de Botanica,
(“NV”), Universidade de
Coimbra XXX 0.89 +/-0.004 436
Aristolochia
gigantea Mart.
& Zucc.
0.89 +/-0.013
S
436
BJB03.01A, PAC
Kartuz Greenhouses,
California XXX 0.88 +/-0.009 431
Aristolochia
grandiflora Sw.
1.19 +/-0.009
S
583
BJB03.02A, PAC
Mario Blanco
(private coll.)
1.20 +/-0.006 588
1.17 +/-0.008 573
1.21 +/-0.012 593
1.13 +/-0.012 554
91
1.26 +/-0.006 617
1.20 +/-0.006 R 588
Wanke & Neinhuis s.n., DR BG Dresden XXX
Aristolochia
goldieana (Hook.f.) Hutch.
& Dalz. 4.28+/-0.113 S 2097 Neinhuis 117, DR BG Dresden XXX
Aristolochia holostylis
(Duchartre) F.
Gonzalez 0.96+/-0.029 S 470 Neinhuis 116, DR BG Dresden
Aristolochia lindneri Berg. 0.67+/-0.018 S
328 Neinhuis s.n., DR
BG Dresden, Bolivia, San Jose de Chiquitos
Aristolochia macrophylla
Lam.
1.54 +/-0.011 S
755
BJB04.07A, PAC Dawes Arboretum, Ohio
1.53 +/-0.033 750
1.52 +/-0.025 R
745
1.52 +/-0.018 745
Neinhuis s.n., DR BG Dresden XXX
Aristolochia maxima Jacq
0.77+/-0.009 S 755 N.Pabon-Mora & F. Gonzalez,
NY NYBG
0.78+/-0.017 R 764
Gonzalez 4018, COL Panama, Parnama, XXX
Aristolochia. sp. 0.74+/-0.017 S 363 Wanke & Neinhuis s.n., DR BG Munich XXX
Aristolochia
passiflorafolia Rich.
0.74 +/-0.006 S
363 BJB06.05A, PAC
Mario Blanco
(private coll.)
Neinhuis s.n., DR Cuba, BG Dresden XXX
Aristolochia
prevenosa
F.Muell. 1.83+/-0.006
S
1793 Neinhuis & Wanke s.n., DR
BG Dresden, Queensland,
Australia, BG Dresden XXX
Aristolochia
promissa
(Mast.) Keay 4.41+/-0.013
S
4321 Neinhuis 118, DR BG Dresden XXX
Aristolochia
ringens Vahl.
0.93 +/-0.004 S
456
BJB06.07A, PAC
Mario Blanco
(private coll.) XXX 0.91 +/-0.003 446
Aristolochia
serpentaria L.
1.69 +/-0.024 S
828
BJB03.03A, PAC
Larry D. Rosen
(private coll.)
1.67 +/-0.019 818
1.57 +/-0.018 S
769
BJB05.01A, PAC B&T World Seeds
1.56 +/-0.013 764
1.61 +/-0.035 R
789
1.58 +/-0.008 774
Priv. coll. B. Westlund USA, Texas, Travis Co. XXX
Aristolochia
tomentosa Sims
1.13 +/-0.008 S
554
BJB03.06A, PAC
Seneca Hill Perennials, New
York
1.39 +/-0.010 681
1.40 +/-0.022 S 686
BJB06.01A, PAC Dawes Arboretum, Ohio
1.44 +/-0.011 R 706
Neinhuis 113, DR BG Dresden XXX
Aristolochia triactina (Hook.
f.) Hutch &
Dalz. 4.39+/-0.059
S
4302 Neinhuis 119, DR BG Dresden XXX
92
Aristolochia
trilobata L.
0.91 +/-0.002
S
446
BJB04.04A, PAC Kartuz Greenhouses, California XXX
0.99 +/-0.010 485
1.01 +/-0.002 495
1.02 +/-0.004 R 500
Asarum canadense L.
10.19 +/-0.040
S
4993
BJB04.03A, PAC Joel McNeal, (private coll.) XXX
9.97 +/-0.083 4885
11.04 +/-0.307 5410
Saruma henryii
Oliv.
6.12 +/-0.044 T 2999 BJB06.08A, PAC
Heronswood Nursery,
Washington
Neinhuis 120, DR BG Dresden XXX
Thottea
siliquosa (Lam.)
Hou 1.25 +/-0.018 S 613 Neinhuis 121, DR India, Kerala, BG Dresden XXX
93
Figure 2S-3: Aristolochia fimbriata genotype and perianth detail A,B VL genotype C, D
NV genotype A, C Presence, absence of leaf variegation B, D Perianth varies in shape
and color E. Perianth is highly modified for insect pollination. Modifications include
limb (li), tube (tu), syrinx (sy), utricle (ut) and gynostemium (gy), which has stamen
locules on the outside and interior stigmatic surfaces. Glass model by Leopold and
Rudolph Blatschka illustrated by Fritz Kredel (reproduced with permission). .
94
Figure 2S-4: Phylogram of Aristolochiaceae relationships, showing minimal variation in
branch lengths within Aristolochiaceae. Only one tree was found in search with
maximum likelihood methods.
Chapter 3
In vitro propagation, shoot regeneration and rooting protocols for
Aristolochia fimbriata, potential model plant for basal angiosperms
This chapter is a manuscript for submission by Barbara J. Bliss, Hong Ma, Claude
dePamphilis, Lena Landherr, Yi Hu, Siela N. Maximova to Plant Cell, Tissue and Organ
Culture.
This work resulted from collaboration among the labs of Drs. Claude
dePamphilis, Hong Ma, and Siela Maximova. Lena Landherr performed experiments
optimizing reduced light/enriched nutrient micropropagation methods (REN2, Table 3-1),
and initial stem/petiole segment regeneration methods (Table 3-6). Yi Hu assisted with
micropropagation and regeneration experiments (Tables 3-3, 3-4). All other results were
generated by Barbara Bliss.
96
Short original article for submission to Plant Cell. Tissue and Organ Culture
Title: Regeneration and plantlet development from somatic tissues of Aristolochia
fimbriata
Authors: Barbara J. Bliss1, Lena Landherr
1, Claude W. dePamphilis
1, Hong Ma
1, Yi Hu
1,
Siela N. Maximova2
1Department of Biology, Institute of Molecular Evolutionary Genetics, and the Huck
Institutes of the Life Sciences, 201 Life Sciences Building, The Pennsylvania State
University, University Park, PA 16802, USA
2The Department of Horticulture, 421 Life Sciences Building, The Pennsylvania State
University, University Park, PA 16802, USA
S. N. Maximova (author for correspondence);
The Department of Horticulture, 421 Life Sciences Building, The Pennsylvania State
University, University Park, PA 16802, USA
e-mail: [email protected]
Tel: 814-863-7286
Fax: 814-865-1357
Suggested running head: Regeneration and plantlet development from somatic tissues of
Aristolochia fimbriata
97
Abstract
Aristolochia fimbriata is a small herbaceous perennial in the basal angiosperm
family Aristolochiaceae. The family contains diverse floral forms ranging from radial to
monosymmetric flowers with a wide variety of insect pollinators. Additionally,
Aristolochia species contain secondary metabolites that are important natural toxins and
traditional medicines, and are critical to the reproduction of swallowtail butterflies. These
characteristics, in combination with the small genome size and short life cycle of
Aristolochia fimbriata, have prompted further development of this species as a model
system to study the evolution of basal angiosperms. As a prerequisite for developing a
genetic transformation procedure for Aristolochia, we have developed protocols for in
vitro plant multiplication, shoot organogenesis, rooting, and acclimation of tissue culture-
derived Aristolochia plants. Nodal cuttings from two different Aristolochia genotypes
were sterilized and cultured for in vitro propagation. Explants were further multiplied in
large numbers and rooted with up to 100% success. Shoot regeneration was achieved
from whole leaf, internodal stem, and petiole explants. Regeneration was observed
starting at 28 days after culture initiation on MS basal medium supplemented with 0.5
mg/L 6-benzoamino-purine (6BA), 1.0 mg/L -naphthalene acetic acid (NAA), and 1.0
mg/L thidiazuron (TDZ), followed by regeneration on MS basal medium supplemented
with 1.75 mg/L 6BA and 1.0 mg/L NAA. Maximal shoot primordia formation occurred at
frequencies up to 97%, with stem explants. Large numbers of regenerated shoots were
rooted and acclimated to greenhouse conditions and developed flowers after 2-4 weeks.
98
Keywords: Aristolochia fimbriata, basal angiosperm, micropropagation, shoot
organogenesis
99
Introduction
Aristolochia fimbriata is a flowering perennial plant cultivated for its interesting
flowers, attraction of butterflies, and traditional medicinal properties. The flowers of
Aristolochia species have a unipartite, monosymmetric perianth adapted for insect
pollination, while other genera in Aristolochiaceae have radially symmetric flowers (e.g.,
Saruma, Asarum) (Gonzalez and Stevenson, 2000b). Aristolochia species have been
studied for their pollination strategies associated with floral aroma and a fly-trapping
perianth (Petch, 1924; Hall and Brown, 1993; Sakai, 2002; Banziger et al., 2006;
Murugan et al., 2006; Trujillo and Sersic, 2006), as well as for species-specific host plant
relationships with swallowtail butterfly larvae. Secondary metabolites produced by
Aristolochia plants are critical for the defense and survival of the butterflies during their
larval feeding stage (Rausher, 1981; Klitzke and Brown, 2000), such that the decline of
particular swallowtail butterfly populations is attributed to the declining distribution of
particular Aristolochia species (Sands et al., 1997). These secondary metabolites include
aristolochic acids and aristolactams produced via alkaloid biosynthesis pathways (Kumar
et al., 2003).
Aristolochia species, particularly root tissues, have been used as traditional
medicines (Reddy et al., 1995) and have been subsequently studied to reveal the
molecular mechanisms underlying their observed human health effects (Lemos et al.,
1993; Levi et al., 1998; Nortier et al., 2000; Qiu et al., 2000; Hranjec et al., 2005; Hwang
et al., 2006; Meinl et al., 2006). The potent toxins found in Aristolochia offer promise in
specific applications, including antivenom (Otero et al., 2000; Jimenez-Ferrer et al.,
100
2005; Abubakar et al., 2006), antibacterial (Gadhi et al., 1999; Gadhi et al., 2001a; Gadhi
et al., 2001b), antifertility (Pakrashi and Chakrabarty, 1978; Pakrashi and Pakrasi, 1979;
Gupta et al., 1996), cytotoxic (Kupchan and Doskotch, 1962; Hinou et al., 1990),
antimicrobial or trypanocidal (Abe et al., 2002; Elizabeth and Raju, 2006; Kumar et al.,
2006), and insecticidal (Lajide et al., 1993; Broussalis et al., 1999; Nascimento et al.,
2004; Jbilou et al., 2006).
In addition to its medicinal value and importance for butterfly reproduction,
Aristolochia occupies an important phylogenetic position. As a basal angiosperm from
the magnoliid clade (Jansen et al., 2007), it offers uncommon opportunities for studying
the evolution of development in flowering plants. Evolutionarily conserved
developmental pathways, such as those controlling floral organ identities in angiosperms,
have been identified via functional studies in model plants including Arabidopsis,
Petunia, Zea, and Oryza f(Kramer et al., 1998; Ma and dePamphilis, 2000; Whipple et
al., 2004; Agrawal et al., 2005; Zahn et al., 2005a) Comparative studies of these plant
species have led to hypotheses about the evolution of gene function in flowering plants
(Jaramillo and Kramer, 2004; Kim et al., 2006). However, the monocot and eudicot
lineages from which current model systems arose diverged only about 113-133 million
years ago (mya), some 28-48 million years after the angiosperm divergence
(approximately 161 mya) (Bell et al., 2005; Leebens-Mack et al., 2005). If pathways
found in monocots and/or core eudicots are also found to be present in a basal
angiosperm such as Aristolochia, this would imply that the conserved components were
present in angiosperms before the origin of these two major lineages of angiosperms.
Comparative gene-functional analysis between basal angiosperms and derived model
101
plants is a powerful approach to test this hypothesis. Therefore, the development of a
basal angiosperm species as an experimental model plant will be of great benefit to the
study of plant evolutionary biology.
Plant genetic manipulation is essential for testing hypotheses about gene function,
and Agrobacterium-mediated transformation offers a convenient and efficient tool for this
purpose. Transformation can be used to generate both loss-of-function and gain-of-
function transgenic plants. The easy-to-use and efficient in planta “dip” transformation
method currently used with Arabidopsis thaliana is rare among plant systems (Bent,
2000). Traditional tissue culture based transformation systems are used in other model
plant species, including Nicotiana tabacum (An et al., 1986), Medicago truncatula
(McKersie et al., 1997), Lotus japonica (Handberg and Stougaard, 1992), Petunia
hybrida (Napoli et al., 1990), Lycopersicon esculentum (Dan et al., 2006), Brachypodium
distachyon (Draper et al., 2001), Oryza (Sallaud et al., 2003) and Zea (Frame et al.,
2002).
Efficient propagation and regeneration systems for Aristolochia fimbriata are
important prerequisites for the development of Agrobacterium tumefaciens-mediated
transformation. Prior to this study, in vitro methods for Aristolochia indica, a species
valued for medicinal use (Shafi et al., 2002), were developed using axillary shoot
multiplication and shoot organogenesis to provide source tissue for the purification of
secondary metabolites (Remashree et al., 1997); (Soniya and Sujitha, 2006). Here we
report the development of methods for micropropagation and shoot regeneration of two
genotypes of A. fimbriata, a species with a small genome size (Bharathan et al., 1994)
and promising characteristics for use as a basal angiosperm model system, such as short
102
generation time and small size. These methods will provide tools for fast propagation of
large number of clonal plants and will support the development of genetic transformation
methods.
Materials and Methods
Plant material
Seeds of Aristolochia fimbriata were provided by Larry D. Rosen, Florida, USA
and by Jardim Botanico, Departamento de Botanica, Universidade de Coimbra, Portugal.
Plants were grown from the seeds and maintained in a greenhouse at The Pennsylvania
State University, University Park, PA. All seeds were germinated in soil-free potting
medium (Pro-Mix BX, Premier Horticulture Inc., Quakertown, PA) in shallow
germination trays with drainage holes, in the greenhouse at 18-27C (varying from night
to day) and 40-70% humidity. The trays were incubated on heating mats operating at
approximately 27C. Natural day length was supplemented with high-pressure sodium
lamps (1000 watt) from October through April to provide twelve-hour days. Plants
received regular watering as needed. Depending on the stage of growth, regular fertilizer
applications were provided, as a drench, alternating Peter's Professional 15-16-17 Peat
Lite Special at 200 PPM nitrogen (once to twice weekly) with Peters Professional 21-7-7
Acid Special (Scotts Horticulture, Marysville, OH) at 200 PPM nitrogen (approximately
every six weeks). The plants were drenched once a month with 100 ppm chelated iron
(Sprint 330 10% iron, RoseCare.com, Santa Barbara, CA). Plants grown from Florida
103
seeds had variegated leaves (VL), while those from Coimbra were not variegated (NV)
(Figure 3-1 A and B).
Micropropagation initiation and multiplication
Six month old, healthy, flowering plants of both NV and VL genotypes grown in
greenhouse were selected for in vitro propagation. Green stems were collected from
newly developed branch terminals (NV: n=30, VL: n=35, VL: n=60). Only the three most
distal (youngest) nodes per stem were used, such that the excised explants were 1 - 2 cm
long, 1.5 – 2.0 mm in diameter, and contained one terminal or axillary bud. Fully
expanded leaves were removed and the resulting green nodal cuttings/explants were
sterilized for 20 minutes in 1% Chlorox solution (Commercial Solutions
; 6.15% sodium
hypochlorite) with 1% Tween 20 detergent (polyoxyethylene sorbitan monolaurate;
Sigma Chemical Co., St. Louis, MO, USA). Explants were rinsed three times in sterile
de-ionized water and stems freshly trimmed to a 45 angle before being inserted into 60-
75 mL micropropagation initiation medium (MI, Table 1) in disposable plastic food cups
(Sweetheart, Owings Mills, MD, #DSD8X and #LDS58). Five explants were placed in
each cup. Cultures were incubated in a Conviron growth chamber (Winnipeg, Canada) at
25C with 300 micromoles m-2
sec-1
light (16 hours a day) for approximately 20 days.
For further in vitro multiplication, single node stem explants 1-2 cm long with a terminal
or axillary shoot were excised from the newly developed tissue cultured A. fimbriata
plants and cultured on different micropropagation media (MP). The MP media evaluated
104
105
contained 6-benzoaminopurine (6BA) and -naphthalene acetic acid (NAA) in eight
combinations (Table 3-1). The number of shoots propagated from each greenhouse
cutting was counted after 21 days and significant differences among the media were
determined by the nonparametric Mann-Whitney test, using Minitab WINSV12.11
software (State College, Pennsylvania, U.S.A.).
Figure 3-1: Aristolochia fimbriata regeneration. A. Variegated leaf (VL) genotype; B.
Non-variegated (NV) genotype; C. Regenerating petiole from whole leaf explant; D.
Elongating shoots; E. Regenerating stem from small section; F. Regenerating petiole
from small section; G. Single shoot, separated and rooted; H. Rooted regenerant after 7
days in soil. Scale bars = 1cm (A, B, C, D, G, H) or 1 mm (E, F).
Table 3-1: Media formulations for micropropagation and shoot organogenesis.
Media Components MI MP RI REN1 REN2 SI SR
MS basal medium
(g/L) 4.4 4.4 2.2 2.2 3.3 4.4 4.4
6BA (mg/L) 2.5 1.0 -
5.0 - - - 0.0 - 0.5 1.5 - 2.0
NAA (mg/L) - 0.0 -
1.0 - - - 0.50 - 1.75 1.0
TDZ (mg/L) - - - - - 0.0 - 1.1 -
IBA (mg/L)
0.25 - 0 - 1 - - - -
Sucrose (g/L) 30.0 30.0 20.0 20.0 20.0 30.0 30.0
PhytaGel (g/L) 2.0 2.5 2.7 2.8 2.7 2.7 2.7
pH 5.5 5.7 5.5 5.7 5.7 5.7 5.7
Abbreviations: MI - micropropagation initiation; MP – micropropagation medium; RI -
root initiation; REN1 - root elongation #1; REN2 - root elongation #2; SI - shoot
induction; SR – shoot regeneration; MS - Murashige and Skoog basal medium; 6BA - 6-
benzoamino-purine; NAA - -naphthalene acetic acid; TDZ - thidiazuron; IBA - indole-
3-butyric acid.
106
In vitro rooting
In vitro propagated apical or axillary shoots with two nodes and two buds were
excised from propagules in MP medium. Fully expanded leaves were removed, the stems
freshly cut at a 45 degree angle and inserted into hormone-free modified root elongation
medium #1 or #2 (REN1 or REN2) in which the agar in the original RE medium
(Maximova et al., 1998), was substituted with PhytaGel (REN1, REN2, Table 3-1).
Additionally, REN2 medium contained 50% more MS salts and vitamins than REN1.
Explants were placed in the medium straight up so that the lowest node and stem below
were fully immersed. Explants were incubated in the dark for 3 days and then transferred
to low-light conditions (20-100 micromoles m-2
sec-1
, 16 hours a day) at 25C. When
roots were visible, each plant was transferred to an individual container with fresh REN1
(cultured under high-light conditions: 300 micromoles m-2
sec-1
light, 16 hours a day) or
REN2 medium (cultured under medium-light conditions: 150 micromoles m-2
sec-1
) at
25C. Rooted plants were maintained on REN1 or REN2 media and every 21-28 days,
plants were trimmed and transferred to fresh medium. Plant/shoot clusters of more than
three shoots were divided at the crown so that individual rooted plants and non-rooted
shoots were separated and transferred to fresh medium. Only rooted plants were used as
stock plants in further rooting and regeneration experiments.
The effect of incubation with different concentrations of indole-3-butyric acid
(IBA) on rooting efficiency was evaluated. For this experiment 8-15 stem explants from
NV and VL genotypes were excised from rooted plantlets in REN1 medium and inserted
into solid root initiation (RI) medium supplemented with 0, 0.5, or 1.0 mg/L IBA (RI,
107
Table 3-1). Explants contained two nodes and fully expanded leaves. Explants in RI
medium were incubated in the dark for 3 days at 25C, then transferred to low light
conditions until roots appeared, at which time the newly rooted plantlets were returned to
culture on fresh hormone-free REN1 medium in a Conviron growth chamber under high-
light conditions. The number of rooted plants was recorded after 30 days. To determine if
any factors or interactions among factors had a significant effect on the number of roots
produced or average root length the percentage rooting data were analyzed with the two-
way general linear ANOVA model of Minitab WINSV12.11.
Shoot regeneration from whole leaf explants
All regeneration media evaluated contained 4.4 g/L Murashige and Skoog (MS)
basal medium salts (Murashige and Skoog 1962) with Gamborg vitamins (Gamborg et al.
1968) (Sigma M5519), 30 g/l sucrose, solidified with PhytaGel (Sigma P8169) (Table 3-
1). Leaf explants were cultured first on shoot induction media (SI) with different
concentrations of 6BA, NAA and thiadiazuron (TDZ) (Table 3-1). SI media (50 ml
aliquots) were poured in 100 x 20 mm Petri dishes (VWR cat. 25382-166, Becton
Dickinson Falcon, Franklin Lakes, NJ, USA). The top two fully expanded, dark green
apical leaves of primary or axillary shoots from in vitro rooted NV plants and VL plants
on REN1 medium were cut into approximately1.5 cm2 explants. The explants from the
base of the leaves included 0.5-1 cm (more than one half) of the petiole. After loosely
chopping up the medium with sterile forceps, the explants were gently pressed, abaxial
108
surface up, into the medium to assure good contact. A total of 25 explants per genotype
(5 explants per Petri dish) were cultured for the individual treatments evaluated. Cultures
were incubated in the dark at 25C for 14 days on SI medium. The explants were then
transferred to shoot regeneration (SR) media with different concentrations of 6BA (Table
3-1), and incubated for an additional 14 days in the dark, at 25C. During the course of
the experiment the leaf explants expanded and were transferred from Petri dishes to
disposable plastic food cups as explant size increased. After a total of 28 days in the dark,
cultures on SR media were transferred to low-light for shoot elongation. The explants
were inspected and transferred to fresh SR every 14 days, 4 more times. Explants
producing shoot primordia were subdivided into clusters of 1-3 shoots as needed, to allow
elongation and additional shoot proliferation. After 21-42 days of elongation, individual
shoots with two extended nodes and leaves were transferred from SR media to rooting
medium (REN1, Table 3-1) as described above. A binary logistic regression model was
applied to all the media treatments producing shoots using Minitab WINSV12.11 to
determine if plant genotype, level of NAA in the SI media, level of 6BA in the SR media,
or interactions had a significant effect on the percent of explants regenerating.
Shoot regeneration from petiole and stem sections
For the first two sets of experiments, explants were generated by excising 2 - 3
mm long stem and petiole segments from the upper three nodes of dark green rooted A.
fimbriata VL and NV plants maintained in REN1. Explants were placed horizontally on
SI medium (Table 3-1) containing 0.5 mg/L 6BA, 1 mg/L NAA, and 1 mg/L TDZ, and
109
pressed lightly into the media to ensure adequate contact (up to 60 explants per plate).
Cultures were incubated in the dark at 25-27C for 14 days, and then transferred to low-
light. Cultures were separated in three different groups and each group was incubated on
SI for a different period of time including: 14, 21 or 40 days. After that, the cultures from
each group were transferred to SR medium (Table 3-1) containing 1.75 mg/L 6BA and
1.0 mg/L NAA. All media were refreshed every 14 days. Number of explants
regenerating was evaluated at 40 days. Using Minitab WINSV12.11, a binary logistic
regression model was applied to determine if genotype, tissue type, length of time on SI
media, or interactions significantly affected regeneration.
In a third set of experiments, stem and petiole explants (2-3 mm long) were
selected from rooted VL plants maintained on REN2 medium (Table 3-1). The explants
were cultured on SI medium (20 to 30 explants per plate) for 14 days as described above
then transferred to fresh SR medium every 14 days. At each transfer, cultures were
evaluated and explants with visible shoot primordia were moved to low-light. The rest of
the explants were incubated in the dark until primordia were developed. When distinct
leaves were visible, the base and sides of the newly regenerated shoots were trimmed to
remove remaining callus tissue and the shoots were transferred to REN2 medium in
sterile cups under low-light to promote shoot elongation. Explants on REN2 were
transferred to fresh medium every 4-5 weeks until roots and shoots with fully expanded
leaves were developed. Plants with two or more roots and minimum 3 cm total root
length were transplanted to into multi-cell plant trays with 4-cell packs (Kord, Canada)
filled with water-saturated soil-free potting mix consisting of one part Metro-Mix 200
(Sun Gro Horticulture. Ltd., Vancouver, B. C., Canada) and four parts Miracle-Gro
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Potting Mix, which contains Miracle-Gro slow-release plant fertilizer (Scotts Miracle-
Gro, Scotts Horticulture, Marysville, OH). Potted plants were covered with clear plastic
lids and placed in the growth chamber under medium-light, shaded with one layer of
white paper towels for one day to reduce transplant shock. Plants were acclimated for one
more week by gradually opening the plastic lids to reduce the humidity. The lids were
completely removed by the end of day three. The acclimated plants already in soil were
then transferred to the greenhouse (conditions previously described) where they were
monitored weekly and the number of healthy plants was recorded.
Results
Micropropagation and in vitro rooting
To generate and maintain a large number of aseptic plants as a source of explant
tissue for regeneration and transformation experiments, we developed protocols for A.
fimbriata micropropagation and subsequent in vitro rooting (Fig. 3-2). Single node green
cuttings from two different genotypes of A. fimbriata were obtained from greenhouse
stock plants and introduced into tissue culture on MI medium (Table 3-1). All cuttings of
both genotypes developed at least two new axillary shoots, doubling the number of shoots
in 21 days. To optimize micropropagation, we evaluated the effect of NAA and 6BA
concentrations in MP medium. At 21 days after culture initiation the mean multiplication
rate for treatments without NAA was significantly greater (P<0.05) than for treatments
containing NAA (Table 3-2). Statistical analysis also indicated that this response was not
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a function of 6BA level (Table 3-2). However, our results demonstrated that explants in
MP containing 1 mg/mL 6BA and no NAA had the highest mean multiplication rate of
3.06 fold (P<0.05) (Table 3-2).
Shoots obtained by multiplication or de novo organogenesis were further cultured
for rooting. To optimize the rooting protocol, we experimented with RI medium
supplemented with different concentrations of IBA (Table 3-1), providing a brief period
of root induction, followed by transfer to hormone free medium (Fig. 3-2). The number
of roots and average root length were recorded for each of the concentrations after 28
days of culture. The data met the normal distribution assumption of the two-way
ANOVA model, but no significant effects of genotype, IBA concentration, or interaction
were detected. Our results indicated that all explants placed in hormone-free medium
Figure 3-2: Schematic representation of micropropagation and regeneration protocols of
A. fimbriata. The number of days at each step is indicated
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(REN1, Table 3-1) with the lowest node submerged, followed by culturing in low-light,
developed roots in 21-30 days (Table 3-3). Therefore we discontinued use of RI medium
and continued to introduce single shoots directly into REN1 or REN2 medium for rooting
and further maintenance. The tissue culture initiation, multiplication, and rooting
protocols reported here (Fig. 3-2) yielded approximately six times more plants than the
original number of explants introduced into culture and have been successfully
implemented with both genotypes (NV, VL) of greenhouse-cultivated plants ranging
from three months to four years in age.
Rooted plants in the initial REN1 media developed large, deep green leaves,
abundant roots, and multiple stems from a single crown, similar to plants grown in the
greenhouse. Compact, rooted stock plants in REN1 media provided an ample supply of
fresh leaves for shoot regeneration experiments. The stock plants were trimmed and
Table 3-2: The effect of plant growth regulators on axillary shoot proliferation at 21 days
after culture initiation.
Combination of plant growth
regulators # New shoots
harvested
# Nodal
cuttings
initiated
Multiplication
factor NAA mg/L 6BA mg/L
1.0 3 36 25 1.44
0.5 3 36 20 1.80
0.1 5 50 22 2.27
0.1 4 48 25 1.92
0.1 3 46 24 1.92
0.1 2 35 20 1.75
0.0 3 47 20 *2.35
0.0 1 52 17 *3.06
*Mean multiplication for treatments without NAA was significantly greater (P<0.05).
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transferred to fresh REN1 medium every 3-5 weeks. To reduce the need for frequent
transfer and to improve the overall health of the stock plants, during the later stages of the
experiment, the rooting medium was modified by increasing the MS basal media
concentration by 50% (REN2) and the light intensity was decreased by about 50% to 150
micromoles m-2
sec-1
. Plants under these conditions produced longer internodes and
reduced number of roots compared to the REN1 plants. The plants on REN2 also
required media replacement less frequently (every 4-5 weeks).
Shoot regeneration
To develop shoot organogenesis methods for A. fimbriata, we subjected leaf
explants to different concentrations of auxins and cytokinins in a two-step protocol
consisting of shoot induction (SI) and shoot regeneration (SR) steps. Results from our
preliminary experiments, titrating TDZ and 6BA in different combinations, indicated that
shoot primordia were initiated only on SI media supplemented with 0.5 mg/L 6BA and
1.0 mg/L TDZ (data not shown). Thus we further optimized the protocol by evaluating
Table 3-3: Effect of indole-3-butyric acid (IBA) on rooting of shoots multiplied in vitro.
Two-way ANOVA indicated no significant differences ( = 0.05) in average number of
roots/shoot or average root length among IBA levels.
IBA (mg/L) % Rooted
shoots (n)
Average number
of roots/shoot
± SD a
Average total root
length/plant (mm)
± SD b
0.0 100 (17) 2.6 ± 1.1 32.8 ± 14.6
0.5 100 (29) 2.6 ± 1.6 29.2 ± 15.8
1.0 100 (27) 2.7 ± 1.6 27.2 ± 14.2
a - Only the number of the primary adventitious roots was counted.
b - All primary and secondary adventitious roots were measured.
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four different concentrations of NAA in the SI medium and three different concentrations
of 6BA in the SR medium. Each NAA treatment in the SI medium was followed by each
6BA treatment in the SR medium, for a total of 24 separate combinations of SI/SR
medium (Table 3-4). Shoot regeneration from whole leaf explants was observed on all SI
media containing 1.0 mg/L NAA (Table 3-4). Regeneration from explants induced with
SI media containing 1.0 mg/L NAA was not significantly different than media containing
0.5 or 1.5 mg/L NAA. However, 1.75 mg/L NAA in the SI media produced significantly
fewer regenerating explants (P<0.05). The SR media supplemented with 1.5 or 2.0 mg/L
Table 3-4: Direct organogenesis of Aristolochia fimbriata from whole leaf explants. The
shoot induction medium (SI) contained 0.5 mg/L 6BA and 1.0 mg/L TDZ in addition to
the NAA concentrations evaluated. The shoot regeneration medium (SR) contained 1.0
mg/L NAA in addition to the 6BA concentrations evaluated.
Induction
medium
Regeneration
medium % Explants regenerating
*NAA (mg/L) *6BA (mg/L) VL
NV Overall
0.50 1.50 20 4 12
0.50 1.75 0 0 0
0.50 2.00 16 4 10
1.00 1.50 20 8 14
1.00 1.75 36 36 36
1.00 2.00 12 4 8
1.50 1.50 0 0 0
1.50 1.75 8 20 14
1.50 2.00 4 16 10
1.75 1.50 0 0 0
1.75 1.75 8 0 4
1.75 2.00 0 0 0
*Binary logistic regression factors with a significant effect on regeneration (P<0.05). The
overall best combination of media is highlighted in bold.
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6BA produced lower number of regenerating explants than 1.75 mg/L 6BA (P<0.05).
Overall, the greatest percentage of regeneration (36%) occurred in explants initiated on
SI medium containing 1.0 mg/L NAA, followed by regeneration on SR media containing
1.75 mg/L 6BA (Table 3-4). The results of binary logistic regression analysis indicated
that the amounts of NAA in the SI media and 6BA in the SR media each independently
had a significant effect on regeneration. Genotype and interactions were not significant
factors in the statistical model. Close observation of whole leaf explants on SR medium
indicated that shoot primordia originated only from the petiole region (Fig. 3-1C). Shoots
elongated and developed 2-3 leaves between 28 and 56 days on SR medium (Fig. 3-1D).
To maximize the efficiency of the protocol, we evaluated the regeneration
potential of small sections of different vegetative organs. Root and leaf blade sections,
and sections containing the base of the leaf without the petiole did not regenerate.
Regeneration from leaf bases with the petiole attached (data not shown) was comparable
to that of the whole leaf explant (36%, Table 3-4). Stem and petiole sections regenerated
more successfully (up to 67.5 %), with minimal callus formation (Table 3-5).
Additionally, stem and petiole explants offered the substantial advantage of reducing the
space required by the regeneration system by 80%.
The culture time of stem and petiole explants on SI medium was studied in two
separate experiments where incubation periods of 14, 21 and 40 days were evaluated. For
this experiment explants were collected from plants cultured on REN1 under high-light
conditions. The results indicated that the percentage of regenerating explants was the
116
highest for explants cultured on SI medium for 14 days only (Table 3-5). The results of
the first experiment demonstrated that explants cultured on SI medium for 21 days
produced a significantly lower percentage of regenerating explants compared to the
explants cultured on SI medium for 14 days (P<0.05). However, there was no significant
difference observed between the 14-day and 21-day treatments in the second experiment.
Explants cultured on SI medium for 40 days in both experiments produced regenerating
explants with significantly lower success (Table 3-5). Statistical analysis of the data
Table 3-5: Shoot regeneration of petiole and stem explants. Explants from source plants
maintained on REN1 medium were incubated on SI medium for varying periods of time.
Data was collected at 40 days after regeneration culture initiation.
Experiment
number
Tissue
type *
Number of
days on SI *
Number of
replicates
Total #
explants
per
replicate
% Explants
regenerating
(Std. dev.)
A petiole 14 3 9-10 57.8 (15.4)
21 3 9-10 21.5 (1.3)
40 3 10 53.3 (5.8)
stem 14 3 14-15 16.7 (10.9)
21 3 14-15 4.8 (8.2)
40 3 14-15 22.5 (15.1)
B petiole 14 6 5-8 58.9 (25.6)
14 6 7-11 50.3 (43.5)
21 6 5-10 49.0 (27.1)
21 6 7-10 67.5 (27.4)
40 6 4-10 37.8 (17.5)
40 6 4-11 49.3 (35.3)
stem 14 6 8-10 44.1 (44.4)
14 6 6-12 51.6 (38.9)
21 6 7-11 35.1 (26.6)
21 6 6-11 36.6 (37.7)
40 6 7-11 32.8 (22.6)
40 6 8-12 29.4 (27.6)
*Binary logistic regression factors with a significant effect on regeneration (P<0.05).
Significantly better treatments are indicated, in bold.
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indicated that stem explants were significantly less likely (P < 0.05) to regenerate than
petiole explants. The tissue type and number of days on SI (14, 21, 40) were significant
factors (P < 0.05) in the statistical model. Interactions and genotype were not significant
factors. Overall, the best regeneration occurred from petiole explants induced on SI
medium for 14 days (Table 3-5).
The regeneration of stem and petiole explants was further evaluated with explants
collected from plants maintained on REN2 medium and under medium-light conditions.
In this experiment, 60% of petiole explants regenerated shoots, which was less than the
80 - 97% regeneration recorded for stem explants (Table 3-6). Both petiole and stem
explants regenerated at higher frequency than the maximum regeneration recorded for
whole leaf, stem and petiole explants collected from plants maintained in vitro on REN1
and in high-light conditions (Tables 3-4, 3-5). Furthermore, on average, each stem or
petiole explant in the last experiment produced 3 shoots per explant (Fig. 3-1E and F),
with the highest number of shoots per explant (7) recorded for stem explants (Table 3-6).
All of the newly regenerated shoots uniformly elongated and developed roots on REN2
medium approximately four weeks after transfer (Table 3-6, Fig. 3-1G). Plants with at
Table 3-6: Shoot regeneration, rooting and acclimation of plants regenerated from
petiole and stem explants from plants maintained in REN2 medium.
Experiment
number
Tissue
type
Total #
explants
Explants
regenerating
(%)
Total
#
shoots
Rooted
shoots
(%)
Acclimated
plant
(%)
1 petiole 10 60 21 100 100
stem 10 80 69 100 100
2 stem 30 97 90 100 100
stem 18 94 46 100 100
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least two roots and minimum 3 cm total root length were transferred to soil and
acclimated to greenhouse conditions (Fig. 3-1H). Acclimation to greenhouse conditions
was 100% successful (Table 3-6).
Discussion
Aristolochiaceae occupies an important phylogenetic position for research on
evolution of plant development. Aristolochia species also produce many secondary
metabolites with valuable medicinal properties and have interesting morphological, and
biochemical features important for different insects (Rulik et al., 2008). This manuscript
describes the first successful protocol for micropropagation, regeneration, in vitro rooting
and acclimation of two genotypes of Aristolochia fimbriata (Fig. 3-2).
During our initial attempts to regenerate shoots from Aristolochia fimbriata leaf
explants, we evaluated shoot organogenesis methods reported for Aristolochia indica
(Manjula et al., 1997; Remashree et al., 1997; Soniya and Sujitha, 2006), tobacco , and
apple (Fisher and Guiltinan, 1995; Maximova et al., 1998). The higher levels of cytokinin
applied under these protocols caused the tissue to blacken and die in 2-3 weeks. Thus, the
concentration of 6BA was reduced in the regeneration media, while the concentration of
TDZ (a synthetic plant growth regulator that has both auxin and cytokinin effects )
remained at 1 mg/l as applied for regeneration from apple leaf tissue (Maximova et al.,
1998; Murthy et al., 1998). Additionally, the results from our study indicated that the
light intensity applied during the micropropagation of A. fimbriata had an influence on
the shoot and plant growth during micropropagation, and also on shoot organogenesis
119
from stem and petiole explants. The initial high-light micropropagation protocol
produced plants closely resembling greenhouse plants with respect to compact form, leaf
size, and root development. The plants propagated under the medium-light developed
slightly elongated stems with smaller, but darker-green leaves compared to the high-light
plants. The cultures under high-light needed to be transferred to fresh medium every 3
weeks due to leaf yellowing, while the plants under medium-light and increased MS
concentration required transfers every 4-5 weeks. Moreover stem and petiole explants
from plants under medium-light regenerated more uniformly and with greater success
then the explants from the high-light plants. The variation observed in the shoot
regeneration response could be explained with different “preconditioning” of the tissues
under the different light condition and increased basal medium concentration (Mohamed
et al., 1992). Plant tissues could be developing different cell sizes or have different
photosynthesis rates, or the effects could be due to changes in the nutrient metabolism or
endogenous hormone production (Saebo et al., 1995; Husaini and Abdin, 2007; Molina et
al., 2007; Tabatabaei et al., 2008). Further analysis of the tissues from plants propagated
under the different micropropagation regimes is necessary to reveal the underlying
physiological differences influencing organogenesis.
Conclusions
The high frequency protocols reported here for in vitro propagation of A.
fimbriata could provide a large quantity of greenhouse ready clonal material in a period
as short as three months, with minimal space requirements. Furthermore, these protocols
120
are currently used for the development of a genetic transformation system for A.
fimbriata VL genotype. The ability to study gene function in vivo in this species will
complete an important requirement for the establishment of A. fimbriata as a model
system for basal angiosperms. To further support these efforts, our group has generated
over 30,000 EST sequences from traditional cDNA libraries constructed of whole plant
tissues and pre-meiotic flower buds of A. fimbriata (B. J. Bliss, L. Landherr, Y. Hu, S.
Clifton, J.H. Leebens-Mack, H. Ma, and C. W. dePamphilis, unpublished data) and
greatly expanded transcriptome sequencing is underway as part of the Ancestral
Angiosperm Genome Project (NSF DEB-0638595). The addition of A. fimbriata to the
increasing selection of plant model systems will provide insight into the immediate
progenitor of monocots and eudicots, the two largest and most successful major
angiosperms clades.
Acknowledgements
This work was supported by National Science Foundation grants to C.
dePamphilis and H. Ma (DBI-0115684 and DBI-0638595) and to M. Guiltinan (NSF
430-47/60A), a DOE grant to H. Ma (DE-FG02-02ER15332), and by the Department of
Biology and Huck Institute of Life Sciences of the Pennsylvania State University. We
thank M. Guiltinan for providing the tissue culture lab and growth facility space, and for
editing this manuscript. We also thank L. Rosen and Jardin Botanico, Universidade de
Coimbra for providing seeds; Anthony Omeis for plant care; Brett Shook, Laura Warg,
and Paula Ralph for assistance with tissue culture experiments; Guanfang Wang, Zhe
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Chen, and Yan Zhang for statistical support, and Dr. Stefan Wanke for valuable
discussion. Our initial efforts in developing a micropropagation system for Aristolochia
fimbriata were aided by the unpublished findings of C. Bravo, G. Yormann, and B.
Llorente, recorded in Acta Horticulturae conference proceedings (1999).
Chapter 4
TCP gene family evolution
This chapter contains data, analyses, and plans for research that may be submitted
for publication by Barbara Bliss.1
1Department of Biology, Institute of Molecular Evolutionary Genetics, and the
Huck Institutes of the Life Sciences, 201 Life Sciences Building, Pennsylvania State
University, University Park, PA 16802, USA
123
Founding members: TB1, CYC, and PCF
The TCP family of transcription factors was named for the initially recognized
members, TEOSINTE-BRANCHED1 (TB1, from Zea mays), CYCLOIDEA (CYC from
Antirrhinum majus) and PROLIFERATING CELL FACTOR genes (PCFs from Oryza
sativa ssp. japonica) (Cubas et al., 1999a). TCP genes participate in cell cycle regulation
and modulating organ outgrowth, affecting the development of plant architecture, floral
form, leaf morphology, and other aspects of plant development. Existing studies have
examined TCP gene family evolution using sequences from the fully sequenced genomes
of Arabidopsis and Oryza (Howarth and Donoghue, 2006; Yao et al., 2007), and traced
TCP genes back to the earliest land plant lineage (Navaud et al., 2007). Recent work on
the role of TCP genes in the development of form in monocots and core eudicots suggests
they play a general role in the evolution of form in flowering plants. An understanding of
TCP sequence evolution in the context of recently available basal angiosperm sequence
data will support future studies of the evolution of function of this important gene family.
A review of the published literature concerning TCP gene function in flowering plants is
followed by my preliminary work to understand how molecular evolution has led to
lineage-specific traits under the influence of TCP family genes.
The TEOSINTE-BRANCHED1 (TB1) was identified as the locus responsible for
the existing tb1-ref maize loss of function mutant (Burnham, 1959). The tb1-ref mutant
produced long lateral branches with tassels (male inflorescences) at some upper nodes,
and tillers (axillary branches) at the basal nodes; a plant architecture resembling that of
teosinte , the wild Mexican grass (Doebley and Stec, 1991). Modern Zea mays produces
124
reduced lateral branches that terminate without branching in ears (female inflorescences).
Quantitative trait loci (QTL) analysis identified the TB1 locus as one of five loci
contributing to the remarkable change in plant and inflorescence architecture between
Zea mays ssp. mays and its teosinte (Zea mays ssp. parviglumis) progenitor (Doebley et
al., 1995) that facilitated the transition to modern agriculture..
CYCLOIDEA (CYC) was found in a semi-peloric mutant of Antirrhinum majus
and was the first gene described affecting flower symmetry (Luo et al., 1996). The
Antirrhinum flower has a single dorsoventral plane of symmetry, with one distinct ventral
(abaxial) petal, two lateral petals, and two larger dorsal (adaxial) petals. The adaxial
stamen is aborted, leaving only four stamens at maturity. Complete peloric mutants have
radially symmetric flowers and can occur in natural populations of zygomorphic species,
or can be induced. Semi-peloric Antirrhinum mutants lacking a functional CYCLOIDEA
(CYC) gene exhibited increased radial symmetry because the lateral, and, to some extent,
dorsal organs, acquired a ventralized form (Luo et al., 1996). CYC mutants also produced
an extra floral organ in the sepal, petal, and stamen whorls (Luo et al., 1996).
The third members of the TCP family, PROLIFERATING CELL FACTOR1
(PCF1) and PCF2 are required for activating proliferating cell nuclear antigen (PCNA) in
Oryza sativa ssp. japonica (Kosugi and Ohashi, 1997). PCNA is essential for regulating
proliferation and cell cycle progression during the transition from G1-to-S phase,
exclusively in meristematic tissues (Waga et al., 1994; Kosugi and Ohashi, 1997;
Warbrick, 2000). PCF1 is expressed in all tissues examined, but PCF2 was found only in
meristematic tissue where PCNA was also expressed (Kosugi and Ohashi, 1997). PCF1
125
and PCF2 may act by formation of homo- or heterodimers, activating PCNA expression
only in tissues where all are expressed (Kosugi and Ohashi, 2002).
TCP gene functions
The TCP family transcription factors act generally to promote elaboration of form
(PCF1 and PCF2) or to repress it (CYC and TB1) by regulating the cell cycle. These
genes have been collected into two broad classes of TCP proteins based on amino acid
similarity in the TCP transcription factor domain, which is composed of approximately
221 bases (Cubas et al., 1999a). The TCP domain contains a unique basic helix loop helix
(bHLH) DNA binding motif, which has a different structure and DNA-binding specificity
than does the bHLH domain found in MyoD (Murre et al., 1989a; Murre et al., 1989b;
Weintraub et al., 1991; Cubas et al., 1999a). The class I proteins contain the proliferating
cell factor proteins. The class II proteins have been subdivided into class II and class III,
with class II remaining the designation for the clade containing CYC and TB1, and
referred to by some as the ECE clade (Howarth and Donoghue, 2006; Koyama et al.,
2007; Navaud et al., 2007; Yao et al., 2007).
Class I TCP proteins
In Arabidopsis, the class I protein TCP20 interacts with the Arabidopsis PCNA2
promoter in a manner similar to that of PCF2 with Oryza PCNA. Both PCNA2 and PCNA
contain a telo box element, and a cis-acting site II motif, although the bases differ slightly
126
(Tremousaygue et al., 1999; Tremousaygue et al., 2003). TCP20 binds the PCNA2 site II
motif, which is required and sufficient to activate PCNA2 expression in cycling cells in
meristematic tissue (Tremousaygue et al., 1999; Tremousaygue et al., 2003). TCP20 also
interacts with AtPur, a protein that binds the telo box on PCNA2, and may stimulate
transcription by opening chromatin and interacting with transactivators, recruiting them
for further control of cell cycle activities (Tremousaygue et al., 1999; Tremousaygue et
al., 2003). Along with PCNA and AtPur, Arabidopsis mitotic cyclin, CYCB1:1 binds
TCP20 at the GCCCR motif, upregulating CYCB1:1 at the G2/M transition (Li et al.,
2005). Many promoters of ribosomal proteins and other proteins required for cell cycling
contain the site II and telo box elements (Tremousaygue et al., 2003), as do nuclear genes
encoding components of the mitochondrial oxidative phosphorylation machinery which is
essential for mitochondrial biogenesis prior to cell division (Welchen and Gonzalez,
2006).
Class II TCP proteins
Class II TCP gene expression generally represses outgrowth. TB1 was shown to
repress branch and female inflorescence outgrowth from secondary axillary meristems
where TB1 mRNA accumulates (Doebley et al., 1997; Hubbard et al., 2002; Howarth and
Donoghue, 2006). Two homologs of TB1 in Arabidopsis, BRC1/TBL1 and BRC2
(formerly TCP18 and TCP12) also repress axillary outgrowth with high accumulations of
mRNA in unelongated buds and low levels in elongating buds (Aguilar-Martinez et al.,
2007; Finlayson, 2007). Genetic analyses indicate they respond to and integrate auxin and
127
the MORE AXILLARY GROWTH (MAX) signals to control bud outgrowth (Aguilar-
Martinez et al., 2007; Finlayson, 2007). In Sorghum, increased exposure to far red light
induces a shade avoidance syndrome, accompanied by reduced levels of phytochrome B,
reduced axillary outgrowth, and increased expression of TB1, suggesting TCP genes play
a role in producing architectural responses of plants to light in the environment (Kebrom
et al., 2006)
CYC, another class II TCP gene, has been shown to regulate cyclins D1, D3a, and
D3b (Gaudin et al., 2000). The D-cyclins are induced by cytokinins, and act to induce
cell division . CYC specifically represses cyclin D3b in the dorsal stamen primordia,
which are aborted in the mature wild-type flower, consistent with termination of cell
division activity (Riou-Khamlichi et al., 1999; Gaudin et al., 2000). TB1 and related
genes may produce their effects by similar interactions with cyclins.
In Arabidopsis, TCP1, the most likely CYC ortholog, is expressed early in the
adaxial (dorsal) part of the floral meristem (as CYC is in Antirrhinum), but expression in
Arabidopsis is not maintained later in development. The difference in timing may
account for the polysymmetry of the mature Arabidopsis flower (Cubas et al., 2001). In
Brassicaceae, the CYC homolog laTCP1 from Iberis amara is expressed differentially
across the corolla when the greatest petal growth occurs, and produces a wild-type
monosymmetric perianth (Busch and Zachgo, 2007). TCP1 (Arabidopsis) and CYC
(Antirrhinum) are also expressed in the adaxial regions of all axillary shoot meristems in
Arabidopsis , and in axillary shoot meristems immediately below the oldest floral
meristem in Antirrhinum (Cubas et al., 2001; Clark and Coen, 2002).
128
In Solanum tuberosum, transcripts of the class II protein, SSTCP1, are
concentrated in dormant buds, which are typically arrested in G1. In sprouting buds,
which consist of cycling cells, the transcripts of SSTCP1 are not detectable (Faivre-
Rampant et al., 2004).
CYC genes have been implicated in floral form in all lineages investigated,
including the bisymmetric flowers of the basal eudicots (Papaveraceae; Ranunculales)
(Damerval et al., 2007; Damerval and Nadot, 2007). Duplications in class II TCP family
genes have provided raw materials for neo- and sub-functionalization, supporting floral
form divergence (Ohno, 1970). The extent to which class II TCP genes have been
involved in independent origins and losses of monosymmetry has been investigated in
large families with highly derived and canalized floral form (Orchidaceae, Fabaceae,
Asteridae, Dipsacaceae) (Cubas, 2004) as well as in several species in the same family
and order as Antirrhinum (Plantaginaceae, Lamiales) (Vieira et al., 1999; Gubitz et al.,
2003; Hileman and Baum, 2003). These analyses indicate a duplication in the CYC
lineage occurred prior to the divergence of the Antirrhinae to generate the CYC/DICH
paralogs, which are now under purifying selection (Hileman and Baum, 2003).
In Antirrhinum, a second locus DICHOTOMA (DICH), is responsible for a semi-
peloric mutant in which organ number is not affected but dorsal petal morphology is
ventralized (Luo et al., 1996). The sequence of DICHOTOMA is 66% identical to that of
CYCLOIDEA. Mutations in both CYC and DICH were required to produce a true peloric
phenotype, with radially symmetric, fully ventralized petals (Luo et al., 1999). In
Mojavea confertifolia, a species closely related to Antirrhinum, expression of CYC and
DICH orthologs differed from those of Antirrhinum majus in a manner correlated with
129
differences in floral form between the two species, particularly with respect to additional
stamen abortion (Hileman and Baum, 2003). DICH orthologs have not been found
outside the Antirrhinae.
In the composite inflorescence of Gerbera, the expression gradient of a CYC
homolog across the inflorescence establishes differences in symmetry and petal fusion
associated with flower type (ray, trans, disc) (Broholm et al., 2008).
In Adoxaceae and Caprifoliaceae, in the Dipscales (Campanuliids clade of
asterids), within the class II (ECE genes), three clades of CYC-like genes were identified
(CYC1, CYC2, CYC3), suggesting two duplications in lineages leading to the
Caprifoliaceae (Howarth and Donoghue, 2005). More CYCLOIDEA orthologs have been
found in other orders and families within Asteridae (Calceolariaceae, Scrophulariaceae,
and Oleaceae in Lamiales; Rubiaceae in Gentianales; Solanaceae and Boraginaceae in
Solanales), consistent with evidence of genome duplications early in the asterid lineage
(Reeves and Olmstead, 2003).
However, in Gesneriaceae, another predominantly zygomorphic family in
Lamiales, several independent reversals to radial symmetry have taken place, generating
actinomorphic species. Amplification of genomic DNA revealed only one clear CYC–like
gene for most members of the family (Citerne et al., 2000; Smith et al., 2004). The
protein-coding regions of the CYC orthologs in the naturally occurring gesneriad
actinomorphs did not suggest that loss of function in the protein-coding sequence was
responsible for reversions to actinomorphy (Citerne et al., 2000; Smith et al., 2004).
Furthemore, individuals within a population displayed no visible phenotype associated
with the numerous single nucleotide polymorphisms in the population, supporting the
130
neutral mutation hypothesis (Kimura, 1968). In addition, there was no evidence of
directional selection on lineages leading to radially symmetric taxa (Smith et al., 2004).
Changes in protein-protein interactions, or in gene regulation (i.e., methylation,
expression in different tissues, loss of cis-regulatory elements, miRNA posttranslational
inactivation) could account for such differences.
Class III TCP proteins
The involvement of TCP proteins in establishing lateral organ boundaries was
first reported for the Antirrhinum cupuliformis (cup) mutant, based on interactions of
CUP with a TCP domain-containing transcript . In Arabidopsis, the class III TCP
proteins TCP3, and to a lesser extent TCP2, TCP4, TCP5, TCP10, TCP13, TCP17,
TCP24 were associated with restricted expression of CUP-SHAPED COTYLEDON
(CUC) genes to boundaries of organ primordia (Weir et al., 2004; Koyama et al., 2007).
Repression of TCP3 induced shoot formation on cotyledons and produced many organ
defects attributable to improperly specified organ boundaries, while overexpression was
mitigated by the endogenous activity of miR319/JAW on TCP 3, TCP2, TCP4, TCP10,
and TCP24. When the miR319/JAW target sequence on these class III transcripts was
eliminated, overexpression of TCP3 or related genes produced fused cotyledons and
shoot developmental defects (Koyama et al., 2007).
Class III TCP genes also play a role in leaf form. Mutations the Antirrhinum gene,
CINCINNATA (CIN), produce reduced petal lobes and leaves with a crinkly phenotype .
Cell size, and expression patterns of CYCLIN D3B and HISTONE4, indicated the cell-
131
cycle arrest front in the mutant leaf is delayed as it moves from leaf tip to base (Nath et
al., 2003; Crawford et al., 2004). Growth of medial leaf cells is arrested in the mutant,
while the marginal cells continue to proliferate (McConnell and Barton, 2003; Nath et al.,
2003).
The homologs of CIN in Arabidopsis are TCP2, TCP3, TCP4, TCP5, and TCP24,
each of which is regulated by miR319 (Palatnik et al., 2003). TCP4 negatively regulates
leaf growth and positively regulates leaf senescence in Arabidopsis, through increasing
JA biosynthesis, particularly at the oxylipin biosynthesis step of the pathway (Schommer
et al., 2008). In Solanum lycopersicon, miR319 regulation of a presumably class III TCP
gene is also required for development of compound leaf (Ori et al., 2007)
Evolution by changing gene interactions
TCP genes appear to be able to interact with a variety of other genes. In
Antirrhinum, at the periphery of the floral meristem, and especially at the base of the
organ primordia and in the ventral petals, CYC and DICH impose a dorsalizing effect by
negatively regulating DIVARICATA (DIV), a myb family gene that confers ventral
identity on wild type petals in a dosage dependent manner (Almeida et al., 1997). This
effect is mediated by RADIALIS (RAD) a myb-like gene. CYC and DICH activate RAD in
the early dorsal domain of the floral meristem, which antagonizes the ventralizing effect
of DIV in those dorsal regions . The activation of RAD by CYC does not occur in
Arabidopsis, indicating the regulatory network in which these genes are involved is
lineage-specific. Although CYC does participate in the development of Arabidopsis floral
132
organ form, it does not utilize the same network as in Antirrhinum (Corley et al., 2005;
Costa et al., 2005).
In the rosids, CYCLOIDEA orthologs have been collected from Brassicaceae
(Arabidopsis) and from Fabaceae, another large family containing zygomorphic flowers
(Citerne et al., 2000; Fukuda et al., 2003; Ree et al., 2004). In an investigation of the
interactions of CYC-like genes Most recently, over- and underexpression studies in the
model plant Lotus japonicus have demonstrated the requirement for the CYC ortholog
LjCYC2 in zygomorphy. Overexpression of LjCYC2 generated adaxial cell types in the
lateral and ventral petals, and dorsalized (adaxialized) form in the lateral petals.
Suppression of LjCYC2 conferred abaxial shape and cell type on the lateral petals, and
affected inflorescence morphology with some shorter internodes and frequent fusion of
flower pairs (Feng et al., 2006). A lateralizing counterpart of RAD (Antirrhinum), Keeled
wing in Lotus (Kew1), was also identified in Lotus. Kew1 shares a similar phenotype to
the KEELED WING (K) mutant in Pisum, and macrosynteny with a region containing the
locus responsible for the keeled wing (K) mutant in Pisum (Wang et al., 2008). Though
the mechanism of CYC regulation is conserved (dorsalizing) between the asterid and
rosid lineages, the interactions of CYC with other members of the pathway have diverged.
Regulation by methylation
In addition to translational control and interactions with other genes, TCP genes
are regulated by transcriptional control. In maize, comparisons of the sequence diversity
of the protein-coding region of the TB1 gene and the non-transcribed regulatory region
133
upstream of the TB1 gene to the same regions in teosinte indicated the regulatory region,
not the protein coding region, was under selection (Wang et al., 1999). Some members of
the TCP family are regulated by methylation (Cubas et al., 1999b; Feng et al., 2006),and
microRNA (miRNA) (Ori et al., 2007; Palatnik et al., 2007).
In the naturally occurring peloric mutant of Linaria vulgaris (Plantaginaceae)
radial symmetry is maintained by hypermethylation of the genomic region containing the
CYC ortholog alone: demethylation can restore bilateral symmetry in this species (Cubas
et al., 1999b). Also, in the naturally occurring allopolyploid, Arabidopsis suecica, TCP
family genes were among the nearly 23% genes exhibiting differential expression of
parental genes (Lee and Chen, 2001). As a result of the polyploidy, only the TCP3 allele
from the A. thaliana parent was expressed. The TCP3 allele from the Cardaminopsis
arenosa parent was silenced by methylation, and could be reactivated by demethylation
(Lee and Chen, 2001). TCP3 is a class III TCP gene (Navaud et al., 2007; Yao et al.,
2007)
The dePamphilis lab has an ongoing commitment to provide informatics tools to
facilitate the use and analysis of millions of ESTs generated by genomics projects. My
contribution has primarily been to test and provide feedback regarding features under
development, initially with curation of TCP and auxin response factor gene family
members, using the LIMS and Phylomine web interfaces for FGP under the direction of
Jim Leebens-Mack. Tools we initially conceived of for gene family curation and analysis
are still under development, but have ample functionality to apply to the evolution of the
TCP gene family. Here, I have evaluated the utility of Tribes and an automated gene
family analysis pipeline developed as a component of the FGP/AAGP bioinformatics
134
resource, to collect sequences, produce an alignment, and to generate phylogenetic trees
using maximum likelihood methods.
135
Materials and Methods
Automated pipeline
All sequences from the fully sequenced genomes of seven species (Populus
trichocarpa (Poptr), Vitis vinifera (Vitvi), Sorghum bicolor (Sorbi), Carica papaya
(Carpa), Medicago truncatula (Medtr), Arabidopsis thaliana (Arath), Oryza sativa ssp.
japonica (Orysa), Physcomitrella patens (Phypa), and Selaginella moellendorfii (Selmo)
were collected into Plant Tribes (Wall et al., 2008). The sequences in the Supertribes
were collected and aligned with MUSCLE (Edgar, 2004)and evaluated using RAxML
(Random accelerated maximum likelihood) (Stamatakis, 2006).
Verification of sequences
The Tribe collection and alignment were evaluated to verify that all known TCP
domain-containing sequences from Arabidopsis and Oryza were included (Table 4-1))
and that all amino acid sequences from other taxa also contained the TCP domain (PFAM
domain 03634, http://www.sanger.ac.uk/Software/Pfam/search.shtml). Sequences having
fewer than 50 characters of the TCP domain were eliminated from the alignment.
136
Table 4-1: Arabidopsis thaliana (At) and Oryza sativa ssp. japonica (Os) TCP genes
from Yao et al. (2007) and Navaud et al. (2007).
1 At4g18390 LOC_Os01g11550
2 At1g53230 LOC_Os01g55750
3 At3g15030 LOC_Os01g69980
4 At5g60970 LOC_Os02g42380
5 At5g41030 LOC_Os02g51280
6 At5g23280 LOC_Os02g51310
7 At1g58100 LOC_Os03g49880
8 At2g45680 LOC_Os03g57190
9 At2g31070 LOC_Os04g11830
10 At2g37000 LOC_Os04g44440
11 At1g68800 LOC_Os05g43760
12 At3g02150 LOC_Os06g12230
13 At3g47620 LOC_Os07g04510
14 At1g69690 LOC_Os07g05720
15 At3g45150 LOC_Os08g33530
16 At5g08070 LOC_Os08g43160
17 At3g18550 LOC_Os09g24480
18 At5g51910 LOC_Os09g34950
19 At3g27010 LOC_Os11g07460
20 At5g08330 LOC_Os12g02090
21 At1g72010 LOC_Os12g07480
22 At1g35560 LOC_Os12g42190
23 At1g30210
137
Results
Sequence collection
Tribes returned five supertribes, of which two had no members with the TCP
domain. The remaining three supertribes, each contained 4-7 tribes (Table 4-2). A subset
of these contained the PFAM domain of interest (Table 4-3).
In supertribe 777, only one of 19 Vitis sequences in tribe 2034 (Table 4-2).
contained the TCP domain (Table 4-3). Neither of the Vitis sequences in tribe 8345 or
Table 4-2: Three supertribes of genomic sequences comprise Arabidopsis and rice genes
of interest.
SUP TRIBE Phypa Selmo Orysa Sorbi Vitvi Poptr Medtr Carpa Arath TOTAL
777 2034 19 3 22
777 8345 5 5
777 9164 1 2 1 4
777 20097 1 1
777 20098 1 1
777 30671 1 1
777 79451 1 1
1148 767 2 3 8 8 4 9 2 4 7 47
1148 3418 2 3 2 3 2 3 15
1148 20099 1 1
1148 20826 1 1
1148 29237 1 1
1148 71899 1 1
2066 282 4 3 11 18 11 20 5 13 13 98
2066 14235 2 2
2066 29203 1 1
2066 59121 1 1
Totals 6 6 24 29 44 35 7 28 24 203
138
79451 contained the TCP domain, and the one Vitis sequence in tribe 9164 contained a
TCP domain. The remaining sequences in tribe 9164 all contained TCP domains
(Table 4-3). Besides the sequences in tribe 9164 the Carica sequences in tribe 20098 had
a TCP domain. None of the Carica sequences in tribe 2034 or 30671 contained the TCP
domain.
In supertribe 1148, all members of tribes 767 and 3418 contained TCP domains,
but tribes 20826, 29237, and 29237 had none (Figure 4-2). In supertribe 2066, all
members of tribe 282 contained TCP domains, but tribes 14235, 29203, and 59121 had
none (Figure 4-3).
Table 4-3: Genomic sequences containing TCP domain (PFAM domain 03634).
SUP TRIBE Phypa Selmo Orysa Sorbi Vitvi Poptr Medtr Carpa Arath TOTAL
777 2034 1 0 1
777 8345 0 0
777 9164 1 2 1 4
777 20097 1 1
777 20098 1 1
777 30671 0 0
777 79451 0 0
1148 767 1 3 8 8 4 9 2 4 7 46
1148 3418 2 3 2 3 2 3 15
1148 20099 1 1
1148 20826 0 0
1148 29237 0 0
1148 71899 0 0
2066 282 3 3 11 18 11 20 5 13 13 97
2066 14235 0 0
2066 29203 0 0
2066 59121 0 0
Totals 4 6 22 29 19 34 7 21 24 166
139
An additional Arabidopsis gene, At1g67620 was returned in the tribes. This gene
had been omitted from recently published studies (Navaud et al., 2007; Yao et al., 2007)
and had once been identified as a pseudogene in Genbank. However, functional data for
At1g67620 have been available for some time . Altogether, the 24 Arabidopsis genes
matched those originally described (Cubas et al., 1999a; Cubas et al., 2001).
Alignments
The alignment produced by the automated pipeline used default values of
MUSCLE and located the TCP domain in those sequences having a TCP domain, but
many sequences lacked it (Figure 4-1, 4-2, 4-3).
Phylogenetic analysis
The phylogenetic tree generated by RAxML (Figure 4-4) included only
sequences containing a TCP domain. Clades corresponding to classes 1-3 as found by
Yao (2007) and Navaud (2007) (Figure 4-5, 4-6) could be identified.
140
Figure 4-1: Supertribe 777, MUSCLE alignment of amino acids.
141
Figure 4-2: Supertribe 1148, MUSCLE alignment of amino acids.
142
Figure 4-3: Supertribe 2066, MUSCLE alignment of amino acids.
143
Figure 4-4: RaxML phylogenetic analysis of TCP domain-containing sequences from
automated pipeline. Pink, blue, and green indicate class 1, 2, and 3 genes, respectively.
Class 3
Class 1
Class 2
Class 2
Class 1
Class 3
144
Discussion
An automated pipeline using Tribes was evaluated for its ability to replicate or
improve upon the findings of Yao et al. and Navaud et al. (2007; Yao et al., 2007)
(Figure 4-5, 4-6). A number of sequences, particularly from Vitis, were automatically
included in the alignment although they had no TCP domain. Arabidopsis class 2 and 3
genes were distributed in supertribes 777 and 1148, class 1 genes were in supertribe
2066. Supertribe 777 was dominated by Vitis sequences. Some similarity, other than that
of a TCP domain, caused additional sequences to be collected by Tribes. I suspect that
that similarity was a consequence of the disproportional representation of Vitis sequences
in the database.
145
Figure 4-5: Neighbor joining tree and expression pattern of TCP genes in rice and
Arabidopsis from Yao et al. (2007).
146
Class 3
Class 1
Class 2
147
The alignment of the TCP domain distinguished between classes I and II but did
not produce monophyletic lineages as per the reference phylogenies (Figure 4-5, 4-6).
The HMM model for the TCP gene family reveals several regions of more or less
conservation (Figure 4-8). TCP genes contain many informative motifs, as reported by
previous researchers. With the abundance of sequence information that is available, it
should be possible to align and investigate the molecular evolution of motifs including,
the miR319 target sequence in the class III genes. It may be possible to identify selection,
or perhaps loss of constraint in a lineage in which the functionality of the TCP gene has
been lost (e.g., class II genes in Gesneriaceae). A complete sequence analysis may
suggest new gene interactions. Functional analyses of TCP genes in the basal
angiosperm, A. fimbriata will provide important insight into the early role of this gene
family in flowering plants, and advance our understanding of the evolution of symmetry
and form in angiosperms.
Figure 4-6: Unrooted ML tree of 126 TCP proteins from land plants (Navaud et al.,
2007). Boot strap values > 50% are indicated as percentages. Names in boldface
correspond to proteins described in the literature; italicized names designate EST
sequences.
148
Figure 4-7: Classes 1, 2, 3 TCP sequences.Pink indicates Class 1 (PCF-like) sequences,
blue indicates Class 2 sequences, green indicates Class 3 sequences.
gnl|Carpa1|supercontig 65.19
gnl|Poptr1|786035
gnl|Poptr1|589460
gnl|Vitvi1|GSVIVP00023221001
gnl|Medtr1|AC153725 4.4
gnl|Arath7|AT2G37000
gnl|Carpa1|supercontig 5.261
Class 1 PCFlike
gnl|Orysa5|Os02g42380
gnl|Sorbi1|Sb04g026970.1
gnl|Orysa5|Os04g44440
gnl|Sorbi1|Sb06g023130.1
gnl|Carpa1|supercontig 60.75
gnl|Arath7|AT5G23280
gnl|Arath7|AT5G08330
gnl|Poptr1|654962
gnl|Poptr1|558669
gnl|Medtr1|AC151824 21.4
gnl|Medtr1|AC160925 12.3
gnl|Vitvi1|GSVIVP00007093001
gnl|Sorbi1|Sb04g038140.1
Class 1 PCFlike
gnl|Orysa5|Os01g11550
gnl|Phypa1|143783
gnl|Selmo1|89227
gnl|Selmo1|28794
gnl|Selmo1|29162
gnl|Vitvi1|GSVIVP00027783001
gnl|Carpa1|supercontig 131.71
gnl|Arath7|AT3G15030
gnl|Arath7|AT1G53230
gnl|Medtr1|AC158498 10.4
gnl|Arath7|AT2G31070
gnl|Carpa1|supercontig 209.13
gnl|Arath7|AT5G60970
gnl|Arath7|AT5G08070
gnl|Vitvi1|GSVIVP00038026001
gnl|Arath7|AT3G02150
gnl|Carpa1|supercontig 14.188
gnl|Poptr1|778619
gnl|Poptr1|556378
gnl|Vitvi1|GSVIVP00000038001
gnl|Poptr1|680833
gnl|Carpa1|supercontig 94.62
gnl|Poptr1|568415
gnl|Poptr1|197397
gnl|Carpa1|supercontig 17.1
gnl|Vitvi1|GSVIVP00028330001
gnl|Arath7|AT1G30210
gnl|Arath7|AT4G18390
gnl|Vitvi1|GSVIVP00017624001
gnl|Poptr1|251155
gnl|Poptr1|422658
gnl|Arath7|AT3G18550
gnl|Carpa1|supercontig 13.48
gnl|Poptr1|658950
gnl|Poptr1|656764
gnl|Vitvi1|GSVIVP00030278001
gnl|Arath7|AT1G67260
gnl|Poptr1|266145
gnl|Carpa1|supercontig 179.29
gnl|Arath7|AT1G68800
gnl|Carpa1|supercontig 1401.1
Class two true CYC
gnl|Vitvi1|GSVIVP00018699001
gnl|Poptr1|574356
gnl|Poptr1|592176
gnl|Medtr1|AC171619 17.3
gnl|Vitvi1|GSVIVP00002995001
gnl|Poptr1|642377
gnl|Poptr1|568617
gnl|Sorbi1|Sb03g001940.1
Class 3 (class 2; non-CYC)
gnl|Sorbi1|Sb03g034918.1
gnl|Orysa5|Os05g43760
gnl|Sorbi1|Sb09g025340.1
gnl|Sorbi1|Sb03g035350.1
gnl|Orysa5|Os01g55750
gnl|Sorbi1|Sb01g006020.1
gnl|Orysa5|Os03g57190
gnl|Sorbi1|Sb02g013020.1
gnl|Sorbi1|Sb02g003070.1
gnl|Orysa5|Os07g05720
gnl|Sorbi1|Sb08g021690.1
gnl|Orysa5|Os12g02090
gnl|Orysa5|Os07g04510
gnl|Orysa5|Os02g51310
Class 3 (class 2 not CYC)
gnl|Orysa5|Os08g33530
gnl|Sorbi1|Sb07g021140.1
gnl|Sorbi1|Sb02g024450.1
gnl|Orysa5|Os09g24480
gnl|Sorbi1|Sb01g010690.1
gnl|Orysa5|Os03g49880
Class two true CYC
gnl|Orysa5|Os01g69980
gnl|Sorbi1|Sb03g019510.1
gnl|Sorbi1|Sb03g044320.1
gnl|Sorbi1|Sb03g013360.1
gnl|Sorbi1|Sb03g019411.1
gnl|Sorbi1|Sb06g016340.1
gnl|Sorbi1|Sb08g021725.1
gnl|Orysa5|Os08g43160
gnl|Sorbi1|Sb02g030260.1
gnl|Orysa5|Os09g34950
gnl|Orysa5|Os06g12230
gnl|Sorbi1|Sb04g027960.1
gnl|Sorbi1|Sb04g027960.2
gnl|Vitvi1|GSVIVP00036994001
gnl|Carpa1|supercontig 128.6
gnl|Carpa1|supercontig 1815.2
gnl|Vitvi1|GSVIVP00000129001
gnl|Vitvi1|GSVIVP00024581001
gnl|Arath7|AT1G58100
gnl|Poptr1|780609
gnl|Poptr1|678125
gnl|Arath7|AT1G35560
gnl|Arath7|AT1G72010
gnl|Carpa1|supercontig 66.26
gnl|Poptr1|783149
gnl|Medtr1|AY515253 13.3
gnl|Vitvi1|GSVIVP00035422001
gnl|Carpa1|supercontig 30.48
gnl|Arath7|AT5G41030
gnl|Arath7|AT3G27010
gnl|Vitvi1|GSVIVP00037889001
gnl|Carpa1|supercontig 70.118
gnl|Poptr1|827565
gnl|Poptr1|549725
gnl|Poptr1|554743
gnl|Poptr1|750930
gnl|Poptr1|253757
gnl|Poptr1|570503
gnl|Arath7|AT5G51910
gnl|Carpa1|supercontig 4.210
gnl|Arath7|AT2G45680
gnl|Poptr1|755220
gnl|Poptr1|774325
gnl|Carpa1|supercontig 18.176
gnl|Vitvi1|GSVIVP00026324001
gnl|Vitvi1|GSVIVP00001076001
gnl|Poptr1|554293
gnl|Poptr1|548532
gnl|Carpa1|supercontig 65.17
gnl|Poptr1|650493
gnl|Arath7|AT3G45150
gnl|Vitvi1|GSVIVP00033898001
gnl|Vitvi1|GSVIVP00013660001
gnl|Arath7|AT1G69690
gnl|Arath7|AT3G47620
gnl|Medtr1|AC146548 4.4
gnl|Poptr1|266526
gnl|Poptr1|197953
gnl|Orysa5|Os02g51280
gnl|Sorbi1|Sb10g008030.1
gnl|Selmo1|441321
gnl|Selmo1|404061
gnl|Selmo1|438214
gnl|Phypa1|164296
gnl|Phypa1|172497
gnl|Phypa1|173346
gnl|Sorbi1|Sb08g004600.1
gnl|Orysa5|Os12g07480
gnl|Sorbi1|Sb08g004610.1
gnl|Sorbi1|Sb08g019710.1
gnl|Sorbi1|Sb05g004800.1
gnl|Orysa5|Os11g07460
Class 1 PCFlike
Class 1
gnl|Orysa5|Os04g11830
gnl|Sorbi1|Sb06g002260.1Class 1 PCFlike
gnl|Carpa1|supercontig 104.84
gnl|Carpa1|supercontig 65.20
gnl|Carpa1|supercontig 65.18
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149
Figure 4-8: Class 1 (PCF-like) TCP sequences. Pink indicates Class 1 (PCF-like)
sequences, blue indicates Class 3 sequences.
gnl|Carpa1|supercontig 65.19
gnl|Poptr1|786035
gnl|Poptr1|589460
gnl|Vitvi1|GSVIVP00023221001
gnl|Medtr1|AC153725 4.4
gnl|Arath7|AT2G37000
gnl|Carpa1|supercontig 5.261
Class 1 PCFlike
gnl|Orysa5|Os02g42380
gnl|Sorbi1|Sb04g026970.1
gnl|Orysa5|Os04g44440
gnl|Sorbi1|Sb06g023130.1
gnl|Carpa1|supercontig 60.75
gnl|Arath7|AT5G23280
gnl|Arath7|AT5G08330
gnl|Poptr1|654962
gnl|Poptr1|558669
gnl|Medtr1|AC151824 21.4
gnl|Medtr1|AC160925 12.3
gnl|Vitvi1|GSVIVP00007093001
gnl|Sorbi1|Sb04g038140.1
Class 1 PCFlike
gnl|Orysa5|Os01g11550
gnl|Phypa1|143783
gnl|Selmo1|89227
gnl|Selmo1|28794
gnl|Selmo1|29162
gnl|Vitvi1|GSVIVP00027783001
gnl|Carpa1|supercontig 131.71
gnl|Arath7|AT3G15030
gnl|Arath7|AT1G53230
gnl|Medtr1|AC158498 10.4
gnl|Arath7|AT2G31070
gnl|Carpa1|supercontig 209.13
gnl|Arath7|AT5G60970
gnl|Arath7|AT5G08070
gnl|Vitvi1|GSVIVP00038026001
gnl|Arath7|AT3G02150
gnl|Carpa1|supercontig 14.188
gnl|Poptr1|778619
gnl|Poptr1|556378
gnl|Vitvi1|GSVIVP00000038001
gnl|Poptr1|680833
gnl|Carpa1|supercontig 94.62
gnl|Poptr1|568415
gnl|Poptr1|197397
gnl|Carpa1|supercontig 17.1
gnl|Vitvi1|GSVIVP00028330001
gnl|Arath7|AT1G30210
gnl|Arath7|AT4G18390
gnl|Vitvi1|GSVIVP00017624001
gnl|Poptr1|251155
gnl|Poptr1|422658
gnl|Arath7|AT3G18550
gnl|Carpa1|supercontig 13.48
gnl|Poptr1|658950
gnl|Poptr1|656764
gnl|Vitvi1|GSVIVP00030278001
gnl|Arath7|AT1G67260
gnl|Poptr1|266145
gnl|Carpa1|supercontig 179.29
gnl|Arath7|AT1G68800
gnl|Carpa1|supercontig 1401.1
Class two true CYC
gnl|Vitvi1|GSVIVP00018699001
gnl|Poptr1|574356
gnl|Poptr1|592176
gnl|Medtr1|AC171619 17.3
gnl|Vitvi1|GSVIVP00002995001
gnl|Poptr1|642377
gnl|Poptr1|568617
gnl|Sorbi1|Sb03g001940.1
Class 3 (class 2; non-CYC)
gnl|Sorbi1|Sb03g034918.1
gnl|Orysa5|Os05g43760
gnl|Sorbi1|Sb09g025340.1
gnl|Sorbi1|Sb03g035350.1
gnl|Orysa5|Os01g55750
gnl|Sorbi1|Sb01g006020.1
gnl|Orysa5|Os03g57190
gnl|Sorbi1|Sb02g013020.1
gnl|Sorbi1|Sb02g003070.1
gnl|Orysa5|Os07g05720
gnl|Sorbi1|Sb08g021690.1
gnl|Orysa5|Os12g02090
gnl|Orysa5|Os07g04510
gnl|Orysa5|Os02g51310
Class 3 (class 2 not CYC)
gnl|Orysa5|Os08g33530
gnl|Sorbi1|Sb07g021140.1
gnl|Sorbi1|Sb02g024450.1
gnl|Orysa5|Os09g24480
gnl|Sorbi1|Sb01g010690.1
gnl|Orysa5|Os03g49880
Class two true CYC
gnl|Orysa5|Os01g69980
gnl|Sorbi1|Sb03g019510.1
gnl|Sorbi1|Sb03g044320.1
gnl|Sorbi1|Sb03g013360.1
gnl|Sorbi1|Sb03g019411.1
gnl|Sorbi1|Sb06g016340.1
gnl|Sorbi1|Sb08g021725.1
gnl|Orysa5|Os08g43160
gnl|Sorbi1|Sb02g030260.1
gnl|Orysa5|Os09g34950
gnl|Orysa5|Os06g12230
gnl|Sorbi1|Sb04g027960.1
gnl|Sorbi1|Sb04g027960.2
gnl|Vitvi1|GSVIVP00036994001
gnl|Carpa1|supercontig 128.6
gnl|Carpa1|supercontig 1815.2
gnl|Vitvi1|GSVIVP00000129001
gnl|Vitvi1|GSVIVP00024581001
gnl|Arath7|AT1G58100
gnl|Poptr1|780609
gnl|Poptr1|678125
gnl|Arath7|AT1G35560
gnl|Arath7|AT1G72010
gnl|Carpa1|supercontig 66.26
gnl|Poptr1|783149
gnl|Medtr1|AY515253 13.3
gnl|Vitvi1|GSVIVP00035422001
gnl|Carpa1|supercontig 30.48
gnl|Arath7|AT5G41030
gnl|Arath7|AT3G27010
gnl|Vitvi1|GSVIVP00037889001
gnl|Carpa1|supercontig 70.118
gnl|Poptr1|827565
gnl|Poptr1|549725
gnl|Poptr1|554743
gnl|Poptr1|750930
gnl|Poptr1|253757
gnl|Poptr1|570503
gnl|Arath7|AT5G51910
gnl|Carpa1|supercontig 4.210
gnl|Arath7|AT2G45680
gnl|Poptr1|755220
gnl|Poptr1|774325
gnl|Carpa1|supercontig 18.176
gnl|Vitvi1|GSVIVP00026324001
gnl|Vitvi1|GSVIVP00001076001
gnl|Poptr1|554293
gnl|Poptr1|548532
gnl|Carpa1|supercontig 65.17
gnl|Poptr1|650493
gnl|Arath7|AT3G45150
gnl|Vitvi1|GSVIVP00033898001
gnl|Vitvi1|GSVIVP00013660001
gnl|Arath7|AT1G69690
gnl|Arath7|AT3G47620
gnl|Medtr1|AC146548 4.4
gnl|Poptr1|266526
gnl|Poptr1|197953
gnl|Orysa5|Os02g51280
gnl|Sorbi1|Sb10g008030.1
gnl|Selmo1|441321
gnl|Selmo1|404061
gnl|Selmo1|438214
gnl|Phypa1|164296
gnl|Phypa1|172497
gnl|Phypa1|173346
gnl|Sorbi1|Sb08g004600.1
gnl|Orysa5|Os12g07480
gnl|Sorbi1|Sb08g004610.1
gnl|Sorbi1|Sb08g019710.1
gnl|Sorbi1|Sb05g004800.1
gnl|Orysa5|Os11g07460
Class 1 PCFlike
Class 1
gnl|Orysa5|Os04g11830
gnl|Sorbi1|Sb06g002260.1Class 1 PCFlike
gnl|Carpa1|supercontig 104.84
gnl|Carpa1|supercontig 65.20
gnl|Carpa1|supercontig 65.18
100
42
47
33
51
83
20
98
27
99
100
19
19
19
96
19
19
34
71
22
0
91
21
15
99
39
100
7
11
82
33
56
97
15
77
30
8
95
81
93
81
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94
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56
74
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48
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1
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80
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18
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150
Figure 4-9: HMM logo PFAM03634 (http://pfam.sanger.ac.uk/family?entry=TCP).
Chapter 5
Retrospective and future direction for studies of evolution of development in
basal angiosperms
Basal angiosperm lineages contain relatively few extant species, but they contain
more diversity than in all other angiosperm lineages combined. Basal angiosperms offer a
direct view of the common ancestor of monocots and eudicots, as well as unique insights
into the ancestor of all angiosperms. The magnoliids contain the most species and much
of the diversity found in basal angiosperm lineages, hence three species from that lineage
were selected by the Floral Genome Project (FGP) for floral transcriptome sequencing
aimed at identifying components of genetic diversity responsible for variations in floral
diversity (Soltis et al., 2002). The three magnoliid species included two tree species
(Liriodendron tulipfera and Persea americana), and an herbaceous perennial (Saruma
henryi). Liriodendron and Persea have two floral whorls of three tepals, and the flower of
Saruma has two well-differentiated outer whorls that appear to be sepals and petals.
However, the petal whorl of Saruma is known to arise from tissue that also gives rise to
stamens (de Craene et al., 2003). Thus, the Saruma flower suggests an alternate origin of
petals from those found in core eudicots. Because of this interesting origin of the petal
whorl, .and because Saruma is herbaceous, it seemed like a potential candidate for a basal
angiosperm model system needed to address hypotheses of evolution of development
(Baum et al., 2002).
152
I joined the FGP and applied my horticultural training and experience to obtaining
and cultivate over one hundred Saruma henryi stock plants needed for generating
sufficient tissue for the flower bud cDNA library. I worked with the PSU Plant Pathology
Dept. to identify and address existing problems of crop failure attributed to fungal
pathogens, then with Tony Omeis to develop and implement prophylactic crop care
protocols. I consulted with Andy Stephenson and the PSU Horticulture Dept. and
developed vernalization methods to insure continuous availability of source tissue for the
FGP consortium. I collected the tissue needed for the flower bud cDNA library, and for
subsequent FGP needs. Plants were obtained from the National Arboretum, Heronswood
Nurseries, Seneca Hill Perennials, and Sunshine Farms, with the greatest contribution of
tissue from the Heronswood plants. I sequenced the internal transcribed spacer region
from each population and all were essentially identical (Soltis and Kuzoff, 1993). FGP
generated 10,274 EST‟s from Saruma, the most deeply sequenced magnoliid species.
Altogether, FGP generated over 100,000 floral bud cDNA sequences from fifteen
angiosperms and gymnosperms, filling critical gaps in sequence and expression data
needed to provide perspective on gene family evolution in the MADS-box family, as well
as others. FGP provided many first glimpses into basal angiosperm sequence and
expression data that continue to generate hypotheses about evolution of angiosperm
development, which can only be tested in a basal angiosperm plant model. Our
cultivation experience with Saruma, including its extended time to flower, subsequent
requirement for vernalization, difficulty propagating, and genome size evidence pointing
to polyploidy (Bharathan et al., 1994) demonstrated its limited utility as a plant
experimental model. Therefore, I surveyed all Aristolochia species available from
153
commercial seed catalogs, nurseries, public botanical gardens, private collectors, and
index semina, using a suite of criteria to select the best candidate for development as a
basal angiosperm experimental model (Bliss et al., in preparation, 2008). Leaf explants
from A. fimbriata and Saruma were easily transformed, but the Saruma tissues evidenced
no capacity for regeneration. Micropropagation and regeneration protocols for A.
fimbriata were developed to support the transformation system (Bliss et al., submitted,
December 2008).
Improving the Aristolochia fimbriata transformation system
Explant type, concentration of basal media salts, co-cultivation period, antibiotics,
and pre-condition of stock material have all been modified since my initial work with the
Aristolochia fimbriata transformation system. Although more work has been done with
the shoot organogenesis system, experiments with somatic embryogenesis were also
promising. Production of transgenic lines via somatic embryogenesis would offer the
advantage of generating non-chimeric secondary somatic embryos which can be
germinated to produce transgenic plants (Dandekar et al., 1989). The shoot organogenesis
system yielded results more quickly, but has the inherent tendency to produce chimeras,
which can be eliminated by improved selection to eliminate non-transformed cells.
However, at the present time the main bottleneck in the shoot organogenesis system is
not the production of chimeras, but in getting the transformed tissues to regenerate.
Problems in the water supply are suspected.
154
In the shoot organogenesis system, reduced light in micropropagation of source
tissue generated significant differences in regeneration of stem and petiole tissues, the
physiological basis for which is not understood, but is not unique to A. fimbriata (for
discussion, see Bliss et al. (submitted, December 2008)). Characterizing the endogenous
hormone production, including describing the molecular structures and production levels
in plant tissues, would inform efforts to optimize the A. fimbriata transformation system
and contribute to the basic science of plant growth regulation in lineages preceding the
divergence of monocots from eudicots. The highest level of regeneration (indicated by
primordia formation) occurred in petiole explants sliced open lengthwise (Bliss et al.,
submitted, December 2008), so a transformation protocol using petiole explants may also
display better regeneration. Ongoing problems achieving elongation of primordia on the
elongation medium have been relieved by decreasing the length of time on the second,
TDZ-free, shoot regeneration medium. Reducing NAA level in that medium may also
improve primordia regeneration. The increase in MS salts that lowered the requirements
for stock plant maintenance may be inhibiting production of additional buds (shoot
primordia) in regenerating explants, so perhaps should be reduced in the third step of
regeneration, before roots have formed (Das et al., 1998).
At present, regeneration problems in the transformation system should be
addressed in several ways. The populations of transformed cells that currently survive
selection are not able to regenerate in the subsequent counter selection regime. This may
be attributable to low survival of transformed cells, excess survival of untransformed
cells, inability of the transformed cells to regenerate under the antibiotic regime, or
inability of the transformed cells to regenerate at all. Optimal selection and counter-
155
selection antibiotics should be identified which can quickly and completely eradicate
non-transformed tissues, yet allow the transformed tissues to regenerate. Increasing the
ratio of surface area to explant size will increase exposure to the selection agent, and
support rapid selection. Application of a liquid medium containing high levels of
antibiotics for a short period may also support rapid elimination of both A. tumefaciens
and non-transformed cells. Reducing the co-cultivation time to 48-72 hours may also
improve overall health of the tissues, and/or allow a different population of cells to
survive in the explant. Another way to alter the population of growing, transformed, but
non-regenerating cells is to alter the exposed cells of the explant so that a different
population of cells – hopefully ones with better ability to regenerate – will be exposed
and transformed. Stem and petiole explants (particularly petiole explants) that were split
lengthwise regenerated more frequently than did less dissected explants (Expt. B.,
Table 3-5), therefore it may be beneficial to initiate transformation using explants split
lengthwise.
A systematic assessment of selection antibiotics can be conducted to identify the
antibiotic having the best ability to select for transformed cells. In the experiment shown
(Figure 5-1), the selection agent kanamycin was not sufficient for seedling selection
(Figure 5-1 B) at the concentration tested (100 mg/L). It also was not adequate for
selection of transformed cells in tissue culture (data not shown). However, subtle growth
effects from the selection agent were evident after several weeks (Figure 5-1 C) and the
tips of seedling roots exposed to the kanamycin appeared yellow (Figure 5-1 D).
156
An optimal counter-selection agent should be identified through similar
experiments. It should permit direct organogenesis in untransformed tissues when used at
levels effective for the control of Agrobacterium. The antibiotic properties of A. fimbriata
may contribute to counter selection, reducing the required level of the optimal counter-
Figure 5-1: Evaluating Kanamycin (Kan) as selection agent. A, B Two tests to evaluate
toxicity. A. Effect on 7 day old seedlings. Six replicates, two individuals per cup (upper
six cups), cultured in 100 mg/L Kan in addition to MS20. Lower two cups, two seedlings
per cup, MS 20 only. B. Effect on germinating seedlings. Ten seeds per plate (1/2 MS, no
sucrose), two plates with 100 mg/L Kan; two without Kan have more germination but not
100% C, D. Results of tests. C. Growth inhibited after 20 days is noticeable; the
seedlings from the cups with Kan are smaller. Two grown in contaminated medium are
not as small. D. Root tips yellowed in 100 mg/mL Kan.
157
selection agent. Rinsing explants with distilled water after co-cultivation and after
selection in liquid medium may further reduce dependency on counter-selection
antibiotics.
A protocol will also be needed for selecting potentially transformed seeds
collected from a hemizygous parent produced from successful transformation. GFP
expression in the seedling may not provide an ideal system for selection, as A. fimbriata
seedlings require light for emergence of true leaves, and light promotes chlorophyll
accumulation, which interferes with visualization of GFP. Furthermore, seedlings are not
tolerant of the desiccating conditions of the microscope stage, so use of GFP to identify
transformed seedlings puts at risk the survival of the transformed plant. Antibiotic
selection can be used if seeds are germinated in moist paper toweling, sterilized with 10%
sodium hypochlorite (bleach) solution, rinsed and inserted into freshly made medium
containing antibiotics for seedling selection. Seedling germination on media was not
reliable (see “No Kan” Figure 5-1 B, and Figures B1-6), and the extended time required
(when germination did occur) may have allowed heat or light to degrade the antibiotics.
A different approach would be to take advantage of the ease with which the plant
can be regenerated from root tissue in the greenhouse. Large roots divided into 5-9 cm
sections and half covered with soil produce new shoots and generate new plants. Many
large, healthy transformed roots were generated in initial transformation experiments
(Figure 5-2). Transformed roots can be grown in vitro until large, then acclimated and
planted in soil for shoot regeneration, in which transformation could be confirmed by
PCR.
158
Figure 5-2: GFP expression in A. fimbriata roots. A, C, E. Light images. B, D, F.
Fluorescent images. A, B. Young root B. Four second exposure C, D. Cluster of older
roots growing away from medium. D. Fifteen second exposure E, F. Aging roots growing
down into medium F. Four second exposure. Root marked with * in E is not expressing
GFP. Scale bar = 1 mm
In vivo methods for transformation are desirable, but promising images from early
experiments could not be confirmed. The PCR protocol available now to confirm
transformation of A. fimbriata tissues will support renewed attempts at in vivo
transformation. Efforts to improve any transformation method (but in vivo methods in
particular) should take into account the effect of temperature on transformation (Dillen et
al., 1997): co-cultivation in planta should be done in an appropriately cool location. In
*
159
addition to temperature, optimizing the acetosyringone and surfactant concentrations may
further improve transformation results (Sunilkumar and Rathore, 2001).
Evolution of gene function in basal angiosperms
The A. fimbriata experimental model offers opportunities for investigating
features of physiology and secondary metabolite production in basal angiosperms (see
(Bliss et al., in preparation, 2008). In Arabidopsis, the 35S-ATG-1 and 35S-ATG-34 AG
overexpression constructs produced the ap2 phenotype, in which sepals and petals were
absent and the floral whorls displayed carpel-stamen-stamen-carpel organ identity from
the outermost to inner whorls (Mizukami and Ma, 1992). Loss of AG function in
Arabidopsis generates loss of meristem determinacy (more than 15 whorls of floral
organs) and loss of floral organ identity (absence of stamens and carpels) in both the ag-1
mutant and with an antisense ATG-1 construct (Mizukami and Ma, 1995).
In A. fimbriata, over expression of AG orthologs from Saruma or Aristolochia
(Kramer et al., 2004; Zahn et al., 2006) would be expected to replace the unipartite
perianth with ectopic stamens and carpels, but it is not clear how that would be arranged
given the wild-type inferior ovary and stamen adnation with the style in the gynostemium
structure. Loss of AG function would be expected to eliminate the gynostemium, but it is
not clear what perianth-like structures would take its place, or to what extent meristem
identity is a C-function role in A. fimbriata. Jaramillo and Kramer (2004) reported
orthologs of B-class genes (AP3, PI) expressed in the inner layer of utricle cells of the
Aristolochia sepalloid perianth (as well as in the anthers), and in the staminoid “petals” of
160
Saruma (as well as in the stamens). It seems likely some sort of perianth tissue would
replace the reproductive organs, but it not clear whether one or two different whorls of
perianth tissue would appear. Although no AP1 ortholog has been reported, an AP2
ortholog was found in the unigenes from A. fimbriata (Luo et al., 1996; Feng et al.,
2006). A transgenic plant over-expressing AP2 would be expected to resemble the A.
fimbriata equivalent of the AG loss of function transgenic plant, described above, and
loss of AP2 function in a transgenic A. fimbriata plant would be expected to display a
phenotype of AG over-expression.
The TCP family has also played a significant role in the evolution of form in
angiosperms. Repression of the TCP CYC subfamily of genes is associated with
inhibition of outgrowth in the perianth of Antirrhinum and Lotus (Luo et al., 1996), in the
potato “eye” (Faivre-Rampant et al., 2004), and in the lateral branches of Zea, Oryza,
and Arabidopsis (Doebley et al., 1997; Takeda et al., 2003; Li et al., 2005; Finlayson,
2007). Overexpression of a CYC subfamily gene in A. fimbriata under a CaMV
(constitutive) promoter might produce an embryonic lethal phenotype, but otherwise
would be expected to inhibit growth in many locations. Expression could be targeted to
particular tissues with different promoters. As some CYC subfamily members are
regulated by miRNA (Busov et al., 2008), constitutive expression of miRNA or
repression (by RNA interference) of a miRNA would also be expected alter morphology.
Studies of molecular evolution of this important gene family will suggest additional
hypotheses to test in the A. fimbriata experimental model.
Appendix A
Whole genome comparisons reveal intergenomic transfers
This appendix contains material that may be submitted for publication by Barbara Bliss1.
1Department of Biology, Institute of Molecular Evolutionary Genetics, and the Huck
Institutes of the Life Sciences, 201 Life Sciences Building, Pennsylvania State
University, University Park, PA 16802, USA
162
Abstract
Whole genome comparison performed between fully sequenced chloroplast and
mitochondrial genomes revealed numerous regions of high identity. In comparisons
between the Arabidopsis chloroplast and mitochondrial genome, and between the
Spinacia chloroplast and Beta mitochondrial genome, over 75 gap-free alignments of at
least 100 bases with at least 70% identity were identified. A computer simulation was
devised to evaluate whether these regions of high identity had occurred by chance, and
found they had not. Five of the high-identity regions, psbA, rbcL, ycf1, petG, and psaA,
were selected for phylogenetic analysis to test the hypotheses that the significant
similarities in sequence were due to horizontal transfer from chloroplast to
mitochondrion. Three of these, psbA, rbcL, ycf1, were verified using PCR. My results
confirm the utility of whole genome comparisons for revealing horizontal transfers,
confirm previously reported horizontal transfers of chloroplast to mitochondrial
sequences, and report evidence for horizontal transfer in the asterid lineage.
163
Introduction
Evolutionary relationships can be inferred from sequence similarity by applying
the principle of descent with modification (Darwin, 1958). Sequence data from any of the
three (mitochondrial, chloroplast, and nuclear) genomes may be selected for use in plant
phylogenetic analysis, depending on the potential of the sequence to distinguish the
species of interest (Soltis and Soltis, 1998; Karol et al., 2001). Horizontal gene transfer
(HGT) contradicts patterns of vertical genome transmission, as has been described in the
community of prokaryotic ancestors at the base of the tree of life, in which evolutionary
relationships more resemble a net than a hierarchy (Doolittle, 1999; Jansen et al., 2007).
The earliest eukaryotic cells also used HGT as endosymbiotic relationships were
established with the prokaryotic precursors of the chloroplast and mitochondrion
(Baldauf and Palmer, 1990; Woese, 1998; Woese, 2000; Dyall et al., 2004). Not to be
relegated to the past, HGT continues as an ongoing process (Bergthorsson et al., 2003;
Stegemann et al., 2003) and may account for 1/16,000 tobacco plants carrying a new
section of chloroplast DNA in its nuclear genome (Huang et al., 2003). HGT provides an
additional source of variability in the genome, one that is particularly important to
understand when tracing phylogenetic relationships.
Historically, horizontal transfers between plant organelle genomes have been
revealed one at a time, using careful hybridization (Stern and Palmer, 1984; Sederoff et
al., 1986; Baldauf and Palmer, 1990) together with phylogenetic analysis of sequence
data (Delwiche and Palmer, 1996). Present-day availability of whole, well-annotated
genomes and the tools to manipulate them allow rapid identification and analysis of
164
HGTs, offering new insights into poorly understood events and screening organelle
genomes for horizontally transferred sequence data, which could be misleading in
reconstructions of organismal phylogenies.
In this study, I used a bioinformatics approach to develop a whole genome
comparison of multiple organelles, revealing numerous putative horizontal transfers,
large and small. Experimental evidence from PCR and sequencing reactions confirmed
the existence of previously reported sequences, as well as new ones. Pairwise
comparisons and phylogenetic analyses of alignments from fully sequenced genomes
support the hypothesis that horizontal gene transfer has extensively invaded organelle
genomes.
Methods
Sequence selection, alignment, and phylogenetic analysis
PipMaker (Adams et al., 2000; Schwartz et al., 2000) was used to generate
pairwise alignments of closely related mitochondrial (mt) and chloroplast (cp) genomes.
The strong-hits program of PipTools (http://bio.cse.psu.edu/pipmaker) was used to
identify alignments having at least 70% identity for 100 bases or more, and interrupted by
four or fewer gaps. MultiPipMaker (Schwartz et al., 2003) was used to align and produce
stacked sets of percent identity plots (pips) comparing chloroplast and mitochondrial
genome to each other (Table A-1). Annotations in GenBank
165
(http://www.ncbi.nlm.nih.gov/Genbank/) were used to derive labels for exons in the
Arabidopsis and Spinacia chloroplast
genomes. RNA-coding genes, which are used by both genomes, were excluded and only
chloroplast-specific sequences (Shimko et al., 2001) were further evaluated. Selected
regions of the multiple pairwise DNA alignments produced by MultiPipMaker were
excised with the slice program of PipTools (http://bio.cse.psu.edu/pipmaker), examined,
Table A-1: List of species and GenBank accessions for 25 organelle genome sequences. .
species Accession
Chloroplast genomes
1 Adiantum capillus-veneris NC004766
2 Amborella tricopoda NC005086
3 Arabidopsis thaliana NC000932
4 Calycanthus fertilis NC004993
5 Chaetospheridium globosum NC004115
6 Chlamydomonas reinhardtii NC005353
7 Lotus japonicus NC002694
8 Marchantia polymorpha NC001319
9 Medicago truncatula NC003119
10 Mesostigma viride NC002186
11 Nicotiana tabacum NC001879
12 Oenothera elata NC002693
13 Oryza sativa ssp. japonica NC001320
14 Pinus thunbergii NC001631
15 Populus trichocarpa NC009143
16 Psilotum nudum NC003386
17 Spinacia oleracea NC002202
18 Triticum aestivum NC002762
19 Zea mays ssp. mays NC001666
Mitochondrial genomes
1 Arabidopsis thaliana NC001284
2 Beta vulgaris NC002511
3 Chaetospheridium globosum NC004118
4 Marchantia polymorpha NC001660
5 Oryza sativa ssp. japonica NC011033
6 Zea mays ssp. mays NC007982
166
and manually adjusted with the GENEDOC sequence analysis program (Nicholas et al.,
1997) and with ClustalX (Thompson et al., 1997). Alignments were analyzed with
PAUP* version 4.10b (Swofford, 2002). Nucleotide sequences were analyzed using
Neighbor-Joining/UPGMA method (version 3.573) with negative branch lengths allowed,
and amino acid sequence alignments were analyzed with parsimony.
Comparing distributions of frequencies of similarity scores
The C programming language was used in the Metrowerks CodeWarrior
Integrated Development Environment (IDE) release 2 to develop a computer simulation
(Bliss, B. J. dissertation 2008 enclosure\Cprogramming\overlap\Console App
Release.exe: contact author for access to program and related files) to compare the
distribution of frequency of similarity scores in alignments of actual organelle genomes
with the distribution of frequency of similarity scores in alignments of a simulated
genome sequence with an actual “target” genome sequence. The simulation reads an
alignment file from MultiPipMaker containing multiple pairwise alignments of one
“target” sequence with two or more “query” sequences. Along the alignment, windows of
a user-specified length are evaluated and scored for percent similarity (0.00-1.00). The
frequency with which each similarity score occurs in the alignment is tallied. For each
“query” sequence in the input alignment, the program calculates the base composition
over the length of the sequence and generates a specified number of replicate, simulated
“query” sequences. Each replicate is composed of randomly specified nucleotides, such
that each simulated “query” sequence has the same base composition as the original
167
“query” genome sequence. The frequency distributions for the alignments of the actual
and simulated genome sequences are output in a text file suitable for use with a
spreadsheet program.
Amplification and sequencing
Total DNA was extracted using the CTAB method of McNeal et al. (2006). After
DNA was rinsed with 70% ethanol, samples were re-suspended and stored in .01M Tris.
MacVector (Rastogi, 1999) was used to develop primers for the regions flanking the
chloroplast genes psbA, rbcL, and ycf1 found in both the Arabidopsis mitochondrial and
chloroplast genomes (Table A-2).
Polymerase chain reaction (PCR) total volume 50 μL contained 5.0 μL Mg-free
Taq buffer (Stratagene: 10X) and 5.0 μL MgCl2 (25 mM) for psbA and rbcL, or 5.0 μL
Taq extender buffer with Mg (Stratagene: 10X) and 0.5 μL Taq X enzyme (Stratagene)
for ycf1; 8.0 μL dNTPs (Pharmacia:dATP, dCTP, dGTP, dTTP each at 1.25 mM); 1 μL
Table A-2: Primers to amplify chloroplast genes from Arabidopsis cp and mt genomes.
Gene Organelle genome
Forward primer (5’-3’) Reverse primer (5’-3’) Annealing temp. °C
psbA cp AAT CCA CTT GGC TAC ATC CG
TTT CCG TCT GGG TAT GCG TC 51
mt CCA ATG GTT CCT TCT CTT AGC GT
GCC AGT AGA AGA CGA CAA TAG GTG AGG T 54
rbcL cp CTT TTT GAA GAA GGT TCG GTT AC
TGA ATA CCC CCT GAA GCC AC 51
mt ATA GCC TTC CTT CGG GTA GGG T
CCT GTG AAC TCA GAT AGC CTT GTG G 54
ycf1 cp CTT GGT CCT GTT TAG TCC CAC
AAA GCC AAG GGG TCA ACA GG 52
mt AAG AAG TTG TCC CCT CCT CTC
GTC TTC CCA TTC ATT CCC AG 51
168
each of left and right primers (MWG Biotech: 20 μM); 0.5 μL Taq (Stratagene: 5U/μL); 1
μL DNA template. PCR conditions were 94ºC for 2 minutes initially, followed by 30
cycles of denaturing at 94ºC for 30 seconds, annealing at primer-specific temperatures
(Table A-2) for 60 seconds (touch-down 1ºC cycle), extension at 72ºC for 2 minutes, and
an additional 5 minutes of extension at 72ºC in the last cycle. Five µls of each PCR
reaction were loaded onto 2.5% high-resolution agarose gel (Sigma-Aldrich Co., St.
Louis, MO, #A-4718) for electrophoresis. Selected PCR products were excised from the
gel, followed by gel extraction (QiaQuick, Qiagen). The chloroplast and mitochondrial
ycf1 sequences were determined by automated DNA sequencing on a Beckman-Coulter
CEQ2000 genetic analyzer according to manufacturer‟s instructions.
Results
In pairwise comparisons of the Arabidopsis and Spinacia chloroplast genomes
with 17 other organelle genomes, MultiPipMaker found numerous regions of high quality
alignment occurring with P<0.01 (red) and P<0.05 (green) (Figure A-1 A, B).
169
Figure A-1: MultiPipMaker plots of organelle genome sequences aligned to chloroplast
target genome sequences. A. Arabidopsis chloroplast genome compared to 17 other
organelle genomes. B. Spinacia chloroplast genome compared to 17 other organelle
genomes. Exon names on upper edge indicate coding sequences recorded in GenBank for
the indicated chloroplast genome. High quality alignments are indicated in red (P<0.01)
and green ( P<0.05).
A
B
170
The shape of curves produced by distributions of similarity scores from pairwise
alignments of closely related mt v. cp genome sequences differed from the shape of
curves produced by distributions of similarity scores from pairwise alignments
constructed with simulated mt sequences (Figure A-2). Alignments were evaluated in
windows of 50, 100, 150, and 300 bases. Regardless of the window size, species or
genome used, alignments containing simulated sequences produced a bell-shaped curve
(random distribution) and alignments using actual sequences produced an L -shaped
curve (Figure A-2).
In alignments containing simulated sequences, as window size increased, the
range of high frequency similarity scores narrowed as the amplitude increased, such that
the center of the distribution remained the same. For example, in the alignment of the
simulated Arabidopsis mt genome v. the Arabidopsis cp genome, when the alignment
was evaluated with short (50 bp) windows, over 5% of the alignments had approximately
5-35% identity (a range of 30 percentage points), but when evaluated with long (300 bp)
windows, the same proportion had approximately 10-30% identity (a range of only 20
percentage points) (Figure A-2 A). Correspondingly, there were fewer short windows
with precisely 20% identity than there were long windows. Regardless of window size,
the greatest proportion of the alignment with the simulated sequences displayed 20%
sequence identity (Figure A-2 A).
In alignments with actual genomes, alignments with over 50 % identity occurred
consistently, although less than 5% of the time for any particular value. In comparison, in
alignments containing simulated genomes, windows with over 50% identity never
occurred (Figure A-2 A). In addition, there were a greater proportion of windows having
171
more than 50% identity in the mitochondrion v. chloroplast genome alignments of Beta
mt v. Spinacia cp, Oryza mt v. Oryza cp, and Zea mt v. Zea cp than in the Arabidopsis mt
v. Arabidopsis cp genome alignments (Figure A-2 A-D). In Zea, over 10% of the aligned
Figure A-2: Frequency of high identity regions in alignments of real and simulated
mitochondrial to chloroplast genome sequences. In simulations, 10,000 simulated (sim)
mitochondrial genome sequences were aligned to the chloroplast genome sequence.
Windows of 50, 100, 150, and 300 bases were evaluated in A. Arabidopsis mt and cp
organelle genomes B. Spinacia cp genome compared to Beta mt genome C. Oryza mt
and cp organelle genomes D. Zea mt and cp organelle genomes.
172
organelle genomes shared 90% identity, and in Oryza and Beta/Spinacia, over 5% of the
aligned organelle genomes shared 90% identity (Figure A-2 B-D).
The shape of distribution of similarity scores in pairwise alignments of cp v. cp
genome sequences differed from the shape of the distributions of similarity scores in
pairwise alignments of mt v. cp genomes (Figure A-3). As in Figure A-2, distributions
from the simulated organelle genome sequence alignments (series with broken lines, all
colors), all displayed bell shaped curves while the distributions from actual organelle
genome sequence alignments (series with solid lines, all colors), displayed L-shaped
curves (Figure A-3). However, distributions of similarity scores in pairwise alignments
of 11 chloroplast genomes (series with solid blue–green lines) to the target Arabidopsis
(Figure A-3 A) and Spinacia (Figure A-3 B) chloroplast genomes all took approximately
the same shape, while distributions of similarity scores in the alignments of six
mitochondrial genomes (series with solid yellow-red lines) to the target cp genomes
overall took a different shape (Figure A-3 A, B). The distributions of scores in the
pairwise alignments of cp genomes, actual or simulated, v. target cp genomes (series with
solid or broken blue–green lines) produced a higher frequency of 100 bp, high identity
alignments (Figure A-3 A, B).
173
Figure A-3: Distribution of similarity scores for 100 bp windows from 17 pairwise
alignments of mt genome sequence or simulated mt genome sequence to cp genome
sequence for A. Arabidopsis and B. Spinacia cp genomes.
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100
Nicotiana_cp Nicotiana_cp (sim)
Medicago_cp Medicago_cp (sim)
Populus_cp Populus_cp (sim)
Spinacia_cp Spinacia_cp (sim)
Amborella_cp Amborella_cp (sim)
Zea_cp Zea_cp (sim)
Triticum_cp Triticum_cp (sim)
Oryza_cp Oryza_cp (sim)
Pinusthun_cp Pinusthun_cp (sim)
Marchantia_cp Marchantia_cp (sim)
Chaeto_cp Chaeto_cp (sim)
Arabidopsis_mt Arabidopsis_mt (sim)
Beta_mt Beta_mt (sim)
Zea_mt Zea_mt (sim)
Oryza_mt Oryza_mt (sim)
Marchantia_mt Marchantia_mt (sim)
Chaeto_mt Chaeto_mt (sim)
% b
ases
% identity
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100
Nicotiana_cp Nicotiana_cp (sim)
Medicago_cp Medicago_cp (sim)
Populus_cp Populus_cp (sim)
Arabidopsis_cp Arabidopsis_cp (sim)
Amborella_cp Amborella_cp (sim)
Zea_cp Zea_cp (sim)
Triticum_cp Triticum_cp (sim)
Oryza_cp Oryza_cp (sim)
Pinusthun_cp Pinusthun_cp (sim)
Marchantia_cp Marchantia_cp (sim)
Chaeto_cp Chaeto_cp (sim)
Arabidopsis_mt Arabidopsis_mt (sim)
Beta_mt Beta_mt (sim)
Zea_mt Zea_mt (sim)
Oryza_mt Oryza_mt (sim)
Marchantia_mt Marchantia_mt (sim)
Chaeto_mt Chaeto_mt (sim)
% identity
% b
ases
A
B
174
Fully sequenced mt genomes were also aligned with closely related cp genome
sequences using PipMaker (Schwartz et al., 2000). The strong-hits program of PipTools
indicated the pairwise alignments of the Arabidopsis and Beta mt genomes to their
closest cp genome sequences (Arabidopsis or Spinacia) had more than 75 gap-free, 100
bp alignments with at least 70% identity in regions annotated with chloroplast-specific
genes (data not shown). These alignments included coding sequences for the photosystem
II reaction center polypeptide D1 (psbA); the large subunit of ribulose bisphosphate
carboxylase (rbcL); the cytochrome b6/f complex subunit 5 (petG); the photosystem II
reaction center protein D2 (psbD); two conserved, large open reading frames frequently
found in angiosperms (ycf1, ycf2); photosystem I P700 apoprotein A1 (psaA); and an
approximately 500 bp open reading frame (orf) (Table A-3). Three of these sequences,
rbcL, psbA and ycf1 were excised from MultiPipMaker multiple sequence alignments
for input to PAUP* for phylogenetic analysis and verification by amplification and
sequencing.
175
Bootstrap (500 replicates) consensus trees generated from analyses of the
MultiPipMaker alignments of the psbA, rbcL, and ycf1 sequences indicated the
Arabidopsis chloroplast and mitochondrial sequences were most closely related to each
other (Figure A-4) than to other chloroplast or mitochondrial sequences.
Table A-3: Locations of chloroplast-specific sequences in pairwise alignments of mt
genome sequences with cp genome sequences. Base location references the pairwise
alignment of Beta mt v. Spinacia cp genome sequences or Arabidopsis mt v. Arabidopsis
cp genome sequences.
Organelle genome base location
exon
(pipname)
Arabidopsis
chloroplast
Spinacia
chloroplast
Arabidopsis
mitochondrion
Beta
mitochondrion
psbA 218-750 334571-334320
rbcL 55493-56072 C78120-77545
petG-1 64599-64791 1430-1241
petG-2 64860-64976 249997-250070 1177-1051
psbD 33567-33674 102181-102074
ycf2 86178- 86608 324676-325076
145081-147267
ycf1
127374-127487 81096-80992
128712-129641 C105953-105021
109008-109937
126568-127489 105021-105951
105956-106911 105951-104993
psaA 39012-39162 234192-234042
orf 105187-105698 364678-365190
176
Figure A-4: Strict consensus trees from NJ analysis with PAUP* (500 bootstrap
replicates) using aligned coding sequences from cp and mt genomes for A. rbcL (13 taxa)
B. psbA (13 taxa) C. ycf1 (15 taxa).
+-NICOTI_CP
!
! +---SPINA_CP
-11--9
! +-OENO_CP
!
! +ARAB_CP
! +--3
! ! +-ARAB_MT
! +--7
! ! ! +-ORYZA_CP
! ! ! +--1
! ! +--2 +TRITI_CP
! ! !
+-10 +-ZEA_CP
!
! +-LOTUS_CP
! +--6
! ! +-MEDICA_CP
+--8
! +-----MARCH_CP
! +--4
+--5 +----PINUS_CP
!
+------PSILO_CP
+LOTUS_CP
+--5
! +MEDICA_CP
!
! +---MARCH_CP
+--8 +--1
! ! +--3 +---PSILO_CP
! ! ! !
! ! ! +----PINUS_CP
! +--6
! ! +ORYZA_CP
+-10 ! +--2
! ! +--4 +ZEA_CP
! ! !
! ! +TRITI_CP
! !
! ! +-NICOTI_CP
! +--9
! +SPINA_CP
!
-11-OENO_cp
!
! +ARAB_cp
+--7
+ARAB_MT
+NICOTI_CP !
-13SPINA_CP ! ! +ARAB_cp! +-11 ! ! +ARAB_MT! ! ! ! +--LOTUS_CP ! ! +--7 +-12 ! +-------MEDICA_CP
! ! ! ! +-------------MARCH_CP ! ! +--2 ! ! ! ! +----------------PINUS_CP ! ! +--3 +--1 +-10 ! ! +MARCH_MT
! +------4 ! ! ! ! +----------PSILO_CP ! ! ! ! +--8 +--BETA_MT! ! ! ! ! ! +ORYZA_CP ! ! +--6 +--9 ! +TRITI_CP
! +--5 ! +ZEA_CP ! +--------OENO_cp
A B
C
177
To verify that the three Arabidopsis sequences analyzed in Figure A-4 were not
misannotations, I used primers designed from the pairwise alignments to separately
amplify the psbA, rbcL, and ycf1 coding sequences from the Arabidopsis chloroplast and
mitochondrial genomes (Figure A-5). The ycf1 chloroplast and mitochondrial genome
products were sequenced and had less than 1% difference from the corresponding
accessions in GenBank (data not shown).
Figure A-5: Successful amplification of psbA (A, C), rbcL (B, D), and ycf1 (E, F), from
the Arabidopsis thaliana (A, B, E) chloroplast and (C, D, F) mitochondrial genomes.
C C C A A A B B B D D D
E E E F F F F F F
178
Bootstrap (500 replicates) consensus trees generated from analyses of the
MultiPipMaker alignments of petG and psaA sequences revealed the Spinacia chloroplast
and Beta mitochondrial sequences are more closely related to each other (Figure A-6)
than to other chloroplast or mitochondrial sequences.
Figure A-6: Strict consensus trees from PAUP* (500 bootstrap replicates) analysis of
aligned coding sequences from cp and mt genomes using A. NJ analysis of petG
nucleotide sequence (15 taxa) B. Parsimony analysis of psaA amino acid sequences (13
taxa).
Beta mtpetG
Arabidopsis cppetG
Medicago cppetG
Populus cppetG
Spinacia cppetG
Pinusthun cppetG
Chaeto cppetG
Adiantum cppetG
Marchantia cppetG
Mesostigma cppetG
Chlamyrein cppetG
Nicotiana cppetG
Zea cppetG
Triticum cppetG
Calycanthes cppetG
Amborella cppetG
Spinacia cppsaA
Beta mtpsaA
Nicotiana cppsaA
Arabidopsis cppsaA
Populus cppsaA
Medicago cppsaA
Calycanthes cppsaA
Amborella cppsaA
Zea cppsaA
Triticum cppsaA
Pinusthun cppsaA
Adiantum cppsaA
Marchantia cppsaA
Mesostigma cppsaA
Chaeto cppsaA
Chlamyrein cppsaA
A B
179
chloroplast or mitochondrial sequences, despite the genome-wide greater similarity of
mitochondrial genomes to other mitochondrial genomes (Figure A-3).
Discussion
Genomic sequences displaying similarity to those of the chloroplast or
mitochondrial genomes may be similar due to horizontal rather than vertical transmission.
In-depth studies have revealed repeated transfers of conserved sequences from the plant
chloroplast and mitochondrial genomes to the plant nucleus (Baldauf and Palmer, 1990;
Adams et al., 2001; Millen et al., 2001; Adams et al., 2002), Bioinformatic methods have
revealed horizontal transfers to the nucleus on a larger scale (Lin et al., 1999; Stupar et
al., 2001; Rice Chromosome 10 Sequencing Consortium, 2003). I sought to determine if
comparisons of fully sequenced organelle genomes could yield additional insight into
horizontal transfers.
In this study, whole genome sequence alignments of multiple chloroplast and
mitochondria genomes revealed numerous regions of high-identity. Those regions
attributable to common, conserved function (e.g., ribosomal RNA genes) were excluded
from the analysis. I hypothesized that the remaining high-identity alignments arose either
by horizontal transfer, or else by chance, due to the base composition of the mt genome
sequence. In simulations conducted to evaluate the latter alternative, gap-free alignments
of 100 bp or more and greater than 50% identity were not observed. Gap-free alignments
of over 100 bp with 70% or greater sequence identity were subjected to phylogenetic
analyses, which demonstrated that the psbA, rbcL, ycf1, petG, and psaA sequences from
180
the fully sequenced mt genomes were most closely related to the corresponding
sequences in the cp genome, reflecting horizontal transfer. Additional analyses could
query nuclear genome sequence data to determine if horizontally transferred sequences
were transferred directly from the chloroplast to the mitochondrion, or if the nuclear
genome served as an intermediate.
The greater proportion of high-identity alignments between the Zea/Zea,
Oryza/Oryza, and Beta/Spinacia organelle genomes than between the
Arabidopsis/Arabidopsis ones suggests transfers from the Arabidopsis chloroplast to
mitochondrion may have happened longer ago than the others, or may otherwise be
diverging more rapidly. Cummings et al. (2003) found that, among the transfers of rbcL
to the Arabidopsis, Brassica, Zea, Oryza, and Ipomoea mt genomes, those in the crucifer
lineage were most ancient. Comparative, whole genome analysis can be used to describe
the timing of large-scale genomic events (Cui et al., 2006). Including additional species
in whole genome analysis of organelle sequence data can help to better estimate the
frequency and timing of large scale horizontal transfers.
Acknowlegements
Xiaomu Wei and Joel McNeal provided technical assistance. Dr. Webb Miller
provided extended access to the PipMaker server http://bio.cse.psu.edu/pipmaker/. The
lab of Dr. Hong Ma provided A. thaliana, Lansberg ecotype. The lab of Dr. Ramesh
Raina provided A. thaliana, Columbia ecotype DNA. Primers and materials were funded
from the Chloroplast Genome grant awarded to Dr. Claude dePamphilis.
Appendix B
Cultivation Supplement
This appendix contains material that may be submitted for publication by Barbara Bliss.1
This Cultivation Supplement describes seed germination experiments and cultivation
methods. Included are detailed, illustrated instructions for hand pollinating Aristolochia
fimbriata and related species (e.g., A. elegans). The system used for recording plant
pedigree is described herein, as are abnormal seedlings initially observed during AAGP
seedling tissue collection efforts.
1Department of Biology, Institute of Molecular Evolutionary Genetics, and the Huck
Institutes of the Life Sciences, 201 Life Sciences Building, Pennsylvania State
University, University Park, PA 16802, USA
182
Seed germination experiments
Reliable seed germination methods are prerequisite for developing inbred lines, for
collecting tissues for seedling expression libraries (etiolated and non-etiolated tissues
used by AAGP), for developing seedling selection protocols for antibiotic selection of
potentially transgenic individuals, and for evaluating seed storage methods. Aristolochia
germinates best in potting medium in the greenhouse, but, some circumstances require
soil-free germination to allow continual access to the germinating seed. In order to
identify an efficient method of germinating seeds, I initially investigated several methods
of germination, using 25 seeds each, placed in varying conditions of light, temperature,
and medium for 42 days (Figure B-1). Seeds germinated with varying success in
different conditions. I experimented with total darkness (D), and with 16 hour days with 8
hour nights (16/8). Light and temperature conditions were provided by varying means.
Agrowth chamber provided30°C days, 16°C nights (GrCham). A Conviron room
programmed for maize provided 25°C days, 25°C nights (cornEnviron). An incubator
set at 30°C, provided no light for seeds incubated on petri dishes (PD) containing
sucrose-free, half-strength MS media (PDmedia), or wet filter paper (PDfp). Seeds were
from open-air, insect-mediated pollinations of the S1 and S2 lines combined (OAL) or
from self-pollinations of individual S1 and S2 plants (S1-30, S2-18). Data collected from
the initial experiment were not appropriate for statistical analysis, but the three samples
with the highest percent germination (mean= 0.96, SD 0.04) were those germinated in
petri dishes containing filter paper (PDfp; Figure B-1). Lack of humidity in the incubator
and in the cornEnviron was problematic, and seeds often dried out.
183
In another experiment to determine if seeds would germinate in the dark as well
as in a light/dark regime (Figure B-2), seeds from open air pollinations of S1-27 flowers
Figure B-1: Seeds planted in varying conditions on 9/28/2006. Conditions of light,
medium, humidity and temperature were varied. Seeds germinated irregularly. Greatest
percent germination results were obtained using petri dishes (PD) containing wet filter
paper (fp) instead of no sucrose media (media).
0
10
20
30
40
50
60
70
80
90
100
7 14 21 28 35 42
% G
erm
inat
ed
Days
LD 28/25 cornEnvir PDfp OAL LD 28/25 cornEnvir PDmedia OAL
LD 28/25 cornEnvir PDmedia S1-30 LD 28/25 cornEnvir PDmedia S2-18
D 28/25 cornEnvir PDfp OAL D 28/25 cornEnvir PDmedia OAL
D 28/25 cornEnvir PDmedia S1-30 D 28/25 cornEnvir PDmedia S2-18
16/8 30/16 GrCham PDfp OAL 16/8 30/16 GrCham PDmedia OAL
16/8 30/16 GrCham PDmedia S1-30 16/8 30/16 GrCham PDmedia S2-18
D 30/16 GrCham PDfp OAL D 30/16 GrCham PDmedia OAL
D 30/16 GrCham PDmedia S1-30 D 30/16 GrCham PDmedia S2-18
16/8 25 GrRoom PDmedia OAL 16/8 25 GrRoom PDmedia S1-30
16/8 25 GrRoom PDmedia S2-18 D 25 GrRoom PDmedia OAL
D 25 GrRoom PDmedia S1-30 D 25 GrRoom PDmedia S2-18
D 25 Incubator PDfp OAL D 30 Incubator PDmedia OAL
D 30 Incubator PDmedia S1-30 D 30 Incubator PDmedia S2-18
184
were planted in petri dishes containing sucrose-free, half-strength MS media in an
attempt to provide continuous moisture. The plates were incubated in a growth chamber
operating at 30°C days, 16°C nights, either in total darkness (D) or 16 hour days/8 hour
nights (16/8). Overall, the percent germination was low (mean 0.30, SD 0.1354) with no
significant difference (two sample T test) between mean percent germinated in 16/8 (n=8,
mean 9.50, SD 2.73) and D (n=8, mean 10.25, SD 4.74) treatments (Figure B-2).
To directly evaluate germination on media compared with wet paper (Figure B-
3), seeds from open air pollinations of S1-35 or S2-01 plants were planted in petri dishes
Figure B-2: Comparing effects of light on germination of seeds planted on ½ MS media.
Seeds from open air pollinations of S1-27 flowers were planted on 10/6/2006 on ½ MS
medium in petri dishes and incubated at 30°C days, 16°C nights, either in total darkness
(D) or 16 hour days/8 hour nights (16/8). There was no significant difference (two sample
T test) in mean percent germination in 16/8 and D treatments.
0
10
20
30
40
50
60
70
80
90
100
7 14 21 28 35 42 49
% G
erm
inat
ed
Days
16/8
16/8
16/8
16/8
16/8
16/8
16/8
16/8
D
D
D
D
D
D
D
D
185
(PD; 25 seeds each) containing sucrose-free, half-strength MS (media) or wet filter paper
(fp) and incubated in total darkness in a growth chamber at 30°C days, 16°C nights.
Mean percent germination at 42 days was significantly better (Student‟s two sample t-
test, P < 0.05) in wet filter paper (n=11, mean=0.6145, SD 0.1572) than in media (n=3,
mean=0.0933, SD 0.0611) (Figure B-3).
To evaluate the effects of light on seeds germinating in wet toweling (Figure B-
4), seeds from open air pollinations of flowers on five different S2 plants were planted in
packets of wet towelling, 25 seeds each, and incubated in total darkness (D, n=10) or in
16 hour days/8 hour nights (16/8, n=10) in a growth chamber at 30°C days, 16°C nights.
There was no significant difference between the two treatments at 42, 70, or 91 days.
Figure B-3: Comparing seed germination on wet filter paper or medium. Seeds from open
air pollinations of flowers on S1-35 or S2-01 plants were planted on 10/13/2006 and
incubated in total darkness in a growth chamber at 30°C days, 16°C nights. Seeds
germinated significantly better on wet filter paper (fp) than in petri dishes (PD) containing
sucrose-free ½ MS media.
0
10
20
30
40
50
60
70
80
90
100
7 14 21 28 35 42
% G
erm
inat
ion
Days
PDfp
PDfp
PDfp
PDfp
PDfp
PDfp
PDfp
PDfp
PDfp
PDfp
PDmedia
PDmedia
PDmedia
PDfp
186
Mean percent germination at 91 days was 0.7940 (SD 0.1103). At 42 days, mean overall
percent germination was 0.4480, SD 0.2131 (Figure B-4).
Results from Figure B-4 were lower than the mean percent germination at 42
days in a more brief germination exercise shown in Figure B-4, conducted with the same
methods using seeds from different open-air pollinated S2 plants. In Figure B-4, the 42
day mean percent germination rate was 0.6171 (SD 0.2416), and percent germination in
16/8 (n=14, mean=0.723, SD 0.179) was significantly better than in D (n=14,
mean=0.511, SD 0.255) (Student‟s t test, P<0.05) (Figure B-5).
Figure B-4: Comparing effect of light on germination of seeds planted in wet toweling.
There was no significant difference in mean percent germination of seeds from open air
pollinations of flowers on S2 plants planted 10/27/2006 in packets of wet toweling,
incubated in D (n=10) or in 16/8 ( n=10) in a growth chamber at 30°C days, 16°C nights.
0
10
20
30
40
50
60
70
80
90
100
7 14 21 28 35 42 49 56 63 70 77 84 91
% G
erm
inat
ion
Days
16/8 S2-02
16/8 S2-08
16/8 S2-14
16/8 S2-14
16/8 S2-14
16/8 S2-14
16/8 S2-10
16/8 S2-10
16/8 S2-10
16/8 S2-10
D S2-02
D S2-14
D S2-14
D S2-14
D S2-14
D S2-14
D S2-10
D S2-10
D S2-10
D S2-16
187
There was considerable variability of the effect of light on seed germination
(Figure B-2, B-4, B-5, B-6). It may be that seeds from S2-16 and S2-17 generally
germinate better that other seeds, but it does not appear that light itself is an important
factor for seed germination. Paula Ralph helped with some of these experiments. We
noted that seeds germinating in total darkness died, and light was required for the true
leaves to emerge.
Figure B-5: Effects of light on germination of seeds planted in wet toweling. Seeds from
open air pollinations of S2 plants planted 12/15/2006 in wet toweling, incubated in a
growth chamber with 30°C days, 16°C nights had significantly higher percent
germination after 42 days in 16/8 (n=14) than did seeds incubated in D (n=14).
0
10
20
30
40
50
60
70
80
90
100
7 14 21 28 35 42
% G
erm
inat
ion
Days
16/8 S2-8 16/8 S2-8
16/8 S2-8 16/8 S2-23
16/8 S2-23 16/8 S2-23
16/8 S2-17 16/8 S2-17
16/8 S2-22 16/8 S2-22
16/8 S2-22 16/8 S2-22
16/8 S2-22 16/8 S2-22
D S2-8 D S2-8
D S2-8 D S2-23
D S2-23 D S2-23
D S2-17 D S2-17
D S2-17 D S2-22
D S2-22 D S2-22
D S2-22 D S2-22
188
Figure B-6: Comparing effect of light on germination of seeds planted on soft media.
Seeds from open air pollinations of flowers on S1-44 plants planted 11/13/2006 on PD
containing soft media and incubated in D (n=5) or in 16/8 (n=5) in a growth chamber at
30°C days, 16°C nights. Mean percent germination at 70 days was significantly greater
in D.
0
10
20
30
40
50
60
70
80
90
100
7 14 21 28 35 42 49 56 63 70
% G
erm
inat
ion
Days
16/8
16/8
16/8
16/8
16/8
D
D
D
D
D
189
Germinating in toweling
Although greenhouse germination in soil, on a heating mat generally produced the
best results (Bliss et al., in preparation, 2008), seeds are germinated in toweling packets
when seedlings must be handled. No other method was found to be superior (see
Figures B-1 through B-6, also Figure 5-1). The toweling method was used exclusively
to germinate seeds for AAGP seeding tissue collection (Figure B-7).
Figure B-7: Toweling germination method. A. Seeds arranged in center third of wet
towel. B. Fold over one side C. Fold over the other, apply more water D. Insert toweling
packet into plastic bag (plastic pedigree label is included in this bag), fold over and
incubate under light.
190
Up to 80 (or more) seeds were arranged in the center third of a wet paper towel
(Scott Hi Wet Strength), each side of which was then folded back over the seed array
(Figure B-7 A, B). The packet was squirted with distilled water to saturate all layers of
the toweling thoroughly. The tri-folded wet (but not dripping) towel packet was put into a
polyethylene plastic storage bag, folded to loosely close it over and around the towel
packet. The outside of the plastic bag was labeled to indicate the contents and placed
under lights in a growth chamber operating at 35C for 16 hour days, 25C for 8 hour
nights. Germination was recorded with the appearance of the hypocotyl emerging from
the seed coat. Seeds also have been germinated on shelves in a room maintained at 70° F
(21°C), approximately 18 inches (50 cm) below fluorescent lights fixtures containing
40W cool-white bulbs operating 16 hours/day.
191
Seed storage experiments
Seeds contain the germline of the individual, so the seeds produced by a
transformed plant or naturally occurring mutant represent an important resource for
studying the genetic basis of the phenotype. For annual plants, including Arabidopsis and
Zea, seed storage is critical because the individual dies after producing seed. For A.
fimbriata, a long-lived herbaceous perennial that can also be vegetatively propagated for
ongoing use as a stock plant, seed longevity is less crucial, but it remains important for
planning future experiments. In order to determine if seeds would remain viable after a
period of time in storage, I looked for evidence of any initial decline in seed viability
after short term (7-150 days; Figure B-8) and after longer-term (522-942 days; Figure B-
9) storage in the workplace, at room temperature.
There were no significant differences in germination among seeds planted in wet
toweling weekly, for 22 weeks (Figure B-8). These findings were consistent with
observations from the greenhouse. After 30 days, 50% of the seeds planted at seven day
intervals each group had germinated, and 90% germination was obtained in 63 days. This
was considerably worse than in the greenhouse, where 90% germination was often
observed in 30 days.
192
In the older seeds (Figure B-9), seeds in the middle group (761 days old), were
significantly more likely to likely to germinate than the other groups, and the youngest
group of seeds (577 days old) was slightly more likely to germinate than the oldest group
of seeds (942 days old) (Figure B-9). The middle group of seeds, which had the highest
percent germination, were collected in November, and the newest and oldest seeds were
collected in May. These results suggest something other than seed age was affecting
Figure B-8: Short term effects of seed age on germination. Successive planting of seeds
from open-air pollinations were germinated in toweling packets at weekly intervals,
seven to 150 days after collection, with no significant differences in germination. Most
packets reached maximum germination by 60 days, with over 50% germination obtained
by 30 days. Mean germination at day 35 (n=10) was 0.7695 (SD 0.0199), at day 63
(n=15) was 0.8987 (SD 0.0537).
0
10
20
30
40
50
60
70
80
90
100
7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 126
% G
erm
inat
ed
Days
11/22/2004
11/29/2004
12/6/2004
12/13/2004
12/20/2004
12/27/2004
1/4/2005
1/10/2005
1/17/2005
1/24/2005
1/31/2005
2/7/2005
2/14/2005
2/21/2005
2/28/2005
3/7/2005
3/14/2005
3/21/2005
3/28/2005
4/4/2005
4/11/2005
4/18/2005
193
germination, perhaps the time of year when pollination and maturation took place, or
conditions in the greenhouse when the capsules were maturing.
Figure B-9: Longer-term effects of seed age on germination. Seeds of different ages
from open air pollinations of flowers on mixed plants (VL, and NV) were planted
6/15/2007 in wet toweling and incubated in 16 hour days/8 hour nights (16/8, n=14) in a
growth chamber with 30°C days, 16°C nights. The midrange seed age (761 days old) was
significantly (P < 0.05 in binary regression analysis) more likely (3.83 times more likely)
to germinate than the newest seeds (577 days old). The oldest seeds 942 days old were
0.90 times as likely to germinate as the newest seeds, not significantly different.
0
10
20
30
40
50
60
70
80
90
100
7 14 21 28 35 42 49 56 63 70 77
% G
erm
inat
ion
Days
942
942
942
942
761
761
761
761
577
577
577
577
194
Inducing flowering
A. fimbriata has a vining habit, and produces numerous indeterminate shoots, 1-3
m long, depending on conditions. Blooming can be induced in the same manner as is
done for many herbaceous perennials,by pruning and feeding, as needed, to stimulate a
flush of new growth. Cut plants back to the rhizome approximately two weeks before
pollination efforts are scheduled to begin (Figure B-10 A). After shoots have begun to
proliferate, set pots containing new shoots into larger pots, and trellis them (Figure B-10
B). Older, larger plants may be potted directly into large pots outfit with large trellises.
Arrange trellised plants for pollination, fruit production, and harvest (Figure B-10 C).
Flower and secondary shoot meristems arise from axils (Figure B-11), so a new
flower will open every day, or nearly so. Shoots will produce flowers indefinitely
(Figure B-12), as they elongate, but productivity declines as shoots lengthen and age.
Pruning can improve productivity. Heading (removal of terminal meristems) will induce
axillary meristem outgrowth. Thinning (removal of shoots at the crown) will induce
production of new shoots from the crown. Selective pruning can be used to maintain
induction of flowers on fresh, lower shoots while fruit matures. Collect fruit after the
capsule opens.
195
Figure B-10: Inducing blooms A. Prune back A. fimbriata to induce new shoot
proliferation, which will be visible in 3-5 days. This plant is approximately two years old.
B. Set pot containing plant into larger pot with trellis, in preparation for hand pollinations
and seed collection C. Arrange all plants for pollination, fruit maturation, fruit harvest..
196
Plants sharing a desirable pedigree may be isolated in a room apart from A.
fimbriata plants of other parentage in order to generate closely related seeds by open air
pollination, effected by insects naturally occurring in the greenhouse (e.g., fungus gnats
and other dipterans). For AAGP, first and second generation inbred plants (S1 and S2
descended from LR08) were isolated and used as the source of tissues for transcriptome
sequencing. Seeds from fruits produced by open-air pollinations in that room were
germinated to yield seedling tissues used by AAGP.
Figure B-11: A. fimbriata produces successive axillary blooms along the shoot.
197
Figure B-12: A. fimbriata in bloom. Four main shoots from the crown producing flowers
at successive stages..
198
Hand pollination
These instructions are written for a novice worker in the lab to begin conducting
hand pollinations with A. fimbriata. A basic knowlege of floral organs and plant anatomy
is required: the student may wish to refer to a plant biology text in the lab if terms are
unfamiliar (Raven et al., 1999; Simpson, 2006; Bliss et al., in preparation, 2008).
Select a trellised plant producing flowers at the shoot terminals (Figure B-12),
and tie a pollination bag around the unopened flowers. The entire end of the shoot may be
enclosed in the bag, selecting three or more flowers for subsequent hand pollination
(Figure B-13). Loosely wrap the excess material of the pollination bag to cushion the
stem and tie it below the petiole of the lowest flower, preventing pollinator access
without crushing the stem (Figure B-14 A). Attach tags just below the pollination bag
(Figure B-14 B). Check pollination bags daily for open flowers (Figure B-15). Note on
the tag the date the flower first appears open (day one; first day of anthesis).
199
Figure B-13: Pollination bag. Four unopened flowers are enclosed at the end of one shoot.
200
Figure B-14: Pollination bag attachment detail. A. Tie pollination bag around stem (a)
below petiole of lowest flower enclosed in bag. B. Tie tag below string of pollination bag.
Hand pollinations should be done on day one or day two flowers, as those are the
days in which the stigma is most receptive (Bliss et al., in preparation, 2008). Pollen can
most easily be collected from day two and day three flowers, but we have routinely done
self-pollination of day one flowers. On the first day of anthesis, the anthers have not yet
dehisced, so it is necessary to scrape open the anther to collect pollen on the tip of the
toothpick, or pry an anther locule (or portion of a locule) from the anther and deposit it on
the receiving stigma. If pollen will be collected from a different flower, it also should
have been bagged to prevent contamination with foreign pollen from visiting insects.
a
b
201
Once the flower has been removed from the pollination bag, take care to prevent
pollinator access. Cut the utricle above the gynostemium (Figure B-16 A) and inspect the
utricle, limb, and gynostemium. If an insect is present the pollination bag has failed, so
abort the hand pollination attempt. On the first day of anthesis, the lobes of the
gynostemium should curve inward slightly (Figure B-16 B).
Figure B-15: Day of anthesis. Flower enclosed in pollination bag. Note backward bent
fimbriae..
202
Collect pollen on a toothpick from the anthers, which are on the outside of the
gynostemium (Figure B-17 A). Transfer pollen on the toothpick to the stigma (Figure B-
17 B). Note that the flower in Figure B-17 is a “day two” flower, so its pollen has
Figure B-16: Gynostemium A. Sever the perianth below the limb, above the
gynostemium. The gynostemium occupies about 2/3 of the utricle. B. On the first day,
the lobes of the gynostemium will curve slightly inward
203
already dehisced. Self pollinations are optimally conducted with day two flowers (Bliss et
al., in preparation, 2008).
Figure B-17: Self-pollination in a day two flower A. Collect pollen from the anthers with
a toothpick. B. Transfer it to the stigmatic surfaces of the receiving flower. (This is self-
pollination: Both A. and B. are the same flower)
204
Use lab tape to tape closed the remnant of the perianth, preventing further access
to the stigmatic surfaces. Fold the edges of the perianth as needed. The perianth,
gynoecium, and tape will abscise above the inferior ovary (Figure B-18 A). If successfully
fertilized, the ovary will begin to grow in a week (Figure B-18 B) and mature in a month.
Figure B-18: Inferior ovary. A. Perianth, taped shut, abscisses where indicated by tip of
arrow. B. Maturing ovary, after perianth with lab tape has abscissed.
205
Recording pedigree
A method for labelling the plants was devised to convey the maximum amount of
information possible on the tag in the pot in the greenhouse. Pedigree information can be
determined from the label in the pot. An alternate method of labelling the plants would
be to assign a serial number to the plant and tag, and record in a notebook the pedigree of
the plant tageed with the serial number. In that system, the serial number would serve to
connect the plant to the notebook and the notebook would connect the serial number to
the pedigree information. Based on the experience I had keeping lineage information for
our tissue culture work, I elected to use labelling system in the greenhouse which would
communicate the pedigree directly from the pot label. Capsules are stored in envelopes
with the pedigree information written on the outside. Here is the labelling and pedigree
recording system, which was used to label the first and second generation lines used for
AAGP tissue collection.
The name is written from left to right, adding on the right end as the depth of the
pedigree increases. The label is interpreted from right to left, as follows.
a. Genotype = LR or CO (equivalent to VL and NV)
b. Seed/plant ID = 01-99. At one time, there were about one hundred plants each
of LR and CO, all from the same parent plant. Eventually, many were
eliminated to reduce maintenance.
c. Type of event generating the capsule. The letters S and O are reserved:
S=selfing, O=open air pollination. Crosses recorded in the experimenters lab
notebook can use other letters.
206
d. Year in which this capsule was collected = e.g., 04, 05, etc.
e. Capsule collected for this type of event, this year, using A-Z, 1-9, beginning
with A.
f. Numerical designation of plants produced from the designated fruit
Letters c-f iterate through generations of descent from the original parent plant.
Example 1:
LR08 – S04A10– S06D01
Reading from thr right-most field, the label printed above Table B-1 indicates the
first (01) seed germinated from the fourth (D) capsule collected in 2006 from selfing
attempts done on plant LR08-S04-A10, which was grown from the tenth seed
germinating from the first (A) capsule (LR08-S04A) collected in 2004 from selfing
attempts done on plant LR08.
Table B-1: Pedigree record, example 1.
(a) (b) (c) (d) (e) (f) (c) (d) (e) (f)
LR 08 S 04 A 10 S 06 D 01
207
Example 2:
LR08 – S03A01 – S04A01 – S04B03 – S05C
The label above Table B-2 designates the third (C) capsule collected in 2005 (05)
from selfing (S) attempts done on plant LR08 – S03A01 – S04A01 – S04B03, which
was, itself, the third (03) seed to germinate from the second (B) capsule collected in
2004 (capsule can be identified as capsule LR08 – S03A01 – S04A01 - S04B), from
selfing attempts done on a plant (LR08 – S04A01 – S04A01) that was, itself, the first
plant to successfully germinate from seeds taken from capsule (LR08 – S03A01 – S04A),
which was the first collected in 2004 from selfing atempts on plant LR08 – S03A01 ,
which was the first (01) seed to germinate from the first capsule (LR08 – S03A) collected
in 2003 from selfing attempts done on LR08, also known as VL08.
In this manner, up to 26 (A-Z) capsules per year can be collected from selfing
events, and open air pollinations and eachother type of event that can be designated by
the other 24 letters in the alphabet or numbers 1-9 . Up to 99 seeds per capsule generating
new plants can be tracked.
Table B-2: Pedigree record, example 2.
(a) (b) (c) (d) (e) (f) (c) (d) (e) (f) (c) (d) (e) (f) (c) (d) (e)
LR 08 S 03 A 01 S 04 A 01 S 04 B 03 S 05 C
208
Possible mutants
During AAGP seedling tissue collection several abnormal seedlings were
observed by Paula Ralph. Two types are illustrated here. One type, we referred to as
“tricot” appears to be producing three cotyledons (Figure B-19 A, B). These seeds
(Table B-3) had three cotyledons when the seedling was extracted from the softened seed
coat. Another unusual seedling type seemed to produce “basal” leaves (Figure B-19 C-
E). and was apparently unable to extend the internode normally until after the earliest true leaves
emerged.
Table B-3: Pedigree and incidence of “tricots.”
Expt. date
Parent Frequency
(n=1)
10/6/06 LR08 S04 A05 2/200
10/27/06 LR05 S04 A01 S05 A01 1/?
12/15/06 LR08 S04 A01 S05 B02 2/50
12/15/06 LR12 S04 A01 S05 C03 1/150
209
Figure B-19: Abnormal seedlings. A, B. “Tricots” C, D, E with basal leaves.
Literature cited
Abe F, Nagafuji S, Yamauchi T, Okabe H, Maki J, Higo H, Akahane H, Aguilar A,
Jimenez-Estrada M, Reyes-Chilpa R (2002) Trypanocidal constituents in plants 1.
Evaluation of some Mexican plants for their trypanocidal activity and active constituents
in Guaco, roots of Aristolochia taliscana. Biol Pharm Bull 25: 1188-1191
Abubakar MS, Balogun E, Abdurahman EM, Nok AJ, Shok M, Mohammed A, Garba M (2006) Ethnomedical treatment of poisonous snakebites: Plant extract neutralized Naja
nigricollis venom. Pharm Biol 44: 343-348
Adams KL, Daley DO, Qiu YL, Whelan J, Palmer JD (2000) Repeated, recent and diverse
transfers of a mitochondrial gene to the nucleus in flowering plants. Nature 408: 354-357
Adams KL, Ong HC, Palmer JD (2001) Mitochondrial gene transfer in pieces: fission of the
ribosomal protein gene rpl2 and partial or complete gene transfer to the nucleus. Mol
Biol Evol 18: 2289-2297
Adams KL, Qiu YL, Stoutemyer M, Palmer JD (2002) Punctuated evolution of mitochondrial
gene content: high and variable rates of mitochondrial gene loss and transfer to the
nucleus during angiosperm evolution. Proc Natl Acad Sci USA 99: 9905-9912
Agrawal GK, Abe K, Yamazaki M, Miyao A, Hirochika H (2005) Conservation of the E-
function for floral organ identity in rice revealed by the analysis of tissue culture-induced
loss-of-function mutants of the OsMADS1 gene. Plant Mol Biol 59: 125-135
Aguilar-Martinez JA, Poza-Carrion C, Cubas P (2007) Arabidopsis BRANCHED1 acts as an
integrator of branching signals within axillary buds. Plant Cell 19: 458-472
Albert VA, Soltis DE, Carlson JE, Farmerie WG, Wall PK, Ilut DC, Solow TM, Mueller
LA, Landherr LL, Hu Y, Buzgo M, Kim S, Yoo MJ, Frohlich MW, Perl-Treves R,
Schlarbaum SE, Bliss BJ, Zhang X, Tanksley SD, Oppenheimer DG, Soltis PS, Ma
H, Depamphilis CW, Leebens-Mack JH (2005) Floral gene resources from basal
angiosperms for comparative genomics research. BMC Plant Biol 5: 15
Alexenizer M, Dorn A (2007) Screening of medicinal and ornamental plants for insecticidal and
growth regulating activity. J Pest Sci 80: 205-215
Almeida J, Rocheta M, Galego L (1997) Genetic control of flower shape in Antirrhinum majus.
Development 124: 1387-1392
211
Ambrose BA, Lerner DR, Ciceri P, Padilla CM, Yanofsky MF, Schmidt RJ (2000)
Molecular and genetic analyses of the silky1 gene reveal conservation in floral organ
specification between eudicots and monocots. Mol Cell 5: 569-579
An G, Watson BD, Chiang CC (1986) Transformation of tobacco, tomato, potato, and
Arabidopsis thaliana using a binary Ti vector system. Plant Physiol 81: 301-305
Arabidopsis GI (2000) Analysis of the genome sequence of the flowering plant Arabidopsis
thaliana. Nature 408: 796-815
Arias T, Williams JH (2008) Embryology of Manekia naranjoana (Piperaceae) and the origin
of tetrasporic, 16-nucleate female gametophytes in Piperales. Am J Bot 95: 272-285
Arumuganathan K, Earle E (1991) Estimation of nuclear DNA content of plants by cell-flow
cytometry. Plant Mol Biol Rep 9: 229-233
Awad M, Young RE (1979) Post-harvest variation in cellulase, polygalacturonase, and
pectinmethylesterase in avocado (Persea americana Mill, cv. Fuerte) fruits in relation to
respiration and ethylene production. Plant Physiol 64: 306-308
Axtell MJ, Snyder JA, Bartell DP (2007) Common functions for diverse small RNAs of land
plants. Plant Cell 19: 1750-1769
Baldauf SL, Palmer JD (1990) Evolutionary transfer of the chloroplast tufA gene to the
nucleus. Nature 344: 262-265
Banziger H, Disney R, Henry L (2006) Scuttle flies (Diptera: Phoridae) imprisoned by
Aristolochia baenzigeri (Aristolochiaceae) in Thailand. Mitteilungen der
Schweizerischen Entomologischen Gesellschaft 79: 29-61
Barakat A, Wall K, Leebens-Mack J, Wang YJ, Carlson JE, Depamphilis CW (2007)
Large-scale identification of microRNAs from a basal eudicot (Eschscholzia californica)
and conservation in flowering plants. Plant J 51: 991 - 1003
Baum DA, Doebley J, Irish VF, Kramer EM (2002) Response: Missing links: the genetic
architecture of flower and floral diversification. Trends Plant Sci 7: 31-34
Becker A, Saedler H, Theissen G (2003) Distinct MADS-box gene expression patterns in the
reproductive cones of the gymnosperm Gnetum gnemon. Dev Genes Evol 213: 567-572
Becker A, Theissen G (2003) The major clades of MADS-box genes and their role in the
development and evolution of flowering plants. Mol Phylogenet Evol 29: 464-489
Behnke HD (2003) Sieve-element plastids and evolution of monocotyledons, with emphasis on
Melanthiaceae sensu lato and Aristolochiaceae Asaroideae, a putative dicotyledon sister
group. Bot Rev 68: 524-544
212
Bell CD, Soltis DE, Soltis PS (2005) The age of the angiosperms: a molecular timescale without
a clock. Evolution 59: 1245-1258.
Bennett M, Leitch I (2005) Angiosperm DNA C-values Database (release 4.0, October 2005)
http://data.kew.org/cvalues/homepage.html
Bent AF (2000) Arabidopsis in planta transformation. Uses, mechanisms, and prospects for
transformation of other species. Plant Physiol 124: 1540-1547
Bergstrom G, Groth I, Pellmyr O, Endress PK, Thien LB, Hubener A, Francke W (1991)
Chemical basis of a highly specific mutualism - chiral esters attract pollinating beetles in
Eupomatiaceae. Phytochemistry 30: 3221-3225
Bergthorsson U, Adams KL, Thomasson B, Palmer JD (2003) Widespread horizontal transfer
of mitochondrial genes in flowering plants. Nature 424: 197-201
Berjano R, De Vega C, Arista M, Ortiz PL, Talavera S (2006) A multi-year study of factors
affecting fruit production in Aristolochia paucinervis (Aristolochiaceae). Am J Bot 93:
599-606
Bharathan G, Lambert G, Galbraith D (1994) Nuclear DNA content of monocotyledons and
related taxa. Am J Bot 81: 381-386
Bliss BJ, Landherr LL, dePamphilis CW, Ma H, Hu Y, Maximova SN (submitted, December
2008) Regeneration and plantlet development from somatic tissues of Aristolochia
fimbriata. Plant Cell Tiss Org Cult
Bliss BJ, Wanke S, Barakat A, Wall PK, Landherr LL, Hu Y, Ma H, Neinhuis C, Leebens-
Mack J, Arumuganathan K, Clifton S, Maximova SN, dePamphilis CW (in
preparation, 2008) Aristolochia fimbriata: a proposed experimental model for basal
angiosperms. Plant Physiol
Bolker JA (1995) Model systems in developmental biology. BioEssays 17: 451-455
Broholm SK, Tahtiharju S, Laitinen RAE, Albert VA, Teeri TH, Elomaa P (2008) A TCP
domain transcription factor controls flower type specification along the radial axis of the
Gerbera (Asteraceae) inflorescence. Proc Natl Acad Sci USA 105: 9117-9122
Broussalis AM, Ferraro GE, Martino VS, Pinzon R, Coussio JD, Alvarez JC (1999)
Argentine plants as potential source of insecticidal compounds. J Ethnopharmacol 67:
219-223
Burgess KS, Singfield J, Melendez V, Kevan PG (2004) Pollination biology of Aristolochia
grandiflora (Aristolochiaceae) in Veracruz, Mexico. Ann Mo Bot Gard 91: 346-356
Burnham CR (1959) teosinte branched. Maize Genet Coop Newsletter 33: 74
213
Busch A, Zachgo S (2007) Control of corolla monosymmetry in the Brassicaceae Iberis amara.
Proc Natl Acad Sci USA 104: 16714-16719
Busov VB, Brunner AM, Strauss SH (2008) Genes for control of plant stature and form. New
Phytol 177: 589-607
Buzgo M, Endress PK (2000) Floral structure and development of Acoraceae and its systematic
relationships with basal angiosperms. Int J Plant Sci 161: 23-41
Buzgo M, Soltis DE, Soltis PS, Ma H (2004a) Towards a comprehensive integration of
morphological and genetic studies of floral development. Trends Plant Sci 9: 164-173
Buzgo M, Soltis P, Soltis DE (2004b) Floral developmental morphology of Amborella
trichopoda (Amborellaceae). International Journal of Plant Sciences 165: 925-947
Cai ZQ, Penaflor C, Kuehl JV, Leebens-Mack J, Carlson JE, dePamphilis CW, Boore JL,
Jansen RK (2006) Complete plastid genome sequences of Drimys, Liriodendron, and
Piper: implications for the phylogenetic relationships of magnoliids. BMC Evol Biol 6:
20
Caplin SM, Steward FC (1948) Effect of coconut milk on the growth of explants from carrot
root. Science 108: 655-657
Carlquist S, Schneider EL (2002) The tracheid-vessel element transition in angiosperms
involves multiple independent features: Cladistic consequences. Am J Bot 89: 185-195
Carmichael JS, Friedman WE (1996) Double fertilization in Gnetum gnemon (Gnetaceae): its
bearing on the evolution of sexual reproduction within the Gnetales and the anthophyte
clade. Am J Bot 83: 767-780
Chanderbali AS, Kim S, Buzgo M, Zheng Z, Oppenheimer DG, Soltis DE, Soltis PS (2006)
Genetic footprints of stamen ancestors guide perianth evolution in Persea (Lauraceae).
Int J Plant Sci 167: 1075-1089
Chen JT, Chang WC (2001) Effects of auxins and cytokinins on direct somatic embryogenesis
on leaf explants of Oncidium 'Gower Ramsey'. J Plant Growth Regul 34: 229-232
Chen X (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower
development. Science 303: 2022-2025
Chung MH, Chen MK, Pan SM (2000) Floral spray transformation can efficiently generate
Arabidopsis transgenic plants. Transgenic Res 9: 471-476
Citerne HL, Moller M, Cronk QCB (2000) Diversity of cycloidea-like genes in Gesneriaceae
in relation to floral symmetry. Ann Bot 86: 167-176
214
Clark JI, Coen ES (2002) The cycloidea gene can respond to a common dorsoventral prepattern
in Antirrhinum. Plant J 30: 639-648
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated
transformation of Arabidopsis thaliana. Plant J 16: 735-743
Coen ES, Meyerowitz EM (1991) The war of the whorls: genetic interactions controlling flower
development. Nature 353: 31-37
Colombo L, Franken J, Koetje E, van Went J, Dons HJM, Angenent GC, van Tunen AJ (1995) The Petunia MADS box gene FBP11 determines ovule identity. Plant Cell 7:
1859-1868
Corley SB, Carpenter R, Copsey L, Coen E (2005) Floral asymmetry involves an interplay
between TCP and MYB transcription factors in Antirrhinum. Proc Natl Acad Sci USA
102: 5068-5073
Costa MM, Fox S, Hanna AI, Baxter C, Coen E (2005) Evolution of regulatory interactions
controlling floral asymmetry. Development 132: 5093-5101
Crawford BC, Nath U, Carpenter R, Coen ES (2004) CINCINNATA controls both cell
differentiation and growth in petal lobes and leaves of Antirrhinum. Plant Physiol 135:
244-253
Cubas P (2004) Floral zygomorphy, the recurring evolution of a successful trait. BioEssays 26:
1175-1184
Cubas P, Coen E, Zapater JM (2001) Ancient asymmetries in the evolution of flowers. Curr
Biol 11: 1050-1052
Cubas P, Lauter N, Doebley J, Coen E (1999a) The TCP domain: A motif found in proteins
regulating plant growth and development. Plant J 18: 215-222
Cubas P, Vincent C, Coen E (1999b) An epigenetic mutation responsible for natural variation
in floral symmetry. Nature 401: 157-161
Cui L, Wall PK, Leebens-Mack JH, Lindsay BG, Soltis DE, Doyle JJ, Soltis PS, Carlson
JE, Arumuganathan K, Barakat A, Albert VA, Ma H, dePamphilis CW (2006)
Widespread genome duplications throughout the history of flowering plants. Genome Res
16: 738-749
Cummings MP, Nugent JM, Olmstead RG, Palmer JD (2003) Phylogenetic analysis reveals
five independent transfers of the chloroplast gene rbcL to the mitochondrial genome in
angiosperms. Curr Genet 43: 131-138
215
Damerval C, Le Guilloux M, Jager M, Charon C (2007) Diversity and evolution of
CYCLOIDEA-like TCP genes in relation to flower development in Papaveraceae. Plant
Physiol 143: 759-772
Damerval C, Nadot S (2007) Evolution of perianth and stamen characteristics with respect to
floral symmetry in Ranunculales. Ann Bot 100: 631-640
Dan Y, Yan H, Munyikwa T, Dong J, Zhang Y, Armstrong CL (2006) MicroTom--a high-
throughput model transformation system for functional genomics. Plant Cell Rep 25:
432-441
Dandekar AM, McGranahan GH, Leslie CA, Uratsu SL (1989) Agrobacterium-mediated
transformation of somatic embryos as a method for the production of transgenic plants. J
Tissue Cult Methods 12: 145-150
Darwin C (1958) The Origin of Species, Ed 6. The New American Library of World Literature,
New York, v, 479 pp
Darwin CR (1903) letter to J. D. Hooker, July 22nd 1879. In F Darwin, AC Seward, eds, More
Letters of Charles Darwin: A Record of His Work in a Series of Hitherto Unpublished
Papers, Vol II. John Murray, London, pp 20-21
Das DK, Prakash NS, Bhalla-Sarin N (1998) An efficient regeneration system of black gram
(Vigna mungo L.) through organogenesis. Plant Sci 134: 199-206
Davies TJ, Barraclough TG, Chase MW, Soltis PS, Soltis DE, Savolainen V (2004) Darwin's
abominable mystery: Insights from a supertree of the angiosperms. Proc Natl Acad Sci
USA 101: 1904-1909
de Craene LPR, Soltis PS, Soltis DE (2003) Evolution of floral structures in basal angiosperms.
Int J Plant Sci 164: S329-S363
de Mayolo GA, Maximova SN, Pishak S, Guiltinan MJ (2003) Moxalactam as a counter-
selection antibiotic for Agrobacterium-mediated transformation and its positive effects on
Theobroma cacao somatic embryogenesis. Plant Sci 164: 607-615
De Morais ABB, Brown KS, Jr. (1991) Larval foodplant and other effects on troidine guild
composition (Papilionidae) in southeastern Brazil. J Res Lepid 30: 19-37
Delwiche CF, Palmer JD (1996) Rampant horizontal transfer and duplication of rubisco genes
in eubacteria and plastids. Mol Biol Evol 13: 873-882
Dickison WC (1992) Morphology and anatomy of the flower and pollen of Saruma henryi Oliv.,
a phylogenetic relict of the Aristolochiaceae. Bull Torrey Bot Club 119: 392-400
216
Dickison WC (1996) Stem and leaf anatomy of Saruma henryi Oliv., including observations on
raylessness in the Aristolochiaceae. Bull Torrey Bot Club 123: 261-267
Dillen W, De Clercq J, Kapila J, Zambre M, Van Montagu M, Angenon G (1997) The effect
of temperature on Agrobacterium tumefaciens-mediated gene transfer to plants. Plant J
12: 1459-1463
Disney RHL, Sakai S (2001) Scuttle flies (Diptera: Phoridae) whose larvae develop in flowers
of Aristolochia (Aristolochiaceae) in Panama. Eur J Entomol 98: 367-373
Doebley J, Stec A (1991) Genetic analysis of the morphological differences between maize and
teosinte. Genetics 129: 285-296
Doebley J, Stec A, Gustus C (1995) teosinte branched1 and the origin of maize evidence for
epistasis and the evolution of dominance. Genetics 141: 333-346
Doebley J, Stec A, Hubbard L (1997) The evolution of apical dominance in maize. Nature 386:
485-488
Doolittle WF (1999) Phylogenetic classification and the universal tree. Science 284: 2124-2128
Doyle JA (2008) Integrating molecular phylogenetic and paleobotanical evidence on origin of
the flower. Int J Plant Sci 169: 816-843
Doyle JA, Endress PK (2000) Morphological phylogenetic analysis of basal angiosperms:
comparison and combination with molecular data. Int J Plant Sci 161: S121-S153
Doyle JJ, Davis JI (1998) Homology in molecular phylogenetics: a parsimony perspective. In
DE Soltis, PS Soltis, JJ Doyle, eds, Molecular Systematics of Plants II: DNA Sequencing.
Kluwer Academic Publishers, Norwell, MA, USA, pp 101-131
Draper J, Mur LAJ, Jenkins G, Ghosh-Biswas GC, Bablak P, Hasterok R, Routledge APM (2001) Brachypodium distachyon. A new model system for functional genomics in
grasses. Plant Physiol 127: 1539-1555
Duvall MR (2000) Seeking the dicot sister group of the monocots. In KL Wilson, DA Morrison,
eds, Monocots: Systematics and Evolution Proceedings of the Second International
Conference on the Comparative Biology of the Monocots. CSIRO, Melbourne, pp 25-32
Dyall SD, Brown MT, Johnson PJ (2004) Ancient invasions: From endosymbionts to
organelles. Science 304: 253-257
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high
throughput. Nucleic Acids Res 32: 1792 - 1797
217
Elizabeth KM, Raju CS (2006) Antimicrobial activity of Aristolochia bracteata. Asian J Chem
18: 207-211
Endress PK (1992) Protogynous flowers in Monimiaceae. Plant Syst Evol 181: 227-232
Endress PK (1994) Diversity and Evolutionary Biology of Tropical Flowers, Ed 1 (pbk., with
corrections). Cambridge University Press, Cambridge, 530 pp
Endress PK (2001a) The flowers in extant basal angiosperms and inferences on ancestral
flowers. Int J Plant Sci 162: 1111-1140
Endress PK (2001b) Origins of flower morphology. Journal of Experimental Zoology 291: 105-
115
Endress PK (2006) Angiosperm floral evolution: Morphological developmental framework. In
Advances in Botanical Research: Incorporating Advances in Plant Pathology, Vol 44.
Academic Press Ltd., London, pp 1-61
Endress PK (2007) Flower development and evolution viewed from a diversity perspective. In
Botany and Plant Biology 2007 (Abstract 1161). American Society of Plant Biologists,
Chicago, IL USA
Endress PK, Igersheim A (2000) Gynoecium structure and evolution in basal angiosperms. Int
J Plant Sci 161: S211-S223
Endress PK, Lorence DH (1983) Diversity and evolutionary trends in the floral structure of
Tambourissa (Monimiaceae). Plant Syst Evol 143: 53-81
Faegri K, Van Der Pijl L (1979) The Principles of Pollination Ecology, Ed 3. Pergamon Press,
Oxford, xii, 244 pp
Faivre-Rampant O, Bryan G-J, Roberts A-G, Milbourne D, Viola R, Taylor M-A (2004)
Regulated expression of a novel TCP domain transcription factor indicates an
involvement in the control of meristem activation processes in Solanum tuberosum. J Exp
Bot 55: 951-953
Feng X, Zhao Z, Tian Z, Xu S, Luo Y, Cai Z, Wang Y, Yang J, Wang Z, Weng L, Chen J,
Zheng L, Guo X, Luo J, Sato S, Tabata S, Ma W, Cao X, Hu X, Sun C, Luo D (2006)
Control of petal shape and floral zygomorphy in Lotus japonicus. Proc Natl Acad Sci
USA 103: 4970-4975
Finlayson SA (2007) Arabidopsis TEOSINTE BRANCHED1-LIKE1 regulates axillary bud
outgrowth and is homologous to monocot TEOSINTE BRANCHED1. Plant Cell Physiol
48: 667-677
218
Fisher DK, Guiltinan MJ (1995) Rapid, efficient production of homozygous transgenic tobacco
plants with Agrobacterium tumefaciens: a seed-to-seed protocol. Plant Mol Biol Rep 13:
278-289
Floyd SK, Friedman WE (2001) Developmental evolution of endosperm in basal angiosperms:
evidence from Amborella (Amborellaceae), Nuphar (Nymphaeaceae), and Illicium
(Illiciaceae). Plant Syst Evol 228: 153-169
Force A, Lynch M, Pickett FB, Amores A, Yan Yl, Postlethwait J (1999) Preservation of
duplicate genes by complementary, degenerative mutations. Genetics 151: 1531-1545
Fordyce JA, Marion ZH, Shapiro AM (2005) Phenological variation in chemical defense of
the pipevine swallowtail, Battus philenor. J Chem Ecol 31: 2835-2846
Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Pegg SE, Li B,
Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated
transformation of maize embryos using a standard binary vector system. Plant Physiol
129: 13-22
Friedman J, Barrett SH (2008) A phylogenetic analysis of the evolution of wind pollination in
the angiosperms. Int J Plant Sci 169: 49-58
Friedman WE (1992) Evidence of a pre-angiosperm origin of endosperm - implications for the
evolution of flowering plants. Science 255: 336-339
Friedman WE (2006) Embryological evidence for developmental lability during early
angiosperm evolution. Nature 441: 337-340
Friedman WE (2008) Hydatellaceae are water lilies with gymnospermous tendencies. Nature
453: 94-97
Friedman WE, Gallup WN, Williams JH (2003) Female gametophyte development in
Kadsura: implications for Schisandraceae, Austrobaileyales, and the early evolution of
flowering plants. Int J Plant Sci 164: S293-S305
Friedman WE, Madrid EN, Williams JH (2008) Origin of the fittest and survival of the fittest:
relating female gametophyte development to endosperm genetics. Int J Plant Sci 169: 79-
92
Friedman WE, Moore RC, Purugganan MD (2004) The evolution of plant development. Am J
Bot 91: 1726-1741
Friedman WE, Williams JH (2003) Modularity of the angiosperm female gametophyte and its
bearing on the early evolution of endosperm in flowering plants. Evolution 57: 216-230
219
Friedman WE, Williams JH (2004) Developmental evolution of the sexual process in ancient
flowering plant lineages. Plant Cell 16: S119-S132
Fukuda T, Yokoyama J, Maki M (2003) Molecular evolution of cycloidea-like genes in
Fabaceae. J Mol Evol: 57(55): 588-597
Gadhi CA, Benharref A, Jana M, Lozniewski A (2001a) Anti-Helicobacter pylori activity of
Aristolochia paucinervis Pomel extracts. J Ethnopharmacol 75: 203-205
Gadhi CA, Hatier R, Mory F, Marchal L, Weber M, Benharref A, Jana M, Lozniewski A (2001b) Bactericidal properties of the chloroform fraction from rhizomes of Aristolochia
paucinervis Pomel. J Ethnopharmacol 75: 207-212
Gadhi CA, Weber M, Mory F, Benharref A, Lion C, Jana M, Lozniewski A (1999)
Antibacterial activity of Aristolochia paucinervis Pomel. J Ethnopharmacol 67: 87-92
Gaudin V, Lunness PA, Fobert PR, Towers M, Riou-Khamlichi C, Murray JA, Coen E,
Doonan JH (2000) The expression of D-cyclin genes defines distinct developmental
zones in snapdragon apical meristems and is locally regulated by the Cycloidea gene.
Plant Physiol 122: 1137-1148
Gelvin SB (2000) Agrobacterium and plant genes involved in T-DNA transfer and integration.
Annu Rev Plant Physiol Plant Mol Biol 51: 223-256
Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the "gene-
jockeying" tool. Microbiol Mol Biol Rev 67: 16-37
Glass flowers from the Ware Collection in the Botanical Museum of Harvard University (1940).
Harcourt, Brace and Company, New York, 58 pp
Gonzales F, Rudall P (2003) Structure and development of the ovule and seed in
Aristolochiaceae, with particular reference to Saruma. Plant Systemat Evol 241: 223-244
Gonzalez F (1999) Inflorescence morphology and the systematics of Aristolochiaceae. Syst
Geogr Pl 68: 159-172
Gonzalez F, Rudall PJ, Furness CA (2001) Microsporogenesis and systematics of
Aristolochiaceae. Bot J Linnean Soc 137: 221-242
Gonzalez F, Stevenson DW (2000a) Gynostemium development in Aristolochia
(Aristolochiaceae). Bot Jahrb Syst 122: 249-291
Gonzalez F, Stevenson DW (2000b) Perianth development and systematics of Aristolochia.
Flora 195: 370-391
220
Grollman AP, Shibutani S, Moriya M, Miller F, Wu L, Moll U, Suzuki N, Fernandes A,
Rosenquist T, Medverec Z, Jakovina K, Brdar B, Slade N, Turesky RJ,
Goodenough AK, Rieger R, Vukelic M, Jelakovic B (2007) Aristolochic acid and the
etiology of endemic (Balkan) nephropathy. Proc Natl Acad Sci USA 104: 12129-12134
Gubitz T, Caldwell A, Hudson A (2003) Rapid molecular evolution of CYCLOIDEA-like genes
in Antirrhinum and its relatives. Mol Biol Evol 20: 1537-1544
Gupta RS, Dobhal MP, Dixit VP (1996) Morphometric and biochemical changes in testes of
Presbytis entellus entellus Dufresne (Langur monkey) following aristolochic acid
administration. Annals of Biology (Ludhiana) 12: 328-334
Haeckel E (1909) Charles Darwin as an Anthropologist. In AC Seward, ed, Darwin and Modern
Science: Essays in commemoration of the centenary of the birth of Charles Darwin and of
the fiftieth anniversary of the publication of "The Origin of Species". Cambridge Univ.
Press, Cambridge, UK, p 595
Hall DW, Brown BV (1993) Pollination of Aristolochia littoralis (Aristolochiales:
Aristolochiaceae) by males of Megaselia spp. (Diptera: Phoridae). Ann Entomol Soc Am
86: 609-613
Handberg K, Stougaard J (1992) Lotus japonicus, an autogamous, diploid legume species for
classical and molecular genetics Plant J 2: 487-496
Hileman LC, Baum DA (2003) Why do paralogs persist? Molecular evolution of CYCLOIDEA
and related floral symmetry genes in Antirrhineae (Veronicaceae). Mol Biol Evol 20:
591-600
Hinou J, Demetzos C, Harvala C, Roussakis C (1990) Cytotoxic and antimicrobial principles
from the roots of Aristolochia longa. International Journal of Crude Drug Research 28:
149-151
Honma T, Goto K (2001) Complexes of MADS-box proteins are sufficient to convert leaves
into floral organs. Nature 409: 525 - 529
Howarth DG, Donoghue MJ (2005) Duplications in CYC-like genes from Dipsacales correlate
with floral form. Int J Plant Sci 166: 357-370
Howarth DG, Donoghue MJ (2006) Phylogenetic analysis of the "ECE" (CYC/TB1) clade
reveals duplications predating the core eudicots. Proc Natl Acad Sci USA 103: 9101-
9106
Hranjec T, Kovac A, Kos J, Mao WY, Chen JJ, Grollman AP, Jelakovic B (2005) Endemic
nephropathy: the case for chronic poisoning by Aristolochia. Croat Med J 46: 116-125
221
Hu S, Dilcher DL, Jarzen DM, Taylor DW (2008) Early steps of angiosperm-pollinator
coevolution. Proc Natl Acad Sci USA 105: 240-245
Huang CY, Ayliffe MA, Timmis JN (2003) Direct measurement of the transfer rate of
chloroplast DNA into the nucleus. Nature 422: 72-76
Hubbard L, McSteen P, Doebley J, Hake S (2002) Expression patterns and mutant phenotype
of teosinte branched1 correlate with growth suppression in maize and teosinte. Genetics
162: 1927-1935
Huber H (1993) Aristolochiaceae. In K Kubitzki, JG Rohwer, V Bittrich, eds, The Families and
Genera of Vascular Plants. Springer, Berlin, pp 129-137
Husaini AM, Abdin MZ (2007) Interactive effect of light, temperature and TDZ on the
regeneration potential of leaf discs of Fragaria x ananassa Duch. In Vitro Cell Dev Biol
Plant 43: 576-584
Hwang MS, Park MS, Moon J-Y, Lee JS, Yum YN, Yoon E, Lee H, Nam KT, Lee BM, Kim
SH, Yang KH (2006) Subchronic toxicity studies of the aqueous extract of Aristolochiae
fructus in Sprague-Dawley rats. J Toxicol Environ Health Part A 69: 2157 - 2165
International-Rice-Genome-Sequencing-Project (2005) The map-based sequence of the rice
genome. Nature 436: 793-800
Irish VF (2003) The evolution of floral homeotic gene function. Bioessays 25: 637-646
Irish VF (2006) Duplication, diversification, and comparative genetics of angiosperm MADS-
box genes. In Advances in Botanical Research: Incorporating Advances in Plant
Pathology, Vol 44. Academic Press Ltd., London, pp 129-161
Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency
transformation of maize (Zea mays L) mediated by Agrobacterium tumefaciens. Nat
Biotechnol 14: 745-750
Jansen RK, Cai Z, Raubeson LA, Daniell H, dePamphilis CW, Leebens-Mack J, Muller
KF, Guisinger-Bellian M, Haberle RC, Hansen AK, Chumley TW, Lee SB, Peery R,
McNeal JR, Kuehl JV, Boore JL (2007) Analysis of 81 genes from 64 plastid genomes
resolves relationships in angiosperms and identifies genome-scale evolutionary patterns.
Proc Natl Acad Sci USA 104: 19369-19374
Jansson S, Douglas CJ (2007) Populus: a model system for plant biology. Annu Rev Plant Biol
58: 435-458
Jaramillo MA, Kramer EM (2004) APETALA3 and PISTILLATA homologs exhibit novel
expression patterns in the unique perianth of Aristolochia (Aristolochiaceae). Evolution
& Development 6: 449-458
222
Jbilou R, Ennabili A, Sayah F (2006) Insecticidal activity of four medicinal plant extracts
against Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). AJB 5: 936-940
Jimenez-Ferrer JE, Perez-Teran YY, Roman-Ramos R, Tortoriello J (2005) Antitoxin
activity of plants used in Mexican traditional medicine against scorpion poisoning.
Phytomedicine 12: 116-122
Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in
plants. Ann Rev Plant Biol 57: 19 - 53
Judd WS, Campbell CS, Kellogg EA, Stevens PF (2002) Plant systematics: a phylogenetic
approach, Ed 2. Sinauer Associates, Sunderland, Mass., USA, 576 pp
Juretic B, Jelaska S (1991) Plant development in long-term embryogenic callus lines of
Cucurbita pepo Plant Cell Rep 9: 623-626
Karol KG, McCourt RM, Cimino MT, Delwiche CF (2001) The closest living relatives of
land plants. Science 294: 2351-2353
Kebrom TH, Burson BL, Finlayson SA (2006) Phytochrome B represses Teosinte Branched1
expression and induces Sorghum axillary bud outgrowth in response to light signals.
Plant Physiol 140: 1109-1117
Kellogg EA, Shaffer HB (1993) Model organisms in evolutionary studies. Syst Biol 42: 409-
414
Kelly LM (1998) Phylogenetic relationships in Asarum (Aristolochiaceae) based on morphology
and ITS sequences. Am J Bot 85: 1454-1467
Kenrick P, Crane PR (1997) The origin and early evolution of plants on land. Nature 389: 33-
39
Kim S, Koh J, Ma H, Hu Y, Endress PK, Hauser BA, Buzgo M, Soltis PS, Soltis DE (2005a)
Sequence and expression studies of A-, B-, and E-class MADS-box homologues in
Eupomatia (Eupomatiaceae): support for the bracteate origin of the calyptra. International
Journal of Plant Sciences 166: 185-198
Kim S, Koh J, Yoo MJ, Kong HZ, Hu Y, Ma H, Soltis PS, Soltis DE (2005b) Expression of
floral MADS-box genes in basal angiosperms: implications for the evolution of floral
regulators. Plant J 43: 724-744
Kim S, Soltis PS, Wall K, Soltis DE (2006) Phylogeny and domain evolution in the
APETALA2-like gene family. Mol Biol Evol 23: 107-120
223
Kim S, Yoo M-J, Albert VA, Farris JS, Soltis PS, Soltis DE (2004) Phylogeny and
diversification of B-function MADS-box genes in angiosperms: evolutionary and
functional implications of a 260-million-year-old duplication. Am J Bot 91: 2102-2118
Kimura M (1968) Evolutionary rate at the molecular level. Nat Aust 217: 624-626
Klitzke CF, Brown KS, Jr. (2000) The occurrence of aristolochic acids in neotropical troidine
swallowtails (Lepidoptera: Papilionidae). Chemoecology 10: 99-102
Kosugi S, Ohashi Y (1997) PCF1 and PCF2 specifically bind to cis elements in the rice
proliferating cell nuclear antigen gene. Plant Cell 9: 1607-1619
Kosugi S, Ohashi Y (2002) DNA binding and dimerization specificity and potential targets for
the TCP protein family. Plant J 30: 337-348
Koyama T, Furutani M, Tasaka M, Ohme-Takagi M (2007) TCP transcription factors control
the morphology of shoot lateral organs via negative regulation of the expression of
boundary-specific genes in Arabidopsis. Plant Cell 19: 473-484
Kramer EM, Dorit RL, Irish VF (1998) Molecular evolution of genes controlling petal and
stamen development: duplication and divergence within the APETALA3 and PISTILLATA
MADS-box gene lineages. Genetics 149: 765-783
Kramer EM, Irish VF (1999) Evolution of genetic mechanisms controlling petal development.
Nature 399: 144-148
Kramer EM, Irish VF (2000) Evolution of the petal and stamen developmental programs:
evidence from comparative studies of the lower eudicots and basal angiosperms.
International Journal of Plant Sciences 161: S29-S40
Kramer EM, Jaramillo MA, Di Stilio VS (2004) Patterns of gene duplication and functional
evolution during the diversification of the AGAMOUS subfamily of MADS box genes in
angiosperms. Genetics 166: 1011-1023
Kramer EM, Zimmer EA (2006) Gene duplication and floral developmental genetics of basal
eudicots. In Advances in Botanical Research: Incorporating Advances in Plant Pathology,
Vol 44, Vol 44. ACADEMIC PRESS LTD, London, pp 353-384
Kumar V, Poonam, Prasad AK, Parmar VS (2003) Naturally occurring aristolactams,
aristolochic acids and dioxoaporphines and their biological activities. Nat Prod Rep 20:
565-583
Kumar VP, Chauhan NS, Padh H, Rajani M (2006) Search for antibacterial and antifungal
agents from selected Indian medicinal plants. J Ethnopharmacol 107: 182-188
224
Kupchan SM, Doskotch RW (1962) Tumor inhibitors. I. Aristolochic acid, the active principle
of Aristolochia indica. Journal of Medicinal and Pharmaceutical Chemistry 5: 657-659
Labandeira CC, Dilcher DL, Davis DR, Wagner DL (1994) Ninety-seven million years of
angiosperm-insect association: paleobiological insights into the meaning of coevolution.
Proc Natl Acad Sci USA 91: 12278-12282
Lahaye R, Civeyrel L, Speck T, Rowe NP (2005) Evolution of shrub-like growth forms in the
lianoid subfamily Secamonoideae (Apocynaceae s.l.) of Madagascar: phylogeny,
biomechanics, and development. Am J Bot 92: 1381-1396
Lajide L, Escoubas P, Mizutani J (1993) Antifeedant activity of metabolites of Aristolochia
albida against the tobacco cutworm, Spodoptera litura. J Agric Food Chem 41: 669-673
Lee HS, Chen ZJ (2001) Protein-coding genes are epigenetically regulated in Arabidopsis
polyploids. Proc Natl Acad Sci USA 98: 6753-6758
Leebens-Mack J, Raubeson LA, Cui L, Kuehl JV, Fourcade MH, Chumley TW, Boore JL,
Jansen RK, dePamphilis CW (2005) Identifying the basal angiosperm node in
chloroplast genome phylogenies: sampling one's way out of the Felsenstein zone. Mol
Biol Evol 22: 1948-1963
Leins P, Erbar C (1985) Contribution to floral development in Aristolochiaceae: a link to the
monocotyledons. Bot Jahrb Syst 107: 343-368
Leins P, Erbar C (1995) The early pattern of differentiation in the flowers of Saruma henryi
Oliv. (Aristolochiaceae). Bot Jahrb Syst 117: 365-376
Leins P, Erbar C, Van Heel WA (1988) Note on the floral development of Thottea
Aristolochiaceae. Blumea 33: 357-370
Lemos VS, Thomas G, Barbosa JM (1993) Pharmacological studies on Aristolochia papillaris
Mast (Aristolochiaceae). J Ethnopharmacol 40: 141-145
Levi M, Guchelaar HJ, Woerdenbag HJ, Zhu YP (1998) Acute hepatitis in a patient using a
Chinese herbal tea - a case report. Pharmacy World & Science 20: 43-44
Li CX, Potuschak T, Colon-Carmona A, Gutierrez RA, Doerner P (2005) Arabidopsis
TCP20 links regulation of growth and cell division control pathways. Proc Natl Acad Sci
USA 102: 12978-12983
Li ZJ, Traore A, Maximova S, Guiltinan MJ (1998) Somatic embryogenesis and plant
regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron. In
Vitro Cell Dev Biol Plant 34: 293-299
225
Liang HY, Fang EG, Tomkins JP, Luo MZ, Kudrna D, Kim HR, Arumuganathan K, Zhao
SY, Leebens-Mack J, Schlarbaum SE, Banks JA, Depamphilis CW, Mandoli DF,
Wing RA, Carlson JE (2007) Development of a BAC library for yellow-poplar
(Liriodendron tulipifera) and the identification of genes associated with flower
development and lignin biosynthesis. Tree Genet Genomes 3: 215-225
Lin X, Kaul S, Rounsley S, Shea TP, Benito MI, Town CD, Fujii CY, Mason T, Bowman
CL, Barnstead M, Feldblyum TV, Buell CR, Ketchum KA, Lee J, Ronning CM,
Koo HL, Moffat KS, Cronin LA, Shen M, Pai G, Van Aken S, Umayam L, Tallon
LJ, Gill JE, Adams MD, Carrera AJ, Creasy TH, Goodman HM, Somerville CR,
Copenhaver GP, Preuss D, Nierman WC, White O, Eisen JA, Salzberg SL, Fraser
CM, Venter JC (1999) Sequence and analysis of chromosome 2 of the plant Arabidopsis
thaliana. Nature 402: 761-768
Lindner E (1928) Aristolochia lindneri Berger und ihre Bestäubung durch Fliegen. Biol Zbl 48:
93-101
Litt A, Irish VF (2003) Duplication and diversification in the APETALA1/FRUITFULL floral
homeotic gene lineage: implications for the evolution of floral development. Genetics
165: 821-833
Lu KL (1982) Pollination biology of Asarum caudatum (Aristolochiaceae) in Northern
California. Syst Bot 7: 150-157
Luo D, Carpenter R, Copsey L, Vincent C, Clark J, Coen E (1999) Control of organ
asymmetry in flowers of Antirrhinum. Cell 99: 367-376
Luo D, Carpenter R, Vincent C, Copsey L, Coen E (1996) Origin of floral asymmetry in
Antirrhinum. Nature 383: 794-799
Lynch M, Conery JS (2000) The evolutionary fate and consequences of duplicate genes.
Science 290: 1151-1155
Ma H (1994) The unfolding drama of flower development: recent results from genetic and
molecular analyses. Gene Dev 8: 745-756
Ma H, dePamphilis C (2000) The ABCs of floral evolution. Cell 101: 5-8
Mabberley DJ (2008) Mabberley's Plant-book: a portable dictionary of the vascular plants Ed
3rd. Cambridge University Press, Cambridge, UK, 1021 pp
Madrid E, Friedman N (2008) Female gametophyte development in Aristolochia labiata Willd.
(Aristolochiaceae). Bot J Linnean Soc 158: 19-29
Magallon S, Crane PR, Herendeen PS (1999) Phylogenetic pattern, diversity, and
diversification of eudicots. Annals of the Missouri Botanical Garden 86: 297-372
226
Malcomber ST, Kellogg EA (2004) Heterogeneous expression patterns and separate roles of the
SEPALLATA gene LEAFY HULL STERILE1 in grasses. Plant Cell 16: 1692 - 1706
Malcomber ST, Kellogg EA (2005) SEPALLATA gene diversification: brave new whorls.
Trends Plant Sci 10: 427-435
Manjula S, Thomas A, Daniel B, Nair GM (1997) In vitro plant regeneration of Aristolochia
indica through axillary shoot multiplication and organogenesis. Plant Cell Tiss Org Cult
51: 145-148
Maximova S, Miller C, Antunez de Mayolo G, Pishak S, Young A, Guiltinan MJ (2003)
Stable transformation of Theobroma cacao L. and influence of matrix attachment regions
on GFP expression. Plant Cell Rep 21: 872-883
Maximova SN, Dandekar AM, Guiltinan MJ (1998) Investigation of Agrobacterium-mediated
transformation of apple using green fluorescent protein: high transient expression and
low stable transformation suggest that factors other than T-DNA transfer are rate-
limiting. Plant Mol Biol 37: 549-559
McConnell JR, Barton MK (2003) Leaf development takes shape. Science 299: 1328-1329
McKersie BD, Murnaghan J, Bowley SR (1997) Manipulating freezing tolerance in transgenic
plants. Acta Physiologiae Plantarum 19: 485-495
McNeal JR, Leebens-Mack JH, Arumuganathan K, Kuehl JV, Boore JL, dePamphilis CW (2006) Using partial genomic fosmid libraries for sequencing complete organellar
genomes. Biotechniques 41: 69-73
Meinl W, Pabel U, Osterloh-Quiroz M, Hengstler JG, Glatt H (2006) Human
sulphotransferases are involved in the activation of aristolochic acids and are expressed in
renal target tissue. Int J Cancer 118: 1090-1097
Mena M, Ambrose BA, Meeley RB, Briggs SP, Yanofsky MF, Schmidt RJ (1996)
Diversification of C-function activity in maize flower development. Science 274: 1537-
1540
Millen RS, Olmstead RG, Adams KL, Palmer JD, Lao-Nga T, Heggie L, Kavanagh TA,
Hibberd JM, Gray JC, Morden CW, Calie PJ, Jermiin LS, Wolfe KH (2001) Many
parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple
independent transfers to the nucleus. Plant Cell 13: 645-658
Mizukami Y, Ma H (1992) Ectopic expression of the floral homeotic gene AGAMOUS in
transgenic Arabidopsis plants alters floral organ identity. Cell 71: 119-131
Mizukami Y, Ma H (1995) Separation of AG function in floral meristem determinancy from
that in reproductive organ identity by expressing antisense AG RNA. Plant Mol Biol
227
Mohamed MF, Read PE, Coyne DP (1992) Dark preconditioning, CPPU, and thidiazuron
promote shoot organogenesis on seedling node explants of common and faba beans. J Am
Soc Hort Sci 117: 668-672
Molina RV, Castello S, Garcia-Luis A, Guardiola JL (2007) Light cytokinin interactions in
shoot formation in epicotyl cuttings of Troyer citrange cultured in vitro. Plant Cell Tiss
Org Cult 89: 131-140
Moore MJ, Bell CD, Soltis PS, Soltis DE (2007) Using plastid genome-scale data to resolve
enigmatic relationships among basal angiosperms. Proc Natl Acad Sci USA 104: 19363-
19368
Morrison DA (2007) Increasing the efficiency of searches for the maximum likelihood tree in a
phylogenetic analysis of up to 150 nucleotide sequences. Syst Biol 56: 988-1010
Müller J, Müller K (2004) TreeGraph: Automated drawing of complex tree figures using an
extensible tree description format. Mol Ecol Notes 4: 786-788
Müller J, Müller K, Neinhuis C, Quandt D (2007) PhyDE- A phylogenetic data editor.
Version 0.92. PhyDE® is a registered trade mark of Jörn Müller Program distributed
by the authors http://www.phyde.de
Müller K (2004) PRAP - computation of Bremer support for large data sets. Mol Phylogenet
Evol 31: 780-782
Murre C, McCaw PS, Baltimore D (1989a) A new DNA binding and dimerization motif in
immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56: 777-
783
Murre C, McCaw PS, Vaessin H, Caudy M, Jan LY, Jan YN, Cabrera CV, Buskin JN,
Hauschka SD, Lassar AB, et al. (1989b) Interactions between heterologous helix-loop-
helix proteins generate complexes that bind specifically to a common DNA sequence.
Cell 58: 537-544
Murthy BNS, Murch SJ, Saxena PK (1998) Thidiazuron: a potent regulator of in vitro plant
morphogenesis. In Vitro Cell Dev Biol Plant 34: 267-275
Murugan R, Shivanna KR, Rao RR (2006) Pollination biology of Aristolochia tagala, a rare
species of medicinal importance. Current Science (Bangalore) 91: 795-798
Nam J, dePamphilis CW, Ma H, Nei M (2003) Antiquity and evolution of the MADS-box
gene family controlling flower development in plants. Mol Biol Evol 20: 1435-1447
Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene
into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell
2: 279-289
228
Nascimento IR, Murata AT, Bortoli SA, Lopes LM (2004) Insecticidal activity of chemical
constituents from Aristolochia pubescens against Anticarsia gemmatalis larvae. Pest
Manage Sci 60: 413-416
Nath U, Crawford BC, Carpenter R, Coen E (2003) Genetic control of surface curvature.
Science 299: 1404-1407
Navaud O, Dabos P, Carnus E, Tremousaygue D, Herve C (2007) TCP transcription factors
predate the emergence of land plants. J Mol Evol 65: 23-33
Negrotto D, Jolley M, Beer S, Wenck AR, Hansen G (2000) The use of phosphomannose-
isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via
Agrobacterium transformation. Plant Cell Rep 19: 798-803
Neinhuis C, Wanke S, Hilu KW, Muller K, Borsch T (2005) Phylogeny of Aristolochiaceae
based on parsimony, likelihood, and Bayesian analyses of trnL-trnF sequences. Plant
Systemat Evol 250: 7-26
Nicholas KB, Nicholas Jr HB, Deerfield II DW (1997) GeneDoc: analysis and visualization of
genetic variation. EMBNEW News 4: 14 [http://www.psc.edu/biomed/genedoc]
Nickrent DL, Parkinson CL, Palmer JD, Duff RJ (2000) Multigene phylogeny of land plants
with special reference to bryophytes and the earliest land plants. Mol Biol Evol 17: 1885-
1895
Nishida R (1994) Oviposition stimulant of a Zeryntiine swallowtail butterfly, Luehdorfia
japonica. Phytochemistry 36: 873-877
Nishida R, Fukami H (1989) Ecological adaptation of an Aristolochiaceae-feeding swallowtail
butterfly Atrophaneura alcinous to aristolochic acids. J Chem Ecol 15: 2549-2564
Nixon KC (1999) The parsimony ratchet, a new method for rapid parsimony analyses. Cladistics
15: 407-414
Norelli JL, Aldwinckle HS (1993) The role of aminoglycoside antibiotics in the regeneration
and selection of neomycin phosphotransferase-transgenic tissue. J Am Soc Hort Sci 118:
311-316
Nortier JL, Martinez MM, Schmeiser HH, Arlt VM, Bieler CA, Petein M, Depierreux MF,
De Pauw L, Abramowicz D, Vereerstraeten P, Vanherweghem JL (2000) Urothelial
carcinoma associated with the use of a Chinese herb (Aristolochia fangchi). New Engl J
Med 342: 1686-1692
Ohi-Toma T, Sugawara T, Murata H, Wanke S, Neinhuis C, Murata J (2006) Molecular
phylogeny of Aristolochia sensu lato (Aristolochiaceae) based on sequences of rbcL,
229
matK, and phyA genes, with special reference to differentiation of chromosome numbers.
Syst Bot 31: 481-492
Ohno S (1970) Evolution by Gene Duplication. Springer, New York, New York, 160 pp
Ori N, Cohen AR, Etzioni A, Brand A, Yanai O, Shleizer S, Menda N, Amsellem Z, Efroni
I, Pekker I, Alvarez JP, Blum E, Zamir D, Eshed Y (2007) Regulation of
LANCEOLATE by miR319 is required for compound-leaf development in tomato.
Nature Genet 39: 787-791
Otero R, Nunez V, Barona J, Fonnegra R, Jimenez SL, Osorio RG, Saldarriaga M, Diaz A (2000) Snakebites and ethnobotany in the northwest region of Colombia Part III:
Neutralization of the haemorrhagic effect of Bothrops atrox venom. J Ethnopharmacol
73: 233-241
Page RDM, Holmes EC (1998) Molecular Evolution: A Phylogenetic Approach. Blackwell
Publishing, Oxford, UK, 346 pp
Pakrashi A, Chakrabarty B (1978) Anti-oestrogenic and anti-implantation effect of
aristolochic acid from Aristolochia indica (Linn). Indian J Exp Biol 16: 1283-1285
Pakrashi A, Pakrasi P (1979) Anti-fertility efficacy of the plant Aristolochia indica (Linn) on
mouse. Contraception 20: 49-54
Palacios SM, Maggi ME, Bazan CM, Carpinella MC, Turco M, Munoz A, Alonso RA,
Nunez C, Cantero JJ, Defago MT, Ferrayoli CG, Valladares GR (2007) Screening of
Argentinian plants for pesticide activity. Fitoterapia 78: 580-584
Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, Weigel D (2003)
Control of leaf morphogenesis by microRNAs. Nature 425: 257-263
Palatnik JF, Wollmann H, Schommer C, Schwab R, Boisbouvier J, Rodriguez R,
Warthmann N, Allen E, Dezulian T, Huson D, Carrington JC, Weigel D (2007)
Sequence and expression differences underlie functional specialization of Arabidopsis
microRNAs miR159 and miR319. Dev Cell 13: 115-125
Petch T (1924) Notes on Aristolochia. Ann Roy Bot Gard, Peradeniya VIII: 1-108
Petri C, Alburquerque N, Burgos L (2005) The effect of aminoglycoside antibiotics on the
adventitious regeneration from apricot leaves and selection of nptII-transformed leaf
tissues. Plant Cell Tiss Org Cult 80: 271-276
Pfeifer HW (1966) Revision of the north and central American hexandrous species of
Aristolochia (Aristolochiaceae). Ann Mo Bot Gard 53: 115-196
230
Pnueli L, Hareven D, Rounsley SD, Yanofsky MF, Lifschitz E (1994) Isolation of the tomato
AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants. Plant Cell
6: 163-173
Preston JC, Kellogg EA (2006) Reconstructing the evolutionary history of paralogous
APETALA1/FRUITFULL-like genes in grasses (Poaceae). Genetics 174: 421-437
Pryer KM, Schneider H, Zimmer EA, Banks JA (2002) Deciding among green plants for
whole genome studies. Trends Plant Sci 7: 550–554
Qiu Q, Liu ZH, Chen HP, Yin HL, Li LS (2000) Long-term outcome of acute renal injury
induced by Aristolochia. Acta Pharmacologica Sinica 21: 1129-1135
Qiu Y-L (2008) Phylogeny and evolution of charophytic algae and land plants. JSE 46: 287-306
Qiu Y-L, Li L, Wang B, Chen Z, Dombrovska O, Lee J, Kent L, Li R, Jobson RW, Hendry
TA, Taylor DW, Testa CM, Ambros M (2007) A nonflowering land plant phylogeny
inferred from nucleotide sequences of seven chloroplast, mitochondrial, and nuclear
genes. Int J Plant Sci 168: 691-708
Qiu YL, Lee J, Bernasconi QF, Soltis-Douglas E, Soltis PS, Zanis M, Zimmer EA, Chen Z,
Savolainen V, Chase MW (1999) The earliest angiosperms: Evidence from
mitochondrial, plastid and nuclear genomes. Nature London 402: 404-407
Raff RA (2000) Evo-devo: the evolution of a new discipline. Nat Rev Genet 1: 74-79
Rastogi PA (1999) MacVector: integrated sequence analysis for the Macintosh. In S Misener,
SA Krawetz, eds, Bioinformatics Methods and Protocols, Vol 132. Oxford Molecular
Group Campbell, CA, pp 47-69
Raubeson LA, Jansen RK (1992) Chloroplast DNA evidence on the ancient evolutionary split
in vascular land plants. Science 255: 1697-1699
Rausher MD (1981) Host plant selection by Battus philenor butterflies: The roles of predation,
nutrition, and plant chemistry. Ecol Monogr 51: 1-20
Raven PH, Evert RF, Eichhorn SE (1999) Biology of plants. WH Freeman and Co, New York,
944 pp
Razdan MK (2003) Introduction to Plant Tissue Culture, Ed 2. Science Publishers, Inc., Enfield
(NH), USA, 375 pp
Razzak MA, Ali T, Ali SI (1992) The pollination biology of Aristolochia bracteolata Lamk.
(Aristolochiaceae). Pakistan Journal of Botany 24: 79-87
231
Reddy RV, Reddy MH, Raju RRV (1995) Ethnobotany of Aristolochia L. Acta Botanica
Indica 23: 291-292
Ree RH, Citerne HL, Lavin M, Cronk QCB (2004) Heterogeneous selection on LEGCYC
paralogs in relation to flower morphology and the phylogeny of Lupinus (Leguminosae).
Mol Biol Evol 21: 321-331
Reeves P-A, Olmstead R-G (2003) Evolution of the TCP gene family in asteridae: cladistic and
network approaches to understanding regulatory gene family diversification and its
impact on morphological evolution. Mol Biol Evol 20: 1997-2009
Reichmann JL, Meyerowitz EM (1997) MADS domain proteins in plant development. Biol
Chem 378: 1079-1101
Remashree AB, Hariharan M, Unnikrishnan K (1997) In vitro organogenesis in Aristolochia
indica (L.). Phytomorphology 47: 161-165
Rensing SA, Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H, Nishiyama T, Perroud
P-F, Lindquist EA, Kamisugi Y, Tanahashi T, Sakakibara K, Fujita T, Oishi K,
Shin-I T, Kuroki Y, Toyoda A, Suzuki Y, Hashimoto S-i, Yamaguchi K, Sugano S,
Kohara Y, Fujiyama A, Anterola A, Aoki S, Ashton N, Barbazuk WB, Barker E,
Bennetzen JL, Blankenship R, Cho SH, Dutcher SK, Estelle M, Fawcett JA,
Gundlach H, Hanada K, Heyl A, Hicks KA, Hughes J, Lohr M, Mayer K,
Melkozernov A, Murata T, Nelson DR, Pils B, Prigge M, Reiss B, Renner T,
Rombauts S, Rushton PJ, Sanderfoot A, Schween G, Shiu S-H, Stueber K,
Theodoulou FL, Tu H, Van de Peer Y, Verrier PJ, Waters E, Wood A, Yang L,
Cove D, Cuming AC, Hasebe M, Lucas S, Mishler BD, Reski R, Grigoriev IV,
Quatrano RS, Boore JL (2008) The Physcomitrella genome reveals evolutionary
insights into the conquest of land by plants. Science 319: 64-69
Renuka C, Swarupanandan K (1986) Morphology of the flower in Thottea siliquosa and the
existence of staminodes in Aristolochiaceae. Blumea 31: 313-318
Rice Chromosome 10 Sequencing Consortium (2003) In-depth view of structure, activity, and
evolution of rice chromosome 10. Science 300: 1566-1569
Rieseberg LH, Welch ME (2002) Gene transfer through introgressive hybridization: history,
evolutionary significance, and evolutionary consequences. In M Syvanen, CI Kado, eds,
Horizontal Gene Transfer, Ed 2nd. Academic Press, London, pp 199-216
Riou-Khamlichi C, Huntley R, Jacqmard A, Murray JA (1999) Cytokinin activation of
Arabidopsis cell division through a D-type cyclin. Science 283: 1541-1544
Roemer RB, Terry LI, Walter GH (2008) Unstable, self-limiting thermochemical temperature
oscillations in Macrozamia cycads. Plant, Cell Environ 31: 769-782
232
Rowe NP, Speck T (2005) Plant growth forms: an ecological and evolutionary perspective. New
Phytol 166: 61-72
Rudall PJ, Remizowa MV, Beer AS, Bradshaw E, Stevenson DW, Macfarlane TD, Tuckett
RE, Yadav SR, Sokoloff DD (2008) Comparative ovule and megagametophyte
development in Hydatellaceae and water lilies reveal a mosaic of features among the
earliest angiosperms. Ann Bot 101: 941-956
Rulik B, Wanke S, Nuss M, Neinhuis C (2008) Pollination of Aristolochia pallida Willd.
(Aristolochiaceae) in the Mediterranean. Flora 203: 175-184
Rutledge R, Regan S, Nicolas O, Fobert P, Cote C, Bosnich W, Kauffeldt C, Sunohara G,
Seguin A, Stewart D (1998) Characterization of an AGAMOUS homologue from the
conifer black spruce (Picea mariana) that produces floral homeotic conversions when
expressed in Arabidopsis. Plant J 15: 625-634
Saebo A, Krekling T, Appelgren M (1995) Light quality affects photosynthesis and leaf
anatomy of birch plantlets in vitro. Plant Cell Tiss Org Cult 41: 177-185
Sakai S (2002) Aristolochia spp. (Aristolochiaceae) pollinated by flies breeding on decomposing
flowers in Panama. Am J Bot 89: 527-534
Sallaud C, Meynard D, van Boxtel J, Gay C, Bes M, Brizard JP, Larmande P, Ortega D,
Raynal M, Portefaix M, Ouwerkerk PB, Rueb S, Delseny M, Guiderdoni E (2003)
Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa
L.) functional genomics. Theor Appl Genet 106: 1396-1408
Samach A, Kohalmi SE, Motte P, Datla R, Haughn GW (1997) Divergence of function and
regulation of class B floral organ identity genes. Plant Cell 9: 559-570
Sands DPA, Scott SE, Moffatt R (1997) The threatened Richmond birdwing butterfly
(Ornithoptera richmondia (Gray)): A community conservation project. Mem Mus Vic
56: 449-453
Sargent RD (2004) Floral symmetry affects speciation rates in angiosperms. Proc R Soc Lond,
Ser B: Biol Sci 271: 603–608
Schommer C, Palatnik JF, Aggarwal P, Chetelat A, Cubas P, Farmer EE, Nath U, Weigel
D (2008) Control of jasmonate biosynthesis and senescence by miR319 targets. PLoS
Biol 6: e230
Schwartz S, Elnitski L, Li M, Weirauch M, Riemer C, Smit A, Program NCS, Green ED,
Hardison RC, Miller W (2003) MultiPipMaker and supporting tools: alignments and
analysis of multiple genomic DNA sequences. Nucl Acids Res 31: 3518-3524
233
Schwartz S, Zhang Z, Frazer KA, Smit A, Riemer C, Bouck J, Gibbs R, Hardison R, Miller
W (2000) PipMaker - A web server for aligning two genomic DNA sequences. Genome
Res 10: 577-586
Schwarz-Sommer Z, Huijser P, Nacken W, Saedler H, Sommer H (1990) Genetic control of
flower development by homeotic genes in Antirrhinum majus. Science 250: 931-936
Sederoff RR, Ronald P, Bedinger P, Rivin C, Walbot V, Bland M, Levings C-S, III (1986)
Maize Zea mays mitochondrial plasmid S-1 sequences share homology with chloroplast
gene psbA. Genetics 113: 469-482
Seymour RS, White CR, Gibernau M (2003) Environmental biology: heat reward for insect
pollinators. Nature 426: 243-244
Shafi PM, Rosamma MK, Jamil K, Reddy PS (2002) Antibacterial activity of the essential oil
from Aristolochia indica. Fitoterapia 73: 439-441
Shan H, Zhang N, Liu C, Xu G, Zhang J, Chen Z, Kong H (2007) Patterns of gene
duplication and functional diversification during the evolution of the AP1/SQUA
subfamily of plant MADS-box genes. Mol Phylogen Evol 44: 26-41
Shimko N, Liu L, Lang BF, Burger G (2001) GOBASE: The organelle genome database.
Nucleic Acids Res 29: 128-132
Simpson MG (2006) Plant systematics. Academic Press 590 pp
Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissue
cultured in vitro. Symp Soc Exp Biol 54: 118-130
Smith JF, Hileman LC, Powell MP, Baum DA (2004) Evolution of GCYC, a Gesneriaceae
homolog of CYCLOIDEA, within Gesnerioideae (Gesneriaceae). Mol Phylogenet Evol
31: 765-779
Soltis DE, Soltis PE, Endress PK, Chase MW (2005) Phylogeny and evolution of
angiosperms. Sinauer Associates, Sunderland, MA, x, 370 pp
Soltis DE, Soltis PS (1998) Choosing an approach and an appropriate gene for phylogenetic
analysis. In DE Soltis, PS Soltis, JJ Doyle, eds, Molecular Systematics of Plants II: DNA
Sequencing. Kluwer Academic Publishers, Norwell, MA, USA, pp 1-42
Soltis DE, Soltis PS (2003) The role of phylogenetics in comparative genetics Plant Physiol
132: 1790-1800
Soltis DE, Soltis PS, Albert VA, Oppenheimer DG, dePamphilis CW, Ma H, Frohlich MW,
Theissen G (2002) Missing links: The genetic architecture of flower and floral
diversification. Trends Plant Sci 7: 22-31
234
Soltis PS, Kuzoff RK (1993) ITS sequence variation within and among populations of
Lomatium grayi and L. laevigatum (Umbelliferae). Mol Phylogen Evol 2: 166-170
Soltis PS, Soltis DE, Kim S, Chanderbali A, Buzgo M (2006) Expression of floral regulators
in basal angiosperms and the origin and evolution of ABC-function. In Advances in
Botanical Research: Incorporating Advances in Plant Pathology, Vol 44. Academic Press
Ltd., London, pp 483-506
Soniya EV, Sujitha M (2006) An efficient in vitro propagation of Aristolochia indica. Biol
Plant 50: 272-274
Speck T, Rowe NP, Civeyrel L, Classen-Bockhoff R, Neinhuis C, Spatz H-C (2003) The
potential of plant biomechanics in functional biology and systematics. In TF Stuessy, V
Mayer, E Hörandl, eds, Deep Morphology: Toward a Renaissance of Morphology in
Plant Systematics. Koeltz, Königstein, pp 241-271
Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with
thousands of taxa and mixed models. Bioinformatics 22: 2688-2690
Stegemann S, Hartmann S, Ruf S, Bock R (2003) High-frequency gene transfer from the
chloroplast genome to the nucleus. Proceedings of the National Academy of Sciences of
the United States of America 100: 8828-8833
Stern DB, Palmer JD (1984) Extensive and widespread homologies between mitochondrial
DNA and chloroplast DNA in plants. Proc Natl Acad Sci USA 81: 1946-1950
Stevens PF (2007) Angiosperm Phylogeny Website. Version 7, May 2006 [and more or less
continuously updated since] http://www.mobot.org/MOBOT/research/APweb/
Stevens PF (2008) Angiosperm Phylogeny Website Version 9, June 2008 [and more or less
continuously updated since] http://www.mobot.org/MOBOT/research/APweb/
Stupar RM, Lilly JW, Town CD, Cheng Z, Kaul S, Buell CR, Jiang J (2001) Complex
mtDNA constitutes an approximate 620-kb insertion on Arabidopsis thaliana
chromosome 2: Implication of potential sequencing errors caused by large-unit repeats.
Proc Natl Acad Sci USA 98: 5099-5103
Sugawara T (1987a) Chromosome number of Saruma henryi Oliver Aristolochiaceae. Bot Mag
Tokyo 100: 99-102
Sugawara T (1987b) Taxonomic studies of Asarum sensu lato III. Comparative floral anatomy.
Bot Mag Tokyo 100: 335-348
Sunilkumar G, Rathore KS (2001) Transgenic cotton: factors influencing Agrobacterium-
mediated transformation and regeneration. Mol Breed 8: 37-52
235
Swofford DL (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Ed
4.0b10. Sinauer Associates, Sunderland, Massachusetts, pp
Tabatabaei SJ, Yusefi M, Hajiloo J (2008) Effects of shading and NO3:NH4 ratio on the yield,
quality and N metabolism in strawberry. Scientia Horticulturae 116: 264-272
Takeda T, Suwa Y, Suzuki M, Kitano H, Ueguchi-Tanaka M, Ashikari M, Matsuoka M,
Ueguchi C (2003) The OsTB1 gene negatively regulates lateral branching in rice. Plant J
33: 513-520
Taylor DW, Hickey LJ (1990) An Aptian plant with attached leaves and flowers: Implications
for angiosperm origin. Science 247: 702-704
Taylor DW, Hickey LJ (1992) Phylogenetic evidence for the herbaceous origin of angiosperms.
Plant Systemat Evol 180: 137-156
Theissen G (2001) Development of floral organ identity: stories from the MADS house. Curr
Opin Plant Biol 4: 75-85
Thien LB, Azuma H, Kawano S (2000) New perspectives on the pollination biology of basal
angiosperms. Int J Plant Sci 161: S225-S235
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX
windows interface: flexible strategies for multiple sequence alignment aided by quality
analysis tools. Nucleic Acids Res 24: 4876-4882
Tremousaygue D, Garnier L, Bardet C, Dabos P, Herve C, Lescure B (2003) Internal
telomeric repeats and 'TCP domain' protein-binding sites co -operate to regulate gene
expression in Arabidopsis thaliana cycling cells. Plant J 33: 957-966
Tremousaygue D, Manevski A, Bardet C, Lescure N, Lescure B (1999) Plant interstitial
telomere motifs participate in the control of gene expression in root meristems. Plant J
20: 553-561
Trujillo CG, Sersic AN (2006) Floral biology of Aristolochia argentina (Aristolochiaceae).
Flora 201: 374-382
Vega JM, Yu WC, Kennon AR, Chen XL, Zhang ZYJ (2008) Improvement of
Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary
vectors. Plant Cell Rep 27: 297-305
Vieira CP, Vieira J, Charlesworth D (1999) Evolution of the cycloidea gene family in
Antirrhinum and Misopates. Mol Biol Evol 16: 1474-1483
Vogel S (1978) Fungus-gnat flowers mimicking fungi Part 1. Flora 167: 329-366
236
Waga S, Hannon GJ, Beach D, Stillman B (1994) The p21 inhibitor of cyclin-dependent
kinases controls DNA replication by interaction with PCNA. Nature 369: 574-578
Wall PK, Leebens-Mack J, Muller KF, Field D, Altman NS, dePamphilis CW (2008)
PlantTribes: a gene and gene family resource for comparative genomics in plants.
Nucleic Acids Res 36: D970-976
Wang RL, Stec A, Hey J, Lukens L, Doebley J (1999) The limits of selection during maize
domestication. Nature 398: 236-239
Wang W, Tanurdzic M, Luo M, Sisneros N, Kim HR, Weng JK, Kudrna D, Mueller C,
Arumuganathan K, Carlson J, Chapple C, dePamphilis C, Mandoli D, Tomkins J,
Wing RA, Banks JA (2005) Construction of a bacterial artificial chromosome library
from the spikemoss Selaginella moellendorffii: a new resource for plant comparative
genomics. BMC Plant Biol 5: 10
Wang Z, Luo Y, Li X, Wang L, Xu S, Yang J, Weng L, Sato S, Tabata S, Ambrose M,
Rameau C, Feng X, Hu X, Luo D (2008) Genetic control of floral zygomorphy in pea
(Pisum sativum L.). Proc Natl Acad Sci USA 105: 10414-10419
Wanke S, Gonzalez F, Neinhuis C (2006a) Systematics of pipevines: Combining
morphological and fast-evolving molecular characters to investigate the relationships
within subfamily Aristolochioideae (Aristolochiaceae). Int J Plant Sci 167: 1215-1227
Wanke S, Jaramillo MA, Borsch T, Samain MS, Quandt D, Neinhuis C (2007) Evolution of
Piperales-matK gene and trnK intron sequence data reveal lineage specific resolution
contrast. Mol Phylogenet Evol 42: 477-497
Wanke S, Quandt D, Neinhuis C (2006b) Universal primers for a large cryptically simple
cpDNA microsatellite region in Aristolochia (Aristolochiaceae). Mol Ecol Notes 6: 1051-
1053
Warbrick E (2000) The puzzle of PCNA's many partners. BioEssays 22: 997-1006
Wei FS, Wing RA (2008) A fruitful outcome to the papaya genome project. Genome Biol 9: 4
Weintraub H, Davis R, Tapscott S, Thayer M, Krause M, Benezra R, Blackwell TK,
Turner D, Rupp R, Hollenberg S, Zhuang Y, Lassar A (1991) The myoD gene family:
nodal point during specification of the muscle cell lineage. Science 251: 761-766
Weir I, Lu JP, Cook H, Causier B, Schwarz-Sommer Z, Davies B (2004) CUPULIFORMIS
establishes lateral organ boundaries in Antirrhinum. Development 131: 915-922
Welchen E, Gonzalez DH (2006) Overrepresentation of elements recognized by TCP-domain
transcription factors in the upstream regions of nuclear genes encoding components of the
mitochondrial oxidative phosphorylation Machinery. Plant Physiol 141: 540-545
237
Wendel JF, Doyle JJ (1998) Phylogenetic incongruence: window into genome history and
molecular evolution. In DE Soltis, PS Soltis, JJ Doyle, eds, Molecular Systematics of
Plants II: DNA Sequencing. Kluwer Academic Publishers, Norwell, MA, USA, pp 263-
296
Weng J-K, Tanurdzic M, Chapple C (2005) Functional analysis and comparative genomics of
expressed sequence tags from the lycophyte Selaginella moellendorffii. BMC Genomics
6: 85
Went FW (1926) On growth accelerating substances in the coleoptile of Avena sativa. Proc Kon
Ned Akad Wet Ser C 30: 10-19
Whipple CJ, Ciceri P, Padilla CM, Ambrose BA, Bandong SL, Schmidt RJ (2004)
Conservation of B-class floral homeotic gene function between maize and Arabidopsis.
Development 131: 6083-6091
Williams JH, Friedman WE (2002) Identification of diploid endosperm in an early angiosperm
lineage. Nature 415: 522-526
Williams JH, Friedman WE (2004) The four-celled female gametophyte of Illicium
(Illiciaceae; Austrobaileyales): Implications for understanding the origin and early
evolution of monocots, eumagnoliids, and eudicots. Am J Bot 91: 332-351
Woese C (1998) The universal ancestor. Proc Natl Acad Sci USA 95: 6854-6859
Woese CR (2000) Interpreting the universal phylogenetic tree. Proc Natl Acad Sci USA 97:
8392-8396
Wolda H, Sabrosky CW (1986) Insect visitors to two forms of Aristolochia pilosa in Las
Cumbres Panama. Biotropica 18: 295-299
Wu IF, Chen JT, Chang WC (2004) Effects of auxins and cytokinins on embryo formation
from root-derived callus of Oncidium 'Gower Ramsey'. Plant Cell Tiss Org Cult 77: 107-
109
Yao X, Ma H, Wang J, Zhang DB (2007) Genome-wide comparative analysis and expression
pattern of TCP gene families in Arabidopsis thaliana and Oryza sativa. JITB 49: 885-897
Zahn LM, Kong HZ, Leebens-Mack JH, Kim S, Soltis PS, Landherr LL, Soltis DE,
dePamphilis CW, Ma H (2005a) The evolution of the SEPALLATA subfamily of
MADS-Box genes: a preangiosperm origin with multiple duplications throughout
angiosperm history. Genetics 169: 2209-2223
Zahn LM, Leebens-Mack J, dePamphilis CW, Ma H, Theissen G (2005b) To B or not to B a
flower: the role of DEFICIENS and GLOBOSA orthologs in the evolution of the
angiosperms. J Hered 96: 225-240
238
Zahn LM, Leebens-Mack JH, Arrington JM, Hu Y, Landherr LL, dePamphilis CW,
Becker A, Theissen G, Ma H (2006) Conservation and divergence in the AGAMOUS
subfamily of MADS-box genes: evidence of independent sub- and neofunctionalization
events. Evol Dev 8: 30-45
Zhang PY, Tan HTW, Pwee KH, Kumar PP (2004) Conservation of class C function of floral
organ development during 300 million years of evolution from gymnosperms to
angiosperms. Plant J 37: 566-577
Zhao Y, Wang G, Zhang J, Yang J, Peng S, Gao L, Li C, Hu J, Li D, Gao L (2006)
Expressed sequence tags (ESTs) and phylogenetic analysis of floral genes from a
paleoherb species, Asarum caudigerum. Ann Bot 98: 157-163
VITA
Barbara J. Bliss 403 Life Science Building University Park, PA 16802 (814) 441-4453 [email protected]
466 Orlando Ave State College, PA 16803 (814) 861-3553 [email protected]
EDUCATION Ph.D. Biology (Molecular Evolutionary Biology) 2008 Pennsylvania State University, University Park, PA M.S. Environmental Science (Toxicology) 1998 Huxley College, Western Washington Univ, Bellingham, WA B. S. Biology magna cum laude 1995 Western Washington University, Bellingham, WA
PUBLICATIONS Bliss BJ, Wanke S, Barkat A, Wall PK, Landherr LL, Hu Y, Ma H, Neinhuis C, Leebens-Mack J,
Arumuganathan K, Clifton S, Maximova SN, dePamphilis CW (in preparation, 2008). Aristolochia fimbriata: A proposed experimental model for basal angiosperms. Plant Physiology
Bliss BJ, Landherr LL, dePamphilis CW, Ma H, Hu Y, and Maximova SN (submitted, 2008). Regeneration and plantlet development from somatic tissues of Aristolochia fimbriata. Plant Cell, Tissue and Organ Culture
Albert VA, Soltis DE, Carlson JE, Farmerie WG, Wall PK, Ilut DC, Solow TM, Mueller LA, Landherr LL, Hu Y, Buzgo M, Kim S, Yoo MJ, Frohlich MW, Perl-Treves R, Schlarbaum SE, Bliss BJ, Zhang X, Tanksley SD, Oppenheimer DG, Soltis PS, Ma H, Depamphilis CW, Leebens-Mack JH (2005) Floral gene resources from basal angiosperms for comparative genomics research. BMC Plant Biol 5: 5
Bliss BJ, Markiewicz AJ, Landis WG (1998) Statistical analysis of toxicity identification evaluation, effluent flow, and composition data to identify sources of whole effluent toxicity. Institute of Environmental Toxicology and Chemistry, Huxley College of Environmental Studies, Western Washington University, Bellingham, WA.
Bliss BJ, Markiewicz AJ, Luxon MG, Landis WG, Harass MC 1998: Utilizing Graphical, Exploratory and Confirmational Statistical Techniques to Evaluate Effluent Toxicity. SETAC annual meeting poster presentation, Charlotte, NC.
Bliss BJ 1998. Estrogenicity of nonylphenol in early life stages in the fathead minnow, Pimephales promelas. Masters thesis. Huxley College of Environmental Studies, Western Washington University, Bellingham, WA.
TEACHING Pennsylvania State University 6/2003-12/2008 Plant Taxonomy (co-instruct field course), Biological
Concepts and Diversity (lab instruction) Whatcom Community College 9/1999 - 6/2002 Biological Diversity, Biological Principles, Environmental
Science, Botany, Human Systems, Human Nutrition, Honors seminar (Toxicology) Western Washington University 9/1995 – 6/1997 Limnology Methods, Water Quality Methods, Toxicology
AWARDS Eberly College of Science Braddock Fellowship 2002-2003, 2008 University Graduate Fellowship, Pennsylvania State University 2002-2003 PNWSETAC annual student presentation award 1998 American Association of University Women Scholarship 1996-1997 Hughes Summer Research Intern Scholarship 1994 Western Foundation Scholarships 1993-1995 Academic Merit Scholarship 1993-1994