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2/26/2014
Organellar genomes, resolution, and support
for deep relationships among the palms
Barrett, Craig1; Baker, William2; Comer, Jason3;
Leebens-Mack, James3; Li, Jeff1, Lim, Gwynne4,6; Mayfield,
Dustin5; Pires, J. Chris5; Santos, Cristian1, Stevenson,
Dennis6; Zomlefer, Wendy3; Davis, Jerrold4
1.
2.
3.
4.
5.
6.
California State University, Los Angeles, Los Angeles, California, USA
Royal Botanic Gardens, Kew, Richmond, Surrey, UK
University of Georgia, Athens, Georgia, USA
Cornell University, Ithaca, New York, USA
University of Missouri, Columbia, Missouri, USA
New York Botanical Garden, Bronx, New York, USA
Acknowledgements
Material, assistance with collections: Staff at NYBG, Fairchild TBG, Huntington BG, Royal BG at
Kew, City of Long Beach, CA
Funding: National Science Foundation MonAToL Grants DEB-0830020 (Cornell U.), DEB0830009 (U. Georgia), DEB-0829849 (U. Missouri), and DEB-0829762 (NY Botanical Garden)
NSF - Research Opportunity Award (ROA) supplement to DEB-0738042 (C.S.U., Los Angeles
and U.C., Berkeley)
Discussion/technical assistance: Eric Antonieu, Raj Ayyampalayam, Connie Asmussen, Thomas
Couvreur, Elena Ghiban, Jeff Doyle, Pat Edger, John Kerry, Sean Lahmeyer, Chang Liu, Michael
McKain, Aakrosh Ratan, Chelsea Specht, Cold Spring Harbor Labs, Cornell Life Sciences
Sequencing Center, University of Georgia Dept. of Plant Biology
Photo credits: emonocot.org, plantillustrations.org, nmh.si.edu
Licuala cordata
Licuala cordata
J. Dransfield
J. Dransfield
Research questions and objectives:
What can phylogenomic data tell us about relationships
and support among subfamilies and tribes of the palms?
1. The importance of palms
Ecologically important: components of tropical/subtropical ecosystems—
emblematic of the tropics
Focus on:
I. ‘Deep’ relationships among 5 palm subfamilies
II. Relationships among tribes of Coryphoideae (fan palms)
Economically important: horticulture, food sources, materials, biofuels
Overview:
I. Brief background on palm systematics & relationships
II. Methods; plastome, mt-gene coverage (NGS data)
III. Molecular evolution of the plastid genome
IV. Trees from complete plastomes (genes, introns,
spacers) + mitochondrial genes + nuclear rDNA
V. Future directions
Structurally remarkable: some rival woody dicots & conifers in height,
without true secondary growth
1. Palms are record-setters among plants
Leaf: Raphia – 20 m
Fruit: Lodoicea – 20 kg
Aerial stem: Calamus – 172 m
Inflorescence: Corypha – ~2 x
107 flowers
Corypha umbraculifera
J. Dransfield
Immense morphological diversity: growth habit, life history, flowers &
inflorescence, pollen, leaves
A well supported palm phylogeny is necessary to understand:
biogeography, morphology & development, comparative GENOMICS,
ecology, physiology...
Licuala cordata
J. Dransfield
1. Rich history of palm systematics
Morphological diversity, importance of palms has
prompted intensive study from 17th - 21st Centuries
Jacquin, Martius, Oersted, Wendland, Wallace…
Burret, Bailey, Moore, Tomlinson, Uhl, Dransfield, many more
Tomlinson (2006) Bot J Linn Soc 151: 5–14
Uhl & Dransfield, 1987; Dransfield & al., 2008
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1. Modern palm taxonomy – 5 subfamilies
1. Molecular data and palm phylogenetics
Calamoideae (21)
Palms display notoriously slow
plastid gene evolutionary rates
Asmussen et al. 2006, Bot J Lin Soc 151: 15–38
Challenges in finding sufficient character
information to resolve relationships
among major palm clades
Coryphoideae (45)
Phylogenetic uncertainty until the most
recent studies
Justifies the need for phylogenomic
data
synonymous s/s/y
Gaut et al.
Arecoideae (112)
Salacca
Nypoideae (1)
Ceroxyloideae (5)
plantillustrations.org
1. Multigene analyses & complete taxon sampling
Combined MP Strict
Consensus:
1. Gathering all available data – supermatrix &
supertree approaches
Supermatrix/supertrees:
most comprehensive
analyses to date
Plastid DNA only
Complete generic sampling: 5
nuclear, 8 plastid, morphology, plastid
RFLP
matK, rbcL, rps16 intron, trnL-trnF
Dense taxon sampling, multiple
outgroups
Support for monophyletic
subfamilies
Generally robust, deep support
among subfamilies
Calamoids sister; (Nyp (Cor (Cer,
Are)))
New subfamilial classification
Asmussen et al. (2006) Bot J Linn Soc
151: 15-38
Support for Nypa lower than in
previous studies, however
Low(er) support for internal structure
of Calamoids and Coryphoids
1. Palm genomic resources – Phoenix dactylifera
Baker et al. (2009) Syst. Biol. 58: 240–256
2. Palm organellar phylogenomics - methods
Taxon sampling: tried to include at least one
representative of each non-Arecoid tribe (with
placeholders in Arecoideae)
Illumina NGS sequencing (SE, PE)
De novo and reference-guided assembly
Annotation, genome alignment
Plastid genome
Yang et al., 2010. PLoS ONE
Combined phylogenetic analyses of whole plastome, mitochondrial
genes, nuclear rDNA (preliminary)
Mitochondrial genome
Fang et al., 2012. PLoS ONE
Nuclear genome and transcriptome
Al-Msallem et al., 2013. Nature Comm.
Maximum Likelihood and bootstrap support values (RAxML)
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2. Sequencing coverage – plastome & mt-genes
from ‘genome skimming’
Plastome
2. Plastid genome assembly & annotation workflow
Process_fastq.sh (concatenate, unzip, quality-trim, shuffle, rename, tarball)
Mito-genes
= 100bp PE, HiSeq
= 71 or 96bp SE, GAIIx
1. VELVET de novo
SE or PE assembly
3.0
3.9
Washingtonia
robusta
3.3
2.5
3. MULAN blastalign de novo
contigs to reference
2.0
3.0
~1000x coverage
2.7
~100x coverage
log mito
log plastid
Log x-coverage
3.6
Washingtonia
robusta
2.4
1.5
2.1
1.0
4. YASRA “draftde novo”
reference guided
assembly
~10x coverage
~100x coverage
1.8
0.5
1.5
1.2
0.0
1.2E07
2.4E07
3.6E07
4.8E07
6E07
7.2E07
8.4E07
9.6E07
1.6E07
1.08E08
3.2E07
4.8E07
6.4E07
8E07
9.6E07
5. SEQUENCHER
“meta-assembly”
9. DOGMA
annotation;
SEQUIN Validation &
GenBank submission
6. UNIX ‘grep’ & PCR
to cross gaps and
correct discrepancies
among contigs
7. SEQUENCHER:
determine IR, LSC,
SSC boundaries;
FINISHED PLASTOME
1.12E08
#reads
#reads
2. YASRA
reference
guided
assembly
# reads
8. SEQUENCHER:
align reference
genes to plastome
Zerbino & Birney, 2008; Ratan, 2009; Ovcharenko et al., 2005; GeneCodes; Drummond et al., 2011; Wyman et al., 2004
3. Molecular evolution of the plastome
3. Molecular evolution of the plastome
Eugeissona tristis  129,442 bp
Length range: 126,031 – 134,078 bp
(excludes second IR copy)
Phoenix dactylifera: 158,462 (incl. IR)
LSC
IR length range: 27,235* – 28,228 bp
Anomalies in:
Eugeissona tristis  129,442 bp
Tahina spectabilis  126,031 bp
LSC
Loss or transfer of
ndhF gene from
SSC of plastome
4. Phylogeny – complete plastome (minus one inverted
repeat)
• Nearly complete
resolution and high
support
• Highly congruent with
most recent analyses
Nypa fruticans
Arecoideae
Coryphoideae
“IRa”
Dasypogonaceae
Calamoideae
Kingia australis
Ceroxyloideae
“IRb” SSC
-Repeated 150 bp piece of
ndhB gene
Otherwise, IR has been
lost!
Calamoideae
Nypoideae
160,922 aligned chars
IRa removed
Maximum likelihood
GTR+GAMMA
1000 Bootstrap
No value  100% support!
IRa
4. Phylogeny – nuclear 5.8S, 18S, & 28S rDNA
Dasypogonaceae
Whole plastomes
SSC
Tahina spectabilis: 126,031 bp
Yang et al., 2010. PLoS ONE
Palms
IRb
Nypoideae
Palms
Nuclear ribosomal DNA
Maximum likelihood
GTR+GAMMA
1,000 Bootstrap
4,838 aligned chars
Ceroxyloideae
Arecoideae
Coryphoideae
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4. Phylogeny – mitochondrial genes
4. Phylogeny – combined plastome, mt-genes, nr-rDNA
Dasypogonaceae
Dasypogonaceae
Calamoideae
Calamoideae
Nypoideae
Nypoideae
Arecoideae
Palms
Palms
Ceroxyloideae
Ceroxyloideae
Arecoideae
Combined
plastome, mtgenes, rDNA
198,967 aligned chars
Maximum likelihood
GTR+GAMMA
1000 Bootstrap
No value = 100%
support
Mitochondrial DNA
33,951 aligned chars
37 protein-coding genes
Coryphoideae
Coryphoideae
4. Tribal relationships – key differences, coryphoids
V. Conclusions/Future Directions
Calectasia
Dasypogon
Combined analysis, 200K positions
Baker et al., 2009 Syst. Biol.
(no value = 100% support)
Supermatrix
Heliconia
Kingia
ChuniophoeniceaeBaxteria (no value = NO support)
Caryota
1. Combined plastome, mt-genes, & rDNA data yield a highly supported
phylogenetic hypothesis of deep relationships
Mauritia
Calamus
Caryoteae
2. High support for tribal relationships among fan palms (Coryphoideae)
Chuniophoe
Corypheae
Borasseae
Sabaleae
Cryosophileae
Phoeniceae
3. High level of congruence among 3 separate genomes
Corypha
Phoenix Trithrinax
Serenoa
4. A few key differences with previous studies, among tribes of
Coryphoideae, Calamoideae, Ceroxyloideae
Sabal
Ravenea
Pseudophoe
Phytelepha
Supertree – equal weights
5. Future directions: Multiple informative nuclear markers
 transcriptomes + gene capture
 coalescent-based species tree approaches
 phylogenomic signature of positive natural selection?
Veitchia
Areca
Trachycarpeae
Thanks!
Licuala
J. Davis
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2. rDNA & mtDNA assembly & workflow
Process_fastq.sh (concatenate, unzip, quality-trim, shuffle, rename, tarball)
rDNA: VELVET de novo PE assembly
(Eugeissona tristis)
mtDNA: Map reads to Phoenix
dactylifera mitogenome in
GENEIOUS
Join contigs in Sequencher
Align to annotated rDNA operon in GENEIOUS
(Asclepias syriaca); transfer annotations to
Eugeissona (a calamoid palm)
Map reads for each accession to
annotated Eugeissona rDNA operon
Extract all CDS (n=37) in GENEIOUS;
build concatenated alignment in MAFFT
RAxML phylogenetic analysis, bootstrap
support
Extract 18S, 5.8S, 28S sequences in
GENEIOUS, build alignment with MAFFT
Zerbino & Birney, 2008; Ratan, 2009; Ovcharenko et al., 2005; GeneCodes; Drummond et al., 2011; Wyman et al., 2004
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