“Molecular mechanisms of speciation in Aquilegia & Pachycladon ”
Palmy, October 2006
University of California Santa Barbara Ecology, Evolution and Marine Biology
Feb 05 - Sep 06
Massey University Allan Wilson Centre
Nov 06 - ???
Claudia Voelckel
1. Convergence, constraint and the role of gene expression during adaptive radiation: Floral anthocyanins in Aquilegia (Whittall et al. 2006, MolEcol early online)
2. The evolution of nectar spurs and staminodia in Aquilegia – a microarray approach (in progress)
3. Ecological speciation in Pachycladon - a microarray approach (about to begin)
Outline
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SHTR
FOEX
FOFL
PUCOOC
COCOCOAL
COOCSp. nov.
EL BAMI
SCDE
CHHICH
PI CHAP
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Bob SkowronBob Skowron
Bob SkowronBob Skowron
Aquilegia As An Evolutionary Model System
Why Aquilegia?
Floral & ecological diversity Recent radiation (interfertility) Small genome (350 Mbp, n=7) Basal lineage in the eudicots
Resource Development
EST database (TIGR Gene Index: 17,800 unique sequences)
Microarrays (NimbleGen) for both expression & genotyping studies
SNPs (ca 3500 assays by Sequenom) Physical map (CUGI) Transformation system (Kramer Lab)
to manipulate candidate gene expression ! Genome Project (JGI 2007) !
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
(Whittall & Hodges, in prep)
Do similar phenotypes evolve by similar molecular mechanisms?convergent/parallel phenotypes as a consequences of similar selection pressures (e.g. succulence, albinisms)
Rapid phenotypic evolution mediated by changes in regulatory rather than enzyme-coding regions?
Floral Anthocyanin Losses In Aquilegia
Loss-of-phenotype mutations more diverse than gain-of-phenotype mutations?
(e.g. insecticide resistance)
floral anthocyanins – phenotypically and moleculary tractable
Aquilegia – multiple losses of floral anthocyanins
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
trit
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ch
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NM
)
hin
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leya
na
ch
rysa
nth
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CH
I)
ch
ap
lin
ei
lon
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sim
a(A
Z)
sk
inn
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sim
a(T
X)
bre
vis
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sax
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nta
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vu
lga
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trit
ern
ata
sh
oc
kle
yi
form
os
a(E
ast)
form
os
a(W
es
t)
ex
imia
fla
ves
cen
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pu
be
sce
ns
co
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lea
va
r.o
ch
role
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(UT
)
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(CO
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sp
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ba
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by
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de
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ch
rysa
nth
a (
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hin
ck
leya
na
ch
rysa
nth
a (
CH
I)
ch
ap
lin
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lon
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sim
a(A
Z)
sk
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X)
bre
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Floral Anthocyanins (A) : How Does Evolution Repeat Itself?
Which genes are mutated in A-?
Structural versus regulatory mutations?
Degree of convergence across lineages?
Any constraints to the evolution of A-?
Phylogenetic Character Mapping:
6 independent losses of floral anthocyanins (A-)
A+ A- A+/A-
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
Coumaroyl CoA + Malonyl CoA
Chalcones
Flavanones
3-OH Flavonols
Leucanthocyanidins
Anthocyanins
Anthocyanidins
The Anthocyanin Biosynthetic Pathway (ABP)
CHS
CHI
F3H
DFR
ANS
UF3GT
6 ABP loci
Experiment:
Monitor expression of these 6 loci in multiple A- species via RT-PCR
Feeding repellents,
UV protectants
…
Hypothesis:
Pleiotropy constrains A-mutations to later stages of the ABP
Floral pigments
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
A. canadensis (A+)
12
1 2 3 4 5
Preliminary Study Or Timing Is Everything!
Peak expression mostly in stages 3 & 4 and tissue 2
CHS
ACTIN Control
CHI
F3H
UF3GT
DFR
ANS
degenerate primers for
6 loci
All loci expressed
Little variation between stages, tissues and individuals
5 stages 2 tissue types3 individuals
highly significant correlation between DFR and ANS (p=0.0001)
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
ABP Gene Expression In 13 Aquilegia Species
2 – expression like in A+ species
2 main patterns in A- species:
1 – reduced expression in one or more loci
LA CA LO PI CH MI BA CO PU FL FO FP OW
CHS
Actin control
CHI
F3H
UF3GT
DFR
ANS
Species
Pattern 2 A+ 1 1 1 2 2 A+ 1 1 A+ 2 1
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
LA CA LO PI CH MI BA CO PU FL FO FP OW
Significant Patterns In A-
X – expressed
– reduced
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
CHS/ACT
CHI/ACT
F3H/ACT
DFR/ACT
ANS/ACT
UGT/ACT
More down-regulated genes in the late part of the ABP (F3H early: p=0.0172, F3H late: p=0.0096)
Pleiotropy
Mutated trans-regulator
Strongly correlated expression of DFR & ANS (p=4.76x10-5)
All genes expressed in A. micrantha lineage Non-functional enzyme(s)
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
expression
ABP GeneRegA+
2 Models To Explain The A-Phenotype
– e.g. A. formosaAncestral
ABP Gene
ABP Gene
Reg
Reg
X
X
A-
A-
ABP GeneA-
no expression
Common – e.g. A. pubescens
Derived 1
- ABP GeneRegXA
impaired function Rare – e.g. A. micrantha
Derived 2
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
What Is Known In Other Systems?
Directing metabolic flux into tannin synthesis turns pink tobacco flowers white
Nicotiana tabacum(Xie et al. 2003)
Anthocyanin polymorphisms caused by different alleles in regulatory loci
(Epperson and Clegg 1988, Quattrocchio et al. 1999, Chang et al. 2005)
Petunia
Ipomoea
Gradual degeneration of the ABPStructural mutation followed by loss of expressionIpomoea(Zufall and Rausher 2004)
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
The role of regulatory loci in the evolution of the A-phenotype Identify ABP regulators, monitor their expression in A+ & A- species,
search for interspecific polymorphisms
Molecular mechanism for A-phenotype in A. pubescens? Do any of the ABP loci map to a QTL for spur color?
Independent origins, repeated fixing of an ancestral polymorphism or introgression via hybridization?
Compare alleles of ABP loci from A+ & A- species
Establish causal links between genotype and A-phenotype Replace A- alleles with A+ alleles to rescue A+ phenotype via genetic
engineering
Next Steps
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
ESTs 85,039
TC sequences 11,985Singleton sequences 5,816Total unique 17,801
Output Sequences
Input Sequences
TIGR Aquilegia Gene Index (Sept 20th, 2005)
A. formosa x pubescens
mixed tissue normalized cDNA
library
High-density oligonucleotide arrays (NimbleGen) for genotyping and expression analysis
Array Research In Aquilegia - Tools
First array: 20 probes/unigene (17,800x20 = 356000 spots)
First experiment: Expression in 5 floral whorls of A. formosa (sepals, petals, anthers, staminodia, carpels)
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
Questions
Whorl-specific gene expression? How conserved is the ABC model? How does Aquilegia compare to
Gerbera and Papaver? Which genes contributed to the
evolution of spurs? What is the function of staminodia?
Sampling
A. formosa, Sierra Nevada5 whorls x 3 replicates = 15 samples = 200 dissected flowers
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
Blue Canyon, Sonora Pass
Mobile lab with liquid nitrogen tank
Array Research In Aquilegia – First Hybridizations
Ecological Speciation In Pachycladon - Intro
Beringia bursifolia
P. novae-zealandiae
P. wallii
P. radicata
P. sp.
P. fastigiata
P. latisiliqua
P. enysii
P. stellata
P. exilis
P. cheesemanii
(after Heenan and Mitchell 2003)
Radiation 2.2 Mya
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
P. fastigiata P. enysii
Ro
sett
e-s
tag
eF
low
erin
g-s
tag
e
monocarpic perennials with serrate leaves and terminal inflorescences
on greywacke
Alpine High alpineGlabrous Hairy
Arabidopsis genome-wide oligonucleotide arrays (DNA hybridizations)
Within species vs between species variation in gene expression (RNA hybridizations, qPCR)
Greenhouse and field studies
Candidates for speciation genes
Integration with QTL studies and physiological studies
Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook
Ecological Speciation In Pachycladon - Ideas
Acknowledgements
Aquilegia
Justen B. WhittallKathleen M. Kay
Ji Yong Yang
Daniel A. Kliebenstein
Scott HodgesJustin BorevitzElena Kramer
Magnus NordborgJeff Tomkins
NSF (EF-0412727)
Pachycladon
Carlos – 7th NZ Meeting of MolEcol
2004, Pohangina Valley
Pete – SMBE Meeting 2005,
Auckland
Kim and Pete –Pachycladon Marsden Grant
Humboldt Foundation –Lynen fellowship
You!