“Molecular mechanisms of speciation in Aquilegia & Pachycladon ”

“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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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


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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-


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


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)


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


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)


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


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)


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


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)


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


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


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


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

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“Molecular mechanisms of speciation in Aquilegia & Pachycladon ”