“Convergence, constraint and the role of regulatory genes during adaptive radiation:

Floral anthocyanins in Aquilegia ”

Evolution, Stony Brook, June 2006

JUSTEN B. WHITTALL, CLAUDIA VOELCKEL DAN J. KLIEBENSTEIN, SCOTT A. HODGES

Ecology, Evolution & Marine BiologyUniversity of California Santa Barbara

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Aquilegia As An Evolutionary Model System

Why Aquilegia?

Floral & ecological diversity Recent & rapid radiation 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) !

Introduction Methods & Results Discussion 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 genes?

Questions Of General Interest…

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

Introduction Methods & Results Discussion Outlook

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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-

Introduction Methods & Results Discussion 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

Introduction Methods & Results Discussion Outlook

A. canadensis (A+)

12

1 2 3 4 5

Preliminary Study Or Timing Is Everything!

Most consistent expression stage 3 and 4 and tissue 2

CHS

ACTIN Control

CHI

F3H

UF3GT

DFR

ANS

degenerate primers for

6 loci

Introduction Methods & Results Discussion Outlook

All loci expressed

Little variation between stages and tissues

5 stages 2 tissue types

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

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CHS

Actin control

CHI

F3H

UF3GT

DFR

ANS

Species

Pattern 2 A+ 1 1 1 2 2 A+ 1 1 A+ 2 1

Introduction Methods & Results Discussion Outlook

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Significant Patterns In A-

Introduction Methods & Results Discussion Outlook

X – expressed

– reduced

X

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CHS/ACT

CHI/ACT

F3H/ACT

DFR/ACT

ANS/ACT

UGT/ACT

More lineages with down-regulation in the late part of the ABP (p=0.0484)

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. formosa

Introduction Methods & Results Discussion Outlook

Ancestral

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

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

Introduction Methods & Results Discussion Outlook

Acknowledgements

Justen B. Whittall

Daniel A. Kliebenstein

Scott A. Hodges

NSF (EF-0412727)

Thank you for your attention!

What Is Known In Other Systems?

Directing metabolic flux into tannin synthesis turns pink tobacco flowers white

Nicotiana tabacum(Xie et al. 2003)

similar mechanism in Aquilegia micrantha?

Anthocyanin polymorphisms caused by different alleles in regulatory loci

(Epperson and Clegg 1988, Quattrocchio et al. 1999, Chang et al. 2005)

Petunia

Ipomoea identify regulators in Aquilegia

Gradual degeneration of the ABPStructural mutation followed by loss of expression

Ipomoea (Zufall and Rausher 2004)

unlikely in Aquilegia

Introduction Methods & Results Discussion Summary

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Χ2 Tests:

More genes down-regulated in the late part of the ABP

More lineages with down-regulation in the late part of the ABP

Strong correlation between expression of DFR and ANS (5 lineages)

Pleiotropy!

Mutated trans-regulator!

Significant Patterns In A-

X – expressed

R – reduced

Introduction Methods & Results Discussion Summary

CHS/ACT X X X X X X X X X X X R XCHI/ACT R X X X R X X X X X X X XF3H/ACT R X R R R X X X X X X X RDFR/ACT R X R R R X X X R R X X RANS/ACT R X R R R R X X R R X X RUGT/ACT X X R R R X X X R X X R X