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

1. “Convergence, constraint and the role of regulatory genes during adaptive radiation: Floral anthocyanins in Aquilegia” JUSTEN B. WHITTALL, CLAUDIA VOELCKEL DAN J. KLIEBENSTEIN, SCOTT A. HODGES Ecology, Evolution & Marine Biology University of California Santa Barbara A. formosa A. pubescens Evolution, Stony Brook, June 2006

2. 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 SH TR FO EX FO FL PU COOC COCO COAL COOC Sp. nov. • 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) ! EL BA * MI Bob Bob Skowron Skowron SC DE * CH HI * CH PI CHAP LO * LO SK CA BR LA JO SA VUL 0.1 Bob Bob Skowron Skowron (Whittall & Hodges, in prep) Introduction Methods & Results Discussion Outlook

3. Do similar phenotypes evolve by similar molecular mechanisms? convergent/parallel phenotypes as a consequences of similar selection pressures (e.g. succulence, albinisms) • 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 Questions Of General Interest… • Rapid phenotypic evolution mediated by changes in regulatory rather than enzyme-coding genes? Introduction Methods & Results Discussion Outlook

4. Phylogenetic Character Mapping: 6 independent losses of floral anthocyanins (A-) 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-? A- A+ A+/A- Introduction Methods & Results Discussion Outlook

5. CHS 6 ABP loci CHI Feeding repellents, UV protectants… Hypothesis: Pleiotropy constrains A-mutations to later stages of the ABP F3H DFR ANS UF3GT Floral pigments The Anthocyanin Biosynthetic Pathway (ABP) Coumaroyl CoA + Malonyl CoA Chalcones Flavanones 3-OH Flavonols Leucanthocyanidins Experiment: Monitor expression of these 6 loci in multiple A- species via RT-PCR Anthocyanidins Anthocyanins Introduction Methods & Results Discussion Outlook

6. A. canadensis (A+) 5 stages 2 tissue types 1 2 3 4 5 1 2 • All loci expressed • Little variation between stages and tissues • Most consistent expression stage 3 and 4 and tissue 2 Preliminary Study Or Timing Is Everything! degenerate primers for 6 loci CHS CHI F3H DFR ANS UF3GT ACTIN Control Introduction Methods & Results Discussion Outlook

7. ABP Gene Expression In 13 Aquilegia Species Species LA CA LO PI CH MI BA CO PU FL FO FP OW CHS 2 main patterns in A- species: CHI F3H 1 – reduced expression in one or more loci DFR ANS 2 – expression like in A+ species UF3GT Actin control Pattern 2 A+ 1 1 1 2 2 A+ 1 1 A+ 2 1 Introduction Methods & Results Discussion Outlook

8. X X X X X X X X X X X X X X X X X X 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 • 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) Non-functional enzyme(s) • All genes expressed in A. micrantha lineage Significant Patterns In A- LA CA LO PI CH MI BA CO PU FL FO FP OW CHS/ACT CHI/ACT F3H/ACT DFR/ACT ANS/ACT UGT/ACT Introduction Methods & Results Discussion Outlook

9. Reg A - X ABP Gene Reg Derived 1 no expression Common – e.g. A. pubescens X A - ABP Gene Derived 2 impaired function Rare – e.g. A. micrantha X A - A - ABP Gene Reg ABP Gene 2 Models To Explain The A-Phenotype Ancestral – e.g. A. formosa A+ Reg ABP Gene expression Introduction Methods & Results Discussion Outlook

10. Next Steps • 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 Introduction Methods & Results Discussion Outlook

11. Acknowledgements Justen B. Whittall Daniel A. Kliebenstein Scott A. Hodges NSF (EF-0412727) Thank you for your attention!

12. Petunia Anthocyanin polymorphisms caused by different alleles in regulatory loci (Epperson and Clegg 1988, Quattrocchio et al. 1999, Chang et al. 2005) identify regulators in Aquilegia Ipomoea Gradual degeneration of the ABP Structural mutation followed by loss of expression Ipomoea (Zufall and Rausher 2004) unlikely in Aquilegia Directing metabolic flux into tannin synthesis turns pink tobacco flowers white (Xie et al. 2003) Nicotiana tabacum similar mechanism in Aquilegia micrantha? What Is Known In Other Systems? Introduction Methods & Results Discussion Summary

13. Figure 3 Wild Hort FO LA CA LO PI CH MI BA CO PU FL FP OW CHS/ACT CHI/ACT F3H/ACT DFR/ACT ANS/ACT UF3GT/ACT

14. Pleiotropy! Mutated trans-regulator! Significant Patterns In A- LA CA LO PI CH MI BA CO PU FL FO FP OW X – expressed R – reduced • 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) Introduction Methods & Results Discussion Summary