“Molecular mechanisms of speciation in Aquilegia & Pachycladon ”

1. “Molecular mechanisms of speciation in Aquilegia & Pachycladon” University of California Santa Barbara Ecology, Evolution and Marine Biology Feb 05 - Sep 06 Massey University Allan Wilson Centre Nov 06 - ??? Claudia Voelckel Palmy, October 2006

2. Outline • Convergence, constraint and the role of gene expression • during adaptive radiation: Floral anthocyanins in Aquilegia • (Whittall et al. 2006, MolEcol early online) • The evolution of nectar spurs and staminodia in Aquilegia • – a microarray approach • (in progress) • Ecological speciation in Pachycladon - a microarray approach • (about to begin)

3. 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 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) Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook

4. 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 Floral Anthocyanin Losses In Aquilegia • Rapid phenotypic evolution mediated by changes in regulatory rather than enzyme-coding regions? Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook

5. 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- Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook

6. 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 Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook

7. A. canadensis (A+) 5 stages 2 tissue types 3 individuals 1 2 3 4 5 1 2 • All loci expressed • Little variation between stages, tissues and individuals • Peak expression mostly in stages 3 & 4 and tissue 2 Preliminary Study Or Timing Is Everything! degenerate primers for 6 loci CHS CHI F3H DFR ANS • highly significant correlation between DFR and ANS (p=0.0001) UF3GT ACTIN Control Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook

8. 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 Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook

9. 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 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) 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 Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook

10. 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 Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook

11. Petunia Anthocyanin polymorphisms caused by different alleles in regulatory loci (Epperson and Clegg 1988, Quattrocchio et al. 1999, Chang et al. 2005) Ipomoea Gradual degeneration of the ABP Structural mutation followed by loss of expression Ipomoea (Zufall and Rausher 2004) Directing metabolic flux into tannin synthesis turns pink tobacco flowers white (Xie et al. 2003) Nicotiana tabacum What Is Known In Other Systems? Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook

12. 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 Aquilegia anthocyanins Aquilegia arrray research Pachycladon outlook

13. High-density oligonucleotide arrays (NimbleGen) for genotyping and expression analysis First array: 20 probes/unigene (17,800x20 = 356000 spots) First experiment: Expression in 5 floral whorls of A. formosa (sepals, petals, anthers, staminodia, carpels) Array Research In Aquilegia - Tools TIGR Aquilegia Gene Index(Sept 20th, 2005) A. formosa x pubescens mixed tissue normalized cDNA library Aquilegia anthocyaninsAquilegia arrray researchPachycladon outlook

14. Blue Canyon, Sonora Pass Mobile lab with liquid nitrogen tank Array Research In Aquilegia – First Hybridizations Sampling A. formosa, Sierra Nevada 5 whorls x 3 replicates = 15 samples = 200 dissected flowers • 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? Aquilegia anthocyaninsAquilegia arrray researchPachycladon outlook

15. Radiation 2.2 Mya P. fastigiata P. enysii monocarpic perennials with serrate leaves and terminal inflorescences on greywacke Alpine High alpine Glabrous Hairy P. fastigiata P. sp. P. stellata P. enysii P. latisiliqua P. wallii Rosette-stage P. novae-zealandiae P. radicata P. cheesemanii P. exilis Beringia bursifolia Flowering-stage (after Heenan and Mitchell 2003) Ecological Speciation In Pachycladon - Intro Aquilegia anthocyaninsAquilegia arrray research Pachycladon outlook

16. 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 Arabidopsis genome-wide oligonucleotide arrays (DNA hybridizations) Aquilegia anthocyaninsAquilegia arrray research Pachycladon outlook

17. Acknowledgements Aquilegia Justen B. Whittall Kathleen M. Kay Ji Yong Yang Daniel A. Kliebenstein Scott Hodges Justin Borevitz Elena Kramer Magnus Nordborg Jeff 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!