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“Convergence, constraint and the role of gene expression during adaptive radiation:

“Convergence, constraint and the role of gene expression 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.

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“Convergence, constraint and the role of gene expression during adaptive radiation:

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  1. “Convergence, constraint and the role of gene expression 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 Chicago, July 2006

  2. 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) 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 regions? 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 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 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 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 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. 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? Introduction Methods & Results Discussion Outlook

  11. 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

  12. High-density oligonucleotide arrays (NimbleGen) for genotyping and expression analysis First array: 20 probes/unigene (17,800x20 = 356000 spots) Expression in 5 floral whorls of 6 A. formosa individuals (30 samples) 1. sepals 2. petals (spurs) 3. anthers (♂) 4. carpels (♀), 5. staminodia (???) Within population genetic variation? Large Scale Array Research in Aquilegia TIGR Aquilegia Gene Index(Sept 20th, 2005) A. formosa x pubescens mixed tissue normalized cDNA library

  13. Matrix of colored dots ? ? What’s the big picture? Georges Seurat1884 • Whorl-specific genes? • Which floral organs are most similar? • How does Aquilegia compare to Gerbera or Poppy? • Intra-specific SFPs? 1. Make sense of data! 2. See the Art Institute! 2 Goals for my Chicago visit... Matrix of sample-dependent expression values (x)

  14. Acknowledgements Justen B. Whittall Daniel A. Kliebenstein Scott Hodges Justin Borevitz Elena Kramer Magnus Nordborg Jeff Tomkins NSF (EF-0412727) Thank you for your attention!

  15. Significant Patterns In A- LA CA LO PI CH MI BA CO PU FL FO FP OW X X X X X X X X X CHS/ACT X X X X X X X X X CHI/ACT X X X X X X X F3H/ACT X X X X X DFR/ACT X X X X X ANS/ACT UGT/ACT X X X X X X X X – expressed – reduced • More down-regulated genes in the late part of the ABP • (F3H early: p=0.0172, F3H late: p=0.0096) Pleiotropy • Strongly correlated expression of DFR & ANS (p=4.76x10-5) Mutated trans-regulator • All genes expressed in A. micrantha lineage Non-functional enzyme(s)

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

  17. 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

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

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