University of California Santa Barbara Ecology, Evolution & Marine Biology Hodges Lab Reproductive isolation & speciation in Columbines (Aquilegia) • Goal: Establish Aquilegia as an evolutionary model system • 70 taxa (diverse in floral morphology & ecology) • recent & rapid radiation (little genetic variation & successful hybrid formation) • diploid (n=7), small genome (320-400Mbp) • basal lineage in the eu-dicots • Requires: Development of molecular resources • (J. Borevitz, University of Chicago, E. Kramer, Harvard University, J. Tomkins, Clemson University, M. Nordborg, University of Southern California) • EST database (TIGR) (microarrays for both expression & genotyping studies) • physical map (CUGI) • transformation system to manipulate candidate gene expression
SH TR FO EX FO FL PU COOC COCO COAL COOC Sp. nov. 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 AFLP-based Phylogeny of North American Columbines Diversity in pollination syndromes Bee-pollinated flowers Hummingbird-pollinated flowers Hawkmoth-pollinated flowers Shifts from blue/red to white/yellow = Shift to hawkmoth pollination (?) (Whitall and Hodges, in prep)
6 independent losses of floral anthocyanins... Are the molecular mechanisms of a convergent phenotype convergent themselves? Phylogenetic Mapping of Floral Anthocyanins TR - A. triternata SH - A. shockleyi FO - A. formosa (East) FO - A. formosa (West) EX - A. eximia FL - A. flavescens PU - A. pubescens COOC - A. coer. ochrol. COAP - A. coer. alpina COCO - A. coer. coerulea COOC - A. coer. ochrol. A. sp. nov. EL - A. elegantula MI - A. micrantha BA - A. barnebyi SC - A. scopulorum DE - A. desertorum PI – A. pinetorum CH - A. chrysantha (NM) HI - A. hinckleyana CH - A. chrysantha (CHI) CHAP - A. chaplinei LO - A. longissima (AZ) SK - A. skinneri LO - A. longissima (TX) BR - A. brevistyla CA - A. canadensis LA - A. laramiensis SA - A. saximontana JO - A. jonesii VUL - A. vulgaris A+ A- A+/A- Which genes drive anthocyanin loss? Relative importance of structural versus regulatory mutations?
6 core loci CHS Isoflavenoids Aurones Many side branches CHI Phlobaphenes Flavones F3H Flavonols DFR Flavan-3-ols ANS Condensed Tannins UF3GT The Anthocyanin Biosynthetic Pathway (ABP) Coumaroyl CoA + Malonyl CoA Chalcones Flavanones • 3 classes of regulators • bHLH (myc) • myb • WD 40 Early 3-OH Flavonols Late Leucanthocyanidins Petunia, Zea, Arabidopsis, Ipomoea, Antirrhynum... Anthocyanidins Anthocyanins
Convergence in ABP Gene Expression in A- species? • Degenerate primers from multiple aa alignments for 6 loci • Clone 6 loci from A. formosa (A+) • Optimise RT-PCR (primers, temperatures, cycle number) • Find the right floral tissue and developmental stage!
Most consistent expression in old bud and pre-anthesis flowers 1 2 3 AN YB OB PrA PoA YB OB PrA PoA YB OB PrA PoA 1 2 3 GB GB GB CHS CHI F3H DFR ANS UF3GT ACT A. canadensis as an A+ Reference Species GB YB OB PrA PoA 5 stages, 3 individuals, 2 tissues • Little variation between individuals and tissues • No expression of DFR and ANS in anthers (A- tissue of an A+ species)
Select OB+PrA for multispecies comparison Comparing kinetics in three species... Petals Anthers YB OB PoA PrA GB PrA CA (A+) CHS FO (A+) CH (A-) • Different kinetics in A. chrysantha CA (A+) CHI FO (A+) A. canadensis (A+) CH (A-) CA (A+) F3H FO (A+) • DFR and ANS missing in • A. chrysantha CH (A-) CA (A+) DFR FO (A+) CH (A-) A. formosa (A+) CA (A+) ANS FO (A+) CH (A-) CA (A+) UF3GT FO (A+) CH (A-) CA (A+) ACT FO (A+) A. chrysantha (A-) CH (A-)
ABP gene expression in 14+2 species - Results Species LA CA LO PI CH SC MI BA CO CO SP PU FL FO FLA OW OC NV PU CHS CHI X X F3H X X X X X DFR X X X X ANS X X UF3GT ACT Pattern 1 A+ 3 2 3 A+ 1 1 A+ 4 4 2 2 1 3 A+
ABP gene expression in 16 species - Summary • 2 main patterns among A- species • 1. all genes expressed comparable to A+ species or • 2. reduction/lack of expression in the second half of the ABP • Hypotheses for • Pattern 1 - structural mutation in one or more ABP loci or side • branches of the ABP „took over“ • Pattern 2 - deficiency in regulation of expression • Convergence within the A. chrysantha clade, the A. micrantha clade and the A. coerulea clade • CHS and CHI always expressed – pleiotropy! • Amplification of ABP loci worked from DNA
A+ anthocyanins present A- anthocyanins absent R regulator ABP anthocyanin biosynthetic pathway X mutation expression Anthocyanin loss in A. pubescens 5 possible scenarios leading to anthocyanin loss:
Down the road... • Expression of regulatory loci in A. pubescens & A. formosa (EST database + phylogenetic analysis for identification of regulatory loci) • Localize ABP & regulatory loci on a genetic map (A. pubescens x A. formosa) (SNPs in EST database) Does any of them map to a QTL for spur chroma? • Sequence alleles of ABP & regulatory loci from A. pubescens and A. formosa (EST database) • Completion of the genetic map (13 microsattelite loci in an F2 population of 250 individuals) • New floral whorls in Aquilegia – Gene expression in Staminodia as compared to other whorls? (high density oligonucleotide arrays, designed by J. Borevitz, Chicago and W. Rensink, TIGR, printed by NimbleGen) • Identify and sequence BACs with loci of interest (e.g. ABP loci)