“Transcriptional & Biochemical Signatures Of Speciation In NZ Alpine Cress”

1. “Transcriptional & Biochemical Signatures Of Speciation In NZ Alpine Cress” Claudia Voelckel Palmy, May 07

2. Expression & Biochemical Studies in Pachycladon greywacke 1. Do Arabidopsis arrays work with Pachycladon? P. fastigiata (1,2) P. sp. P. stellata 2. Within-clade experiment – What differences evolved between two greywacke species? P. enysii (1,2,3) P. latisiliqua schist P. wallii P. novae-zeal. (1,3) 3. Across-clade experiment – Is soil adaptation driving schist/greywacke split? generalist P. radicata P. cheesemanii P. exilis Beringia bursifolia (after Heenan & Mitchell 2003)

3. Within-Clade Experiment – What Expression & Biochemical Differences Evolved Between 2 Greywacke Species? greywacke P. fastigiata monocarpic perennials with serrate leaves and terminal inflorescences on greywacke Alpine (1485m) High alpine (1885m) Glabrous Hairy P. sp. P. stellata P. enysii P. latisiliqua schist P. wallii P. novae-zeal. generalist P. radicata P. cheesemanii P. exilis P. fastigiata P. enysii Beringia bursifolia (after Heenan & Mitchell 2003)

4. Within-Clade Experiment – Sampling F1 E1 F2 E2 F3 E3

5. cy3 cy5 Within-Clade Experiment – Gene Expression 1. Global Expression Patterns Goal: Measure inter-specific differences How: Field experiment – 3 pop/species (F1, F2, F3, E1, E2, E3; 15 plants/pop) Hybridization scheme: 12 arrays • 2. Individual Gene Expression • Goal: • Separate phenotypic plasticity from genetic diversity as a cause for inter-specific differences • How: • Common garden experiment • e.g. grow 30 descendants of F1, F2, F3, E1, E2, E3 in the glasshouse and measure expression of genes identified in step 1 using qPCR (& arrays/Solexa?) • Time course? • Different tissues? F3 F2 F3 F1 F2 F1 E1 E2 E2 E3 E3 E1 • Status: planning stage • seeds available in September? • from which populations? • where to do the experiment? Status: done

6. Expression Analysis – 111 PF vs 147 PE genes • different approaches to data analysis (background subtraction, normalization, data trimming) • two channel analysis evaluates only species differences • single channel analysis evaluates population differences • the 111 vs 147 genes are common to all analyses – most conservative list of differentially expressed genes FO FM FT EB EP EH

7. Expression Analysis – 111 PF vs 147 PE genes

8. Expression Analysis – Population-level analysis Example list (1 of 7) • Preliminary conclusions: • P. enysii populations with more distinct gene expression • among-population differences higher in P. enysii • within-species differences lower than between species differences

9. Glucosinolate Analysis –Population & Species Differences 3MSOP 4MSOB Allyl • on average 15 samples/pop • 90 total • HPLC (MPI Chemical Ecology, Jena) detected 12 individual compounds • significant differences between species and populations 5MSOP 3Butenyl 6MSOH 3MTP 7MSOH 5Pentenyl 4MTB 8MSOO 4MOI3M

10. Glucosinolate Analysis – Clustering identifies 5 Profiles 1 11x EP, 1x EH • except for FM each population dominated by one profile • due to local adaptation? • genetic diversity or phenotypic plasticity? 2 14x FO, 8x FM 3 13x EB 4 14x EH, 5x EP, 2x EB 5 16x FT, 6x FM

11. Glucosinolate Analysis – C3 vs C4 & MS vs AL C3 Alkenyl C4 Methylsulfinyl

12. Conclusions transcriptional + biochemical data • P. enysii populations more heterogeneous in expression and glucosinolate profiles than P. fastigiata populations – due to genetic structure or environmental heterogeneity/phenotypic plasticity? • Higher expression of AOP2 and MAM1 in P. enysii consistent with higher C4- and Alkenyl- glucosinolate levels in P. enysii • Higher expression of F3’H and FAH1 predict higher levels of quercetin and sinapates (flavanoid analysis needed!) Next: • Gene Ontology Annotation of differentially expressed genes • Greenhouse studies to separate G from E • Molecular evolution studies with candidate genes (AOP2, MAM1, F3’H, FAH1..) • Characterizing field sites in abiotic (microclimate) and biotic (e.g. herbivory) conditions

13. Across-Clade Experiment – Is Soil Adaptation Driving Schist/Greywacke Split? greywacke lobed leaves polycarpic lateral inflorescences schist P. fastigiata P. sp. P. stellata P. enysii P. latisiliqua schist P. wallii P. novae-zeal. generalist P. radicata P. cheesemanii P. exilis P. novae-zealandiae Beringia bursifolia (after Heenan & Mitchell 2003)

14. Across-Clade Experiment – Overview Goal: Monitor fitness & expression patterns of a schist species (P. novae-zealandiae) on both schist and greywacke How: Jan07 n_sn_g 100 pl/group Jan08 n_sn_g 0 pl/group • Measure growth, physiology, secondary metabolism, gene expression, dry mass, male and female fitness over time • Harvest subsets of each group at different time points for above analyzes • Status: • 100 plants per soil type potted • one seedling harvest done, next harvest soon?

15. Other ideas/plans • Gene Ontology Analysis of differentially expressed genes (with Leslie) • Flavonoid variation in field samples (??) • Schist/greywacke soil analyses • (Landcare Palmy) • Glucosinolate data for reciprocal transplant with P. novae-zealandiae (MPI Jena) • Scanning electron microscopy of trichomes • (Massey)

16. Acknowledgements Pachycladon folks - Pete, Simon, Vaughan, Trish, Richard… Landcare botanists – Peter Heenan & Kerry Ford Array specialist from HortResearch – Bart Janssen Sponsors – Marsden & Humboldt Foundation You!