Transcriptional & Biochemical Signatures Of Speciation In NZ Alpine Cress Palmy, May 07 Claudia Voelckel 2. Within-clade experiment What differences evolved between two greywacke species? 3. Across-clade experiment Is soil adaptation driving schist/greywacke split? Expression & Biochemical Studies in Pachycladon Beringia bursifolia P. novae-zeal. (1,3) P. wallii P. radicata P. sp. P. fastigiata (1,2) P. latisiliqua P. enysii (1,2,3) P. stellata P. exilis P. cheesemanii greywacke schist generalist 1. Do Arabidopsis arrays work with Pachycladon? (after Heenan & Mitchell 2003) P. fastigiataP. enysii monocarpic perennials with serrate leaves and terminal inflorescences on greywacke Alpine (1485m) High alpine (1885m) Glabrous Hairy Within-Clade Experiment What Expression & Biochemical Differences Evolved Between 2 Greywacke Species? Beringia bursifolia P. novae-zeal. P. wallii P. radicata P. sp. P. fastigiata P. latisiliqua P. enysii P. stellata P. exilis P. cheesemanii greywacke schist generalist (after Heenan & Mitchell 2003) Within-Clade Experiment Sampling F1 F3 F2 E1 E3 E2 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 Within-Clade Experiment Gene Expression F1 E1 F2 E3 F1 E2 F2 E2 F3 E3 F3 E1 cy3cy5 Status: done 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? Status: planning stage seeds available in September? from which populations? where to do the experiment? 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 FOFMFTEBEPEH Expression Analysis 111 PF vs 147 PE genes 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 3MSOP4MSOBAllyl 5MSOP3Butenyl6MSOH 3MTP7MSOH5Pentenyl 4MTB8MSOO4MOI3M Glucosinolate Analysis Population & Species Differences on average 15 samples/pop 90 total HPLC (MPI Chemical Ecology, Jena) detected 12 individual compounds significant differences between species and populations 11x EP, 1x EH 14x FO, 8x FM 13x EB 14x EH, 5x EP, 2x EB 16x FT, 6x FM Glucosinolate Analysis Clustering identifies 5 Profiles except for FM each population dominated by one profile due to local adaptation? genetic diversity or phenotypic plasticity? C3AlkenylC4Methylsulfinyl Glucosinolate Analysis C3 vs C4 & MS vs AL 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 F3H and FAH1 predict higher levels of quercetin and sinapates (flavanoid analysis needed!) Gene Ontology Annotation of differentially expressed genes Greenhouse studies to separate G from E Molecular evolution studies with candidate genes (AOP2, MAM1, F3H, FAH1..) Characterizing field sites in abiotic (microclimate) and biotic (e.g. herbivory) conditions Next: P. novae- zealandiae Across-Clade Experiment Is Soil Adaptation Driving Schist/Greywacke Split? Beringia bursifolia P. novae-zeal. P. wallii P. radicata P. sp. P. fastigiata P. latisiliqua P. enysii P. stellata P. exilis P. cheesemanii greywacke schist generalist lobed leaves polycarpic lateral inflorescences schist (after Heenan & Mitchell 2003) Status: 100 plants per soil type potted one seedling harvest done, next harvest soon? Goal: Monitor fitness & expression patterns of a schist species (P. novae-zealandiae) on both schist and greywacke How: Across-Clade Experiment Overview Jan07n_sn_g100 pl/group Jan08n_sn_g0 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 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) Other ideas/plans 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!