1. Macroevolutionary patterns of plant phenolic metabolites in the Onagraceae Jeffrey Ahern 1, Daniel Anstett 2, Marc Johnson 2, Juha-Pekka Salminen 1 1 University of Turku, Finland 2 University of Toronto Mississauga, Canada

2. Variation in plant defenses

3. Tradeoffs Defensive Syndromes (Agrawal, 2011)

4. Oenothera (Onagraceae) Evening Primroses 147 species in genus New World genus: S. America – Boreal Canada Herbaceous Annual - Perennial

5. Plant Samples Used 26 species of Oenothera Grew in growth chambers Performed Excised-leaf bioassays to examine resistance Collected leaf material for secondary metabolite analysis

6. Chemical Analysis Measured water soluble phenolics with UPLC-UV-MS Identified via UV and MS spectra Quantified with UV 3 Ellagitannins Oenothein B Oenothein A Oxidised Oenothein A Flavonoid Glycosides (20 different compounds) Caffeic Acid Derivatives Caffeoyl tartaric acid Feruloyl tartaric acid

7. Is the composition and concentrations of plant phenolics evolutionarily conserved? Do traits covary with one another – Negative covariation – tradeoffs in defense strategies – Positive covariation – defensive syndromes hypothesis Do individual traits or suites of traits predict resistance to herbivores?

8. Is the composition and concentrations of plant phenolics evolutionarily conserved?

9. TraitBlomberg's K No. of peaks at 280 nm0.35 No. of peaks at 349 nm0.45 Oenothein B0.22 Oenothein A0.74 Oxidised Oenothein A0.39 Total ellagitannins0.53 Total caffeic acid derivatives0.34 Quercetin Glycoside mw 4640.50 Total kaempferol glycosides0.53 Total quercetin glycosides0.34 Totel other flavonoids0.47 Total flavonoid glycosides0.37 PC10.49 PC20.36

10. To series of traits positively or negatively covary? peaks 349 oeno Boeno A ox-oeno A total ETtotal CA FG 4- querc total querc total kaemp other FGtotal FG toughnes s % H 2 0SLAPPC trichome s peaks 280 0.39-0.19-0.08-0.56-0.54-0.170.04-0.07-0.010.240.310.030.450.19-0.45-0.27 peaks 349 -0.210.18-0.22-0.25-0.43-0.14-0.12-0.020.360.6-0.06-0.17-0.18-0.17-0.16 oeno B-0.130.010.540.250.30.16-0.05-0.29-0.34-0.170.080.150.290.23 oeno A0.060.17-0.110.05-0.130.120.08-0.170.59-0.62-0.630.58-0.06 ox-oeno A 0.530.04-0.21-0.160.05-0.2-0.50.45-0.41-0.240.53-0.09 total ET-0.03 -0.230.02-0.4-0.620.29-0.29-0.180.880.05 total CA0.260.42-0.3-0.08-0.25-0.38-0.040.50.120.27 FG 4- querc 0.86-0.33-0.190.09-0.190.430.22-0.060.16 total querc -0.39-0.130.28-0.330.390.35-0.230.42 total kaemp -0.240.220.3-0.19-0.38-0.11-0.01 other FG0.34-0.11-0.3-0.11-0.17-0.05 total FG-0.310.07-0.08-0.650.2 toughnes s -0.32-0.660.44-0.18 % H 2 00.63-0.6-0.05 SLA-0.36-0.1 PPC0.03

11. To series of traits positively or negatively covary? Hierarchical cluster analysis Identified associations between groups of metabolites

15. Do individual traits or suites of traits predict resistance to herbivores? Beet armyworm Spodoptera exigua Two spotted spidermite Tetranychus urticae “Specialist” flea beetle Altica foliacea

16. Summary So Far Plant phenolics are not highly evolutionary conserved, suggesting they are evolutionary labile Individual traits predict resistance to herbivores, but not really apparent with suites

17. Summary So Far Is the composition and concentrations of plant phenolics evolutionarily conserved? Do traits covary with one another – Negative covariation – tradeoffs in defense strategies – Positive covariation – defensive syndromes hypothesis Do individual traits or suites of traits predict resistance to herbivores?

18. 76 Oenothera species, plus additional in Onagraceae Examining leaf, flower, and fruit tissues independently.

19. Analytic Approach Prelim Analysis Identification of target metabolites Development and selection of MRM methods

20. Detection by DAD Dimer-specific MRM Heptamer-specific MRM Pentamer-specific MRM Trimer-specific MRM Tetramer-specific MRM

21. Dimer Oenothein B Trimer Oenothein A Oxidized Trimer

22. Traitλp Total Ellagitannins (mg/g)0.630.06 Proportion Dimer (Oenothein B)0.480.15 Proportion Trimer (Oenothein A)0.61<0.01 Proportion Oxidized Trimer0.72<0.01 Caffeoyl tartaric acid0.550.04 Total Phenolics (Folin-Ciocalteu)0.320.61 Pro-Oxidant Phenolics (pH 10)0.540.25 Protein Precipitation Capacity (RDA)0.380.23

23. Acknowledgements and Funding Juha-Pekka Salminen Daniel Ansettt Marc Johnson Anthony R. Ives Anne Koivuniemi Funding Academy of Finland NSERC Canada Canadian Foundation for Innovation University of Toronto

24. Questions?