Macroevolutionary patterns of plant phenolic metabolites in the Onagraceae Jeffrey Ahern 1, Daniel...

Macroevolutionary patterns of plant phenolic metabolites in the Onagraceae

Jeffrey Ahern1, Daniel Anstett2, Marc Johnson2, Juha-Pekka Salminen1

1 University of Turku, Finland2 University of Toronto Mississauga, Canada

Variation in plant defenses

Variation in plant defenses

Tradeoffs

Defensive Syndromes (Agrawal, 2011)

Oenothera (Onagraceae)

• Evening Primroses

• 147 species in genus

• New World genus:S. America – Boreal Canada

• Herbaceous

• Annual - Perennial

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

O. elata – SEX iO. grandiflora – SEX iO. biennis – PTH iO. rhombipetala – SEX i/iiO. clelandii – PTH iiO. longituba – SEX iO. versicolor – SEX iiiO. sandiana – PTH iiiO. affinis – SEX iiiO. villaricae – PTH iiiO. laciniata – PTH iiiO. humifusa – PTH iiiO. drummondii – SEX iiiO. grandis – SEX iiiO. heterophylla – SEX iiO. berlandieri – SEX iiiO. serrulata – PTH iiO. acutissima – SEX i/iiO. acaulis – PTH iiO. gaura – PTH iO. suffulta – SEX iiO. triangulata – PTH iiO. perennis – PTH iiiO. fruticosa – SEX iiiO. rosea – PTH iiiO. speciosa – SEX ii

Oeno.specOeno.roseOeno.frut

Oeno.pereGaur.tria

Gaur.suffGaur.bien

Oeno.acauOeno.acut

Caly.serr

Caly.berlOeno.hete

Oeno.graDOeno.drumOeno.humi

Oeno.laciOeno.vill

Oeno.affiOeno.rubi

Oeno.versOeno.lonT

Oeno.clelOeno.rhom

Oeno.bien

Oeno.graFOeno.elat

0.04 0.03 0.02 0.01 0


Oeno.pereGaur.tria

Gaur.suffGaur.bien

Oeno.acauOeno.acut

Caly.serr

Caly.berlOeno.hete


Oeno.laciOeno.vill

Oeno.affiOeno.rubi

Oeno.versOeno.lonT

Oeno.clelOeno.rhom

Oeno.bien

Oeno.graFOeno.elat

0.04 0.03 0.02 0.01 0

0 0.01 0.02 0.03 0.04

Phylogenetic covariance

*

*

*

*85

51

53

**

*

85

*

*

61 *

*

** *

*

O. elata – SEX iO. grandiflora – SEX iO. biennis – PTH iO. rhombipetala – SEX i/iiO. clelandii – PTH iiO. longituba – SEX iO. versicolor – SEX iiiO. sandiana – PTH iiiO. affinis – SEX iiiO. villaricae – PTH iiiO. laciniata – PTH iiiO. humifusa – PTH iiiO. drummondii – SEX iiiO. grandis – SEX iiiO. heterophylla – SEX iiO. berlandieri – SEX iiiO. serrulata – PTH iiO. acutissima – SEX i/iiO. acaulis – PTH iiO. gaura – PTH iO. suffulta – SEX iiO. triangulata – PTH iiO. perennis – PTH iiiO. fruticosa – SEX iiiO. rosea – PTH iiiO. speciosa – SEX ii


Oeno.pereGaur.tria

Gaur.suffGaur.bien

Oeno.acauOeno.acut

Caly.serr

Caly.berlOeno.hete


Oeno.laciOeno.vill

Oeno.affiOeno.rubi

Oeno.versOeno.lonT

Oeno.clelOeno.rhom

Oeno.bien

Oeno.graFOeno.elat

0.04 0.03 0.02 0.01 0


Oeno.pereGaur.tria

Gaur.suffGaur.bien

Oeno.acauOeno.acut

Caly.serr

Caly.berlOeno.hete


Oeno.laciOeno.vill

Oeno.affiOeno.rubi

Oeno.versOeno.lonT

Oeno.clelOeno.rhom

Oeno.bien

Oeno.graFOeno.elat

0.04 0.03 0.02 0.01 0

0 0.01 0.02 0.03 0.04


*

*

*

*85

51

53

**

*

85

*

*

61 *

*

** *

*

Chemical Analysis

• Measured water soluble phenolics with UPLC-UV-MS

• Identified via UV and MS spectra• Quantified with UV

Flavonoid glycosidesKaempferol

Kaempferolglycosides

Photosynthesis(Calvin cycle)

GlycolysisOxidative pentose

phosphate pathway

Acetate/malonatepathway

Shikimate pathway

Hydrolyzable tannin pathway

Ellagitannins

Phenylpropanoid pathway

Flavonoid pathway

Caffeic acid derivatesQuercetin

Quercetinglycosides

Myricetinglycosides

Myricetin

3 Ellagitannins• Oenothein B• Oenothein A• Oxidised Oenothein A

Flavonoid Glycosides(20 different compounds)

Caffeic Acid Derivatives• Caffeoyl tartaric acid• Feruloyl tartaric acid

• 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?

Is the composition and concentrations of plant phenolics evolutionarily conserved?

O. elata – SEXO. grandiflora – SEXO. Biennis - PTHO. rhombipetala – SEXO. clelandii - PTHO. longituba – SEXO. versicolor – SEXO. sandiana - PTHO. affinis – SEXO. villaricae - PTHO. laciniata - PTHO. humifusa - PTHO. drummondii – SEXO. grandis – SEXO. heterophylla – SEXO. berlandieri – SEXO. serrulata - PTHO. acutissima – SEXO. acaulis - PTHO. gaura - PTHO. suffulta – SEXO. triangulata - PTHO. perennis - PTHO. fruticosa – SEXO. rosea - PTHO. speciosa – SEX


Oeno.pereGaur.tria

Gaur.suffGaur.bien

Oeno.acauOeno.acut

Caly.serr

Caly.berlOeno.hete


Oeno.laciOeno.vill

Oeno.affiOeno.rubi

Oeno.versOeno.lonT

Oeno.clelOeno.rhom

Oeno.bien

Oeno.graFOeno.elat

0.04 0.03 0.02 0.01 0

Oeno.spec

Oeno.rose

Oeno.frut

Oeno.pere

Gaur.tria

Gaur.suff

Gaur.bien

Oeno.acau

Oeno.acut

Caly.serr

Caly.berl

Oeno.hete

Oeno.graD

Oeno.drum

Oeno.humi

Oeno.laci

Oeno.vill

Oeno.affi

Oeno.rubi

Oeno.vers

Oeno.lonT

Oeno.clel

Oeno.rhom

Oeno.bien

Oeno.graF

Oeno.elat

0.04 0.03 0.02 0.01 0

0 0.01 0.02 0.03 0.04


Is the composition and concentrations of plant phenolics evolutionarily conserved?

Trait Blomberg's K

No. of peaks at 280 nm 0.35

No. of peaks at 349 nm 0.45

Oenothein B 0.22

Oenothein A 0.74

Oxidised Oenothein A 0.39

Total ellagitannins 0.53

Total caffeic acid derivatives 0.34

Quercetin Glycoside mw 464 0.50

Total kaempferol glycosides 0.53

Total quercetin glycosides 0.34

Totel other flavonoids 0.47

Total flavonoid glycosides 0.37PC1 0.49

PC2 0.36

O. elata – SEXO. grandiflora – SEXO. Biennis - PTHO. rhombipetala – SEXO. clelandii - PTHO. longituba – SEXO. versicolor – SEXO. sandiana - PTHO. affinis – SEXO. villaricae - PTHO. laciniata - PTHO. humifusa - PTHO. drummondii – SEXO. grandis – SEXO. heterophylla – SEXO. berlandieri – SEXO. serrulata - PTHO. acutissima – SEXO. acaulis - PTHO. gaura - PTHO. suffulta – SEXO. triangulata - PTHO. perennis - PTHO. fruticosa – SEXO. rosea - PTHO. speciosa – SEX


Oeno.pereGaur.tria

Gaur.suffGaur.bien

Oeno.acauOeno.acut

Caly.serr

Caly.berlOeno.hete


Oeno.laciOeno.vill

Oeno.affiOeno.rubi

Oeno.versOeno.lonT

Oeno.clelOeno.rhom

Oeno.bien

Oeno.graFOeno.elat

0.04 0.03 0.02 0.01 0

Oeno.spec

Oeno.rose

Oeno.frut

Oeno.pere

Gaur.tria

Gaur.suff

Gaur.bien

Oeno.acau

Oeno.acut

Caly.serr

Caly.berl

Oeno.hete

Oeno.graD

Oeno.drum

Oeno.humi

Oeno.laci

Oeno.vill

Oeno.affi

Oeno.rubi

Oeno.vers

Oeno.lonT

Oeno.clel

Oeno.rhom

Oeno.bien

Oeno.graF

Oeno.elat

0.04 0.03 0.02 0.01 0

0 0.01 0.02 0.03 0.04


To series of traits positively or negatively covary?

peaks 349 oeno B oeno A ox-oeno

A total ET total CA FG 4-querc

total querc

total kaemp other FG total FG toughnes

s % H20 SLA PPC trichomes

peaks 280 0.39 -0.19 -0.08 -0.56 -0.54 -0.17 0.04 -0.07 -0.01 0.24 0.31 0.03 0.45 0.19 -0.45 -0.27

peaks 349 -0.21 0.18 -0.22 -0.25 -0.43 -0.14 -0.12 -0.02 0.36 0.6 -0.06 -0.17 -0.18 -0.17 -0.16

oeno B -0.13 0.01 0.54 0.25 0.3 0.16 -0.05 -0.29 -0.34 -0.17 0.08 0.15 0.29 0.23

oeno A 0.06 0.17 -0.11 0.05 -0.13 0.12 0.08 -0.17 0.59 -0.62 -0.63 0.58 -0.06ox-oeno

A 0.53 0.04 -0.21 -0.16 0.05 -0.2 -0.5 0.45 -0.41 -0.24 0.53 -0.09

total ET -0.03 -0.03 -0.23 0.02 -0.4 -0.62 0.29 -0.29 -0.18 0.88 0.05

total CA 0.26 0.42 -0.3 -0.08 -0.25 -0.38 -0.04 0.5 0.12 0.27

FG 4-querc 0.86 -0.33 -0.19 0.09 -0.19 0.43 0.22 -0.06 0.16

total querc -0.39 -0.13 0.28 -0.33 0.39 0.35 -0.23 0.42

total kaemp -0.24 0.22 0.3 -0.19 -0.38 -0.11 -0.01

other FG 0.34 -0.11 -0.3 -0.11 -0.17 -0.05

total FG -0.31 0.07 -0.08 -0.65 0.2toughnes

s -0.32 -0.66 0.44 -0.18

% H20 0.63 -0.6 -0.05

SLA -0.36 -0.1

PPC 0.03

A

B

C

r=-0.54(-0.84,-0.17)

r=0.60(0.25,0.83)

r=-0.62(0.25,0.83)

0 50 100 150

75808590

Total ellagitannins (mg/g)

No. peaks at 280nm

0 5 10 15

10

15

20

Total flavonoids (mg/g)

No. peaks at 349nm

0 50 100 150

05

10

15


Total flavonoids (m

g/g)

0 50 100 150

75808590


No. peaks at 280nm

0 5 10 15

10

15

20


No. peaks at 349nm

0 50 100 150

05

10

15


Total flavonoids (m

g/g)

0 50 100 150

75808590


No. peaks at 280nm

0 5 10 15

101520


No. peaks at 349nm

0 50 100 150

05

1015


Total flavonoids (m

g/g)

A

B

C

r=-0.54(-0.84,-0.17)

r=0.60(0.25,0.83)

r=-0.62(0.25,0.83)

0 50 100 150

75808590


No. peaks at 280nm

0 5 10 15

10

15

20


No. peaks at 349nm

0 50 100 150

05

10

15


Total flavonoids (m

g/g)

0 50 100 150

75808590


No. peaks at 280nm

0 5 10 15

10

15

20


No. peaks at 349nm

0 50 100 150

05

10

15


Total flavonoids (m

g/g)

0 50 100 150

75808590


No. peaks at 280nm

0 5 10 15

101520


No. peaks at 349nm

0 50 100 150

05

1015


Total flavonoids (m

g/g)

A

B

C

r=-0.54(-0.84,-0.17)

r=0.60(0.25,0.83)

r=-0.62(0.25,0.83)

0 50 100 150

75808590


No. peaks at 280nm

0 5 10 15

10

15

20


No. peaks at 349nm

0 50 100 150

05

10

15


Total flavonoids (m

g/g)

0 50 100 150

75808590


No. peaks at 280nm

0 5 10 15

10

15

20


No. peaks at 349nm

0 50 100 150

05

10

15


Total flavonoids (m

g/g)

0 50 100 150

75808590


No. peaks at 280nm

0 5 10 15

101520


No. peaks at 349nm

0 50 100 150

05

1015


Total flavonoids (m

g/g)

To series of traits positively or negatively covary?

• Hierarchical cluster analysis

• Identified associations between groups of metabolites

Do individual traits or suites of traits predict resistance to herbivores?


Cate

rpill

ar su

rviva

lBe

etle

her

bivo

ry(c

m2 )

Total kaempferol (mg/g)

No. flavonoid compounds

Mite

surv

ival

A

B

C

slopeOU = -0.003, P = 0.039, R2 = 0.17

slopeOU = 0.024, P = 0.005, R2 = 0.28

slopeOU = 0.021, P = 0.028, R2 = 0.25

0 20 40 60 80 100 120 140

0.00.20.40.60.81.0


Caterpillar survival

10 15 20

0.20

.40

.60

.81

.0


Mite survival

0 1 2 3 4

0.000.100.200.30


Beetle herbivory (cm

^2)

0 20 40 60 80 100 120 140

0.00

.20

.40

.60

.81

.0



10 15 20

0.20

.40.60.81

.0


Mite survival

0 1 2 3 4

0.000

.100

.200

.30



^2)

0 20 40 60 80 100 120 140

0.00.20.40.60.81.0



10 15 20

0.20

.40

.60

.81

.0


Mite survival

0 1 2 3 4

0.000.100.200.30



^2)


Cate

rpill

ar su

rviva

lBe

etle

her

bivo

ry(c

m2 )



Mite

surv

ival

A

B

C

slopeOU = -0.003, P = 0.039, R2 = 0.17

slopeOU = 0.024, P = 0.005, R2 = 0.28

slopeOU = 0.021, P = 0.028, R2 = 0.25

0 20 40 60 80 100 120 140

0.00.20.40.60.81.0



10 15 20

0.20

.40

.60

.81

.0


Mite survival

0 1 2 3 4

0.000.100.200.30



^2)

0 20 40 60 80 100 120 140

0.00

.20

.40

.60

.81

.0



10 15 20

0.20

.40.60.81

.0


Mite survival

0 1 2 3 4

0.000

.100

.200

.30



^2)

0 20 40 60 80 100 120 140

0.00.20.40.60.81.0



10 15 20

0.20

.40

.60

.81

.0


Mite survival

0 1 2 3 4

0.000.100.200.30



^2)


Cate

rpill

ar su

rviva

lBe

etle

her

bivo

ry(c

m2 )



Mite

surv

ival

A

B

C

slopeOU = -0.003, P = 0.039, R2 = 0.17

slopeOU = 0.024, P = 0.005, R2 = 0.28

slopeOU = 0.021, P = 0.028, R2 = 0.25

0 20 40 60 80 100 120 140

0.00.20.40.60.81.0



10 15 20

0.20

.40

.60

.81

.0

No. flavonoid compoundsM

ite survival

0 1 2 3 4

0.000.100.200.30



^2)

0 20 40 60 80 100 120 140

0.00

.20

.40

.60

.81

.0



10 15 20

0.20

.40.60.81

.0


Mite survival

0 1 2 3 4

0.000

.100

.200

.30



^2)

0 20 40 60 80 100 120 140

0.00.20.40.60.81.0



10 15 20

0.20

.40

.60

.81

.0


Mite survival

0 1 2 3 4

0.000.100.200.30



^2)

Beet armywormSpodoptera exigua

Two spotted spidermiteTetranychus urticae

“Specialist” flea beetleAltica foliacea

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

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?

76 Oenothera species, plus additional in Onagraceae

Examining leaf, flower, and fruit tissues independently.

Analytic Approach

• Prelim Analysis• Identification of target

metabolites• Development and

selection of MRM methods

Detection by DAD

Dimer-specific MRM

Heptamer-specific MRM

Pentamer-specific MRM

Trimer-specific MRM

Tetramer-specific MRM

OOH

O

OO

O

O

O

O

HO

HO

HO

HO

OH

O

OH

OH

OH

OH

OH

O

O

HOOH

OH

HO

OO

O

O

O

O

O

HO

HO OH

O

O

OH

OH

OH

OH

HO

HO

Oenothein B

OOH

O

OO

O

O

O

O

HO

HO

HO

HO

O

O

OH

OH

OH

OH

OH

O

O

HOOH

OH

HO

OO

O

O

O

O

O

HO

HO OH

O

O

OH

OH

OH

OH

HO

HO

Oenothein A

OH

OH

OH

OH

OH

OH

OHO

OO

O

O

O

O

O

HO

HO OH

O

HO

HO

OH

Time-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

8.0e-1

1.0

1.2

1.4

1.6

1.8

130521 Oenothera 183 16: Diode Array 280

Range: 2.1282.67254.3

3.21227.3

2.88222.3

2.96222.3

4.31265.3

3.59224.3

3.53222.3

3.66222.3 3.98

222.33.85222.3

DimerOenothein B

TrimerOenothein A Oxidized Trimer

Time-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

8.0e-1

1.0

1.2

1.4

1.6

1.8


Range: 2.1363.21258.3

2.94222.32.35

251.33.01222.3

3.58224.3

3.53223.3

3.32223.3

3.63223.3

3.98255.33.85

223.3

4.31265.3

4.50266.3

Time-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

8.0e-1

1.0

1.2


Range: 1.4642.88234.3

4.46224.3

3.98255.3

2.95222.3

3.48224.3

3.14223.3

3.21223.3

3.89225.3

4.13224.3

OOH

O

OO

O

O

O

O

HO

HO

HO

HO

O

O

OH

OH

OH

OH

OH

O

O

HOOH

OH

HO

OO

O

O

O

O

O

HO

HO OH

O

O

OH

OH

OH

OH

HO

HO

Oenothein A

OH

OH

OH

OH

OH

OH

OHO

OO

O

O

O

O

O

HO

HO OH

O

HO

HO

OH

Trait λ pTotal Ellagitannins (mg/g) 0.63 0.06Proportion Dimer (Oenothein B) 0.48 0.15Proportion Trimer (Oenothein A) 0.61 <0.01Proportion Oxidized Trimer 0.72 <0.01Caffeoyl tartaric acid 0.55 0.04Total Phenolics (Folin-Ciocalteu) 0.32 0.61Pro-Oxidant Phenolics (pH 10) 0.54 0.25Protein Precipitation Capacity (RDA) 0.38 0.23

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

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