macroevolutionary patterns of plant phenolic metabolites in the onagraceae jeffrey ahern 1, daniel...
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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.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
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.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.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
0 0.01 0.02 0.03 0.04
Phylogenetic covariance
*
*
*
*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.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.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
Phylogenetic covariance
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.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.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
Phylogenetic covariance
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 ellagitannins (mg/g)
Total flavonoids (m
g/g)
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 ellagitannins (mg/g)
Total flavonoids (m
g/g)
0 50 100 150
75808590
Total ellagitannins (mg/g)
No. peaks at 280nm
0 5 10 15
101520
Total flavonoids (mg/g)
No. peaks at 349nm
0 50 100 150
05
1015
Total ellagitannins (mg/g)
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
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 ellagitannins (mg/g)
Total flavonoids (m
g/g)
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 ellagitannins (mg/g)
Total flavonoids (m
g/g)
0 50 100 150
75808590
Total ellagitannins (mg/g)
No. peaks at 280nm
0 5 10 15
101520
Total flavonoids (mg/g)
No. peaks at 349nm
0 50 100 150
05
1015
Total ellagitannins (mg/g)
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
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 ellagitannins (mg/g)
Total flavonoids (m
g/g)
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 ellagitannins (mg/g)
Total flavonoids (m
g/g)
0 50 100 150
75808590
Total ellagitannins (mg/g)
No. peaks at 280nm
0 5 10 15
101520
Total flavonoids (mg/g)
No. peaks at 349nm
0 50 100 150
05
1015
Total ellagitannins (mg/g)
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?
Total ellagitannins (mg/g)
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
Total ellagitannins (mg/g)
Caterpillar survival
10 15 20
0.20
.40
.60
.81
.0
No. flavonoid compounds
Mite survival
0 1 2 3 4
0.000.100.200.30
Total kaempferol (mg/g)
Beetle herbivory (cm
^2)
0 20 40 60 80 100 120 140
0.00
.20
.40
.60
.81
.0
Total ellagitannins (mg/g)
Caterpillar survival
10 15 20
0.20
.40.60.81
.0
No. flavonoid compounds
Mite survival
0 1 2 3 4
0.000
.100
.200
.30
Total kaempferol (mg/g)
Beetle herbivory (cm
^2)
0 20 40 60 80 100 120 140
0.00.20.40.60.81.0
Total ellagitannins (mg/g)
Caterpillar survival
10 15 20
0.20
.40
.60
.81
.0
No. flavonoid compounds
Mite survival
0 1 2 3 4
0.000.100.200.30
Total kaempferol (mg/g)
Beetle herbivory (cm
^2)
Total ellagitannins (mg/g)
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
Total ellagitannins (mg/g)
Caterpillar survival
10 15 20
0.20
.40
.60
.81
.0
No. flavonoid compounds
Mite survival
0 1 2 3 4
0.000.100.200.30
Total kaempferol (mg/g)
Beetle herbivory (cm
^2)
0 20 40 60 80 100 120 140
0.00
.20
.40
.60
.81
.0
Total ellagitannins (mg/g)
Caterpillar survival
10 15 20
0.20
.40.60.81
.0
No. flavonoid compounds
Mite survival
0 1 2 3 4
0.000
.100
.200
.30
Total kaempferol (mg/g)
Beetle herbivory (cm
^2)
0 20 40 60 80 100 120 140
0.00.20.40.60.81.0
Total ellagitannins (mg/g)
Caterpillar survival
10 15 20
0.20
.40
.60
.81
.0
No. flavonoid compounds
Mite survival
0 1 2 3 4
0.000.100.200.30
Total kaempferol (mg/g)
Beetle herbivory (cm
^2)
Total ellagitannins (mg/g)
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
Total ellagitannins (mg/g)
Caterpillar survival
10 15 20
0.20
.40
.60
.81
.0
No. flavonoid compoundsM
ite survival
0 1 2 3 4
0.000.100.200.30
Total kaempferol (mg/g)
Beetle herbivory (cm
^2)
0 20 40 60 80 100 120 140
0.00
.20
.40
.60
.81
.0
Total ellagitannins (mg/g)
Caterpillar survival
10 15 20
0.20
.40.60.81
.0
No. flavonoid compounds
Mite survival
0 1 2 3 4
0.000
.100
.200
.30
Total kaempferol (mg/g)
Beetle herbivory (cm
^2)
0 20 40 60 80 100 120 140
0.00.20.40.60.81.0
Total ellagitannins (mg/g)
Caterpillar survival
10 15 20
0.20
.40
.60
.81
.0
No. flavonoid compounds
Mite survival
0 1 2 3 4
0.000.100.200.30
Total kaempferol (mg/g)
Beetle herbivory (cm
^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
130516 Oenothera 140 16: Diode Array 280
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
130521 Oenothera 240 16: Diode Array 280
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
Questions?