New Phytologist Supporting Information The betrayed ...

New Phytologist Supporting Information

The betrayed thief – the extraordinary strategy of Aristolochia rotunda to deceive its pollinators Birgit Oelschlägel, Matthias Nuss, Michael von Tschirnhaus, Claudia Pätzold, Christoph Neinhuis, Stefan Dötterl and Stefan Wanke Article acceptance date: 6 November 2014

The following supporting information is available for this article: Table S1 Pherobase survey on chemical substances reported for true bugs of the family Miridae Table S2 Composition of headspace scent samples Table S3 Components used as references in scent analyses and for bioassays Table S4 Amount of EAD-active components Table S5 Attractiveness of synthetic scent mixtures and Heteroptera to pollinators Table S6 Attractiveness of synthetic scent mixture and single components thereof to pollinators

1

Table S1 Pherobase survey on chemical substances reported for true bugs of the family Miridae

Aliphatic esters

Aliphatic alcohols Aliphatic aldehydes Aliphatic ketones other

(E)-2-Butenyl butyrate; Butyl acetate; Butyl butyrate; Ethyl butyrate; Ethyl hexanoate; Ethyl myristate; Ethyl-2-hexenoate; Heptyl acetate; Heptyl butyrate*; Heptyl hexanoate; (E)-2-Hexenyl acetate; (Z)-3-Hexenyl acetate; (E)2-Hexenyl butyrate*; (Z)-3-Hexenyl butyrate*; (E)-2-Hexenyl hexanoate*; (E)2-Hexenyl (E)-2-hexenoate; (E)-2-Hexenyl pentanoate; Hexyl acetate; Hexyl (E)-2-butenoate; Hexyl butyrate*; Hexyl hexanoate*; Hexyl (E)-2-hexenoate; Hexyl propionate; (E)-2-Octenyl acetate; (Z)-3-Octenyl acetate; (E)-2-Octenyl butyrate; (E)-2-Octenyl hexanoate; Octyl acetate; Octyl butyrate*; Octyl hexanoate; Pentyl butyrate; Pentyl hexanoate 1-Hexanol; (E)-2-Hexen-1-ol; (Z)-3-Hexen-1-ol; (E)-2-Octen-1-ol; (Z)-3-Octen1-ol 2,4-Dimethyl pentanal; (E)-2-Heptenal; (E)-2-Hexenal; Nonanal; Octanal; (E)-2Octenal; (E)-4-Oxo-2-hexenal 2-Hexanone; 3-Hexanone Benzothiazole; γ-Caprolactone; Indole; Methyl salicylate; Phenylacetaldehyde

The table comprises altogether 51 chemical components described from 12 mirid species of 10 genera (pherobase.com, 03/26/2012). Components are sorted alphabetically within their respective substance class. Components co-occurring in A. rotunda flower scent (see Table S2) are highlighted in bold, asterisks indicate components being EAD-active in T. ruficeps.

2

Table S2 Composition of headspace scent samples

1♂

2♂

1♂

2♀

1♂2♀

1♂

s

1♂

1488 1532 1542

s

29 ♀ flowers*

Unknown (MS: 69, 39, 41, 55, 83, 111) Unknown (MS: 67, 57, 82, 41, 123, 97) Hexyl valerate

2

24 ♀ ,✝ flowers*

1472 1472 1478 1481

a, s 1, l 4, a, s 4, a, s s

41 ♀ flowers

Hexyl 2-methylbutyrate Unknown (MS: 83, 57, 55, 39, 41, 67,) Hexyl (E)-2-butenoate (E)-2-Hexenyl valerate / isovalerate (MS: 57, 85, 39, 41, 67, 100)

a, s

38 ♀ flowers

1465

b, l, s

X. quadratus

P. gracilicornis

25 ♀ flowers

(Z)-3-Hexenyl valerate / isovalerate (MS: 67, 57, 82, 41, 39, 103)

b, l, s s s

C. ater

50 ♀ flowers

1024 1097 1112 1124 1129 1174 1254 1271 1331 1333 1340 1341 1380 1393 1397 1405 1408 1431 1436

Lygaeidae N. elongata

36 ♀ flowers

Aliphatic esters Unknown (MS: 43, 71, 89, 117, 56, 103) Butyl butyrate (Z)-3-Hexen-1-ol acetate Hexyl acetate (E)-2-Hexen-1-ol acetate Butyl (E)-2-butenoate Pentyl butyrate Hexyl propionate (Z)-3-Hexenyl isobutyrate Unknown (MS: 43, 41, 89, 39, 69, 56) Hexyl isobutyrate (E)-2-Hexenyl isobutyrate Unknown (MS: 41, 69, 39, 87, 56, 97) (Z)-3-Hexenyl butyrate (E)-3-Hexenyl butyrate Hexyl butyrate (E)-2-Hexenyl butyrate (E)-2-Octenyl acetate + Octyl acetate tent. Hexyl (Z)-2-butenoate (MS: 69, 86, 39, 41, 87, 56)

Content / Comments

Component

Retention time (sec)

Miridae A. rotunda

tr 0.2 0.7 0.1 tr tr tr 0.6 0.1 tr tr tr 2.7 0.5 0.2 -

tr 0.1 0.5 0.1 tr tr tr 0.6 0.2 tr tr tr 3.1 1.5 0.1 -

0.1 0.1 tr tr tr 0.2 0.1 tr tr 0.9 0.5 -

0.2 0.7 0.3 tr tr 0.6 0.3 tr 0.1 4.4 1.8 -

0.1 0.1 0.1 0.1 tr 1.1 0.6 0.1 0.2 17.9 15.2 -

0.1 tr 0.1 0.1 tr tr 0.0 0.1 0.3 0.1 0.1 0.2 13.2 23.0 0.1 tr

tr 0.1 tr tr tr 0.1 tr tr tr 3.1 1.5 tr tr

0.6 tr tr tr tr 22.5 1.9 -

0.5 0.2 tr tr 0.1 45.6 3.3 -

0.6 tr tr tr 0.1 26.6 1.9 -

0.5 tr tr 0.1 1.9 -

0.1 tr -

tr tr -

0.1 tr -

0.1

0.1

tr

0.1

0.1

tr

tr

-

-

-

-

-

-

-

0.9 1.9 0.2 0.1

0.8 1.6 0.1 tr

0.1 1.1 tr tr

0.3 2.9 0.2 0.1

5.5 1.1 0.1 0.1

1.8 0.5 0.1

1.0 0.4 tr

tr -

tr -

tr -

-

-

-

-

0.1 tr -

tr tr -

0.1 -

0.1 -

0.2 -

1.2 0.1

0.7 tr

-

-

-

-

-

-

-

3

2♀

1♂2♀

1♂

s

1♂

1, l, s

2♂

1

1♂

l, s

1♂

884 1950

3, s

29 ♀ flowers*

877 883

1


Aliphatic alcohols (E)-2-Hexenol (Z)-3-Hexen-1-ol + (E)-2-Hexen-1-ol + 1Hexanol 1-Hexanol tent. 1-Hexadecanol (MS: 43, 55, 69, 83, 97, 111)

1921 1928 1929 1952 2008 2013 2043

3, s 1, s 4, a, s 2, s

41 ♀ flowers

1673 1677 1681 1682 1707 1747 1755 1786 1803 1832 1875

38 ♀ flowers

Geranyl acetate Hexyl hexoate (E)-2-Hexenyl hexanoate Octyl butyrate Decyl acetate Hexyl (E)-2-hexenoate (E)-2-Hexenyl (E)-2-hexenoate Unknown (MS: 71, 43, 114, 143, 89, 99) Unknown (MS: 69, 43, 87, 103, 129, 55) Undecyl acetate tent. Decyl isobutyrate (MS: 89, 41, 71, 55, 97, 111) Unknown (MS: 99, 71, 43, 81, 55, 110) Decyl butyrate Hexyl benzoate Dodecyl acetate Unknown (MS: 69, 43, 87, 103, 55, 129) Unknown (MS: 69, 41, 55, 99, 81, 125) tent. Dodecyl butyrate (MS: 41, 69/71, 55/57, 97, 89, 83, 111, 125)

X. quadratus

P. gracilicornis

25 ♀ flowers

1672

C. ater

50 ♀ flowers

tent. (E)-3-Hexenyl hexoate (MS: 67, 82, 43, 55, 99, 117)

a, s


36 ♀ flowers

1544 1612 1619 1668

Content / Comments

Component Heptyl butyrate (E)-2-Hexenyl tiglate Unknown (MS: 41, 71, 57, 89, 129) tent. (Z)-3-Hexenyl hexoate (MS: 67, 82, 41, 55, 99, 117)


Miridae A. rotunda

tr tr tr tr

tr tr tr tr

tr tr

tr tr

tr tr

tr tr 0.1

tr tr

tr -

tr -

tr -

tr

-

-

-

tr

tr

tr

tr

0.1

0.1

tr

-

-

-

tr

-

-

-

1.1 0.7 0.1 0.3 tr tr tr 0.4 -

0.7 0.3 0.1 0.4 tr tr tr 0.1 -

tr 0.6 0.5 0.1 0.6 tr tr 0.7 -

2.1 1.6 0.4 0.2 tr tr 0.1 -

4.1 4.6 1.2 0.4 tr 0.1 tr -

5.3 6.5 2.2 0.2 0.1 0.1 0.1 tr -

1.7 1.2 0.4 0.6 tr tr tr 0.3 tr

tr tr tr -

tr tr tr -

-

0.9 41.7 -

-

-

-

0.1 tr 0.1 0.2 tr

0.1 tr tr tr tr

0.1 tr tr tr tr

0.1 tr tr 0.1 tr

0.2 tr 0.1 0.2 tr

1.1 2.0 3.3 tr

0.9 0.9 1.3 0.1

-

tr -

tr -

1.9 -

-

-

-

0.6

0.3

0.1

0.5

2.2

0.1

0.2

-

-

-

39.0 -

-

-

-

tr

tr

0.1

-

-

-

0.1

67.6 -

48.8 -

70.5 -

-

-

-

-

4

Aliphatic acids Butanoic acid Aliphatic hydrocarbons Nonane Decane Undecane (E)-3-Nonene Dodecane a Tridecene (MS: 41, 55, 69, 83, 97, 111) Tridecane Tetradecane tent. Pentadecadiene (MS: 67, 81, 95, 39, 55, 109) a Pentadecene (MS: 55, 69, 41, 83, 97, 111) Pentadecane Heptadecene (MS: 67, 81, 95, 39, 55, 109) Heptadecadiene or Heptadecine (MS: 55, 41, 67, 79, 93, 108) Heptadecene (MS: 41, 55, 69, 97, 83, 111) Unknown aliphatics Unknown (MS: 67, 39, 41, 55, 82, 97) Unknown (MS: 56, 41, 69, 85, 105, 120) Unknown (MS: 55, 112, 83, 97, 39, 67/69) Unknown (MS: 83, 55/57, 41, 71, 113, 95/97) Unknown (MS: 57, 67, 39, 83, 100, 127) Unknown (MS: 55, 41, 69, 83, 97, 111)

X. quadratus

38 ♀ flowers

41 ♀ flowers


29 ♀ flowers*

1♂

1♂

2♂

1♂

2♀

1♂2♀

1♂

P. gracilicornis

25 ♀ flowers

C. ater

50 ♀ flowers

859 1033


36 ♀ flowers

Aliphatic aldehydes (E)-2-Hexenal (E)-4-oxo-2-Hexenal

Content / Comments

Component


Miridae A. rotunda

-

-

-

-

-

-

-

tr

0.2

tr

9.0 1.1

0.3 86.1

0.6 82.8

0.7 95.6

747

s

-

-

-

-

-

-

-

7.1

1.0

-

-

-

-

-

935 1103 1266 1310 1418 1552 1560 1696 1797

s s 3, s l l, s

1.5 2.6 61.6 tr 0.7 0.1 14.3 0.3 tr

1.2 2.5 67.2 tr 0.7 tr 13.5 0.1 tr

0.3 4.2 81.2 tr 1.7 tr 2.8 0.1 -

0.7 2.8 58.1 0.1 0.7 tr 15.9 0.1 -

1.1 6.1 27.0 0.2 0.1 0.1 4.3 0.2 -

0.1 1.5 tr tr 2.2 0.3 -

0.1 1.4 tr 0.1 8.5 0.9 -

-

-

-

-

-

-

-

2, l, s l, s

1802

3, l

0.1

0.1

tr

0.1

0.2

0.7

1.0

-

-

-

-

-

-

-

1823 2027 2033

4, s 2 2

5.2 tr 0.1

2.7 tr tr

3.0 tr tr

3.2 tr tr

3.0 tr tr

23.9 1.7 0.8

64.7 2.4 0.7

-

-

-

-

-

-

-

2035

2

tr

tr

tr

tr

0.1

3.1

2.8

-

-

-

-

-

-

-

864 985 1058 1170

0.2 -

0.2 -

tr -

0.3 -

1.2 -

tr -

tr -

0.3 -

0.2 -

0.2 -

tr 1.2

2.1 11.5

3.9 12.8

tr 3.7

1275 1287

tr

tr tr

tr -

tr -

0.1 -

-

-

-

-

-

-

-

-

-

5


X. quadratus

25 ♀ flowers

38 ♀ flowers

41 ♀ flowers


29 ♀ flowers*

1♂

1♂

2♂

1♂

2♀

1♂2♀

1♂

P. gracilicornis

50 ♀ flowers

C. ater

36 ♀ flowers

Unknown (MS: 55, 41, 67, 85, 97, 111) Unknown (MS: 41, 55, 67/69, 79, 97, 111) Unknown (MS: 55, 41, 69, 83, 97, 111) Unknown (MS: 43, 55, 83, 67, 101, 127) Unknown (MS: 55, 41, 69, 97, 83, 111) Unknown (MS: 67, 43, 81, 54, 95, 110) Unknown (2 substances, MS: 55, 41, 69, 83, 97, 111) Unknown (MS: 55, 41, 69, 83, 97, 111) Unknown (MS: 57, 41, 67, 82, 103, 157) Unknown (MS: 69, 43, 87, 103, 56, 159) Unknown (MS: 41, 55, 69, 83, 97, 111) Unknown (MS: 41, 55, 69, 83, 97, 111) Unknown (MS: 67, 81, 39/43, 54, 96, 110) Unknown (MS: 69, 41/43, 57, 87, 103, 127) Unknown (MS: 69, 57, 39, 81, 99, 110) Unknown (MS: 67, 79/81, 55, 41, 95, 123) Unknown (2 substances, MS: 55, 41, 69, 83, 97, 111) Unknown (MS: 67, 43, 81, 55, 95, 110) Unknown (MS: 41, 55, 97, 67, 81, 111) Unknown (MS: 41, 69, 109, 55, 95, 79/81) Unknown (MS: 67, 81, 95, 41, 55, 109) Unknown (MS: 67, 81, 95, 39/41, 55, 109) Unknown (MS: 41, 55, 69, 83, 97, 111)

Content / Comments

Component Unknown (MS: 67, 39, 81, 55, 95, 110) Unknown (MS: 43, 99, 71, 55, 87, 115) Unknown (MS: 69, 39, 55, 82, 97, 111) Unknown (MS: 55, 41, 69, 83, 97, 109) Unknown (MS: 81, 71, 43, 55, 96, 125) Unknown (MS: 69, 39, 79, 55, 99, 87) Unknown (MS: 70, 41, 89, 55, 98, 129) Unknowns (multiple substances, MS: 41, 56, 69, 85, 103, 129)


Miridae A. rotunda

1304 1337 1442 1461 1483 1492 1506 1543

tr 0.1

tr 0.1

tr tr tr

tr tr 0.1

0.1 tr 0.2

tr 0.1 tr 0.1 0.1

tr tr tr

tr tr -

tr tr -

tr tr -

2.1 -

-

-

-

1548 1553 1602 1616 1660 1665 1686

0.1 tr tr tr tr tr

0.1 0.1 tr tr tr tr

0.1 tr tr tr

0.1 tr tr

0.1 tr tr

0.1 0.1 tr -

0.1 0.1 0.1 tr tr 0.1

-

-

-

-

-

-

-

tr tr tr 0.4 tr tr 0.1

tr tr tr 0.1 tr tr tr 0.1

0.1 tr -

tr tr -

tr tr -

tr tr 0.1 1.4 0.1 0.1 0.1 0.1 1.0

0.1 tr tr 0.7 0.1 0.1 tr 0.6

-

-

-

-

-

-

-

-

-

-

-

-

0.1 tr tr tr

tr tr tr tr tr tr

-

-

-

-

-

-

-

1735 1740 1781 1810 1862 1907 1909 1915 1919 1922 1933 2038 2069 2083 2088 2095

l

2

6


X. quadratus

25 ♀ flowers

38 ♀ flowers

41 ♀ flowers


29 ♀ flowers*

1♂

1♂

2♂

1♂

2♀

1♂2♀

1♂

P. gracilicornis

50 ♀ flowers

C. ater

36 ♀ flowers

Content / Comments


Miridae A. rotunda

-

-

-

-

-

tr tr 0.1

0.2

-

-

-

-

-

-

-

l l, s

tr tr

tr tr

tr -

tr -

tr -

0.1

tr -

-

-

-

-

-

-

-

Total absolute amount (ng) per flower/true bug per 20 min:

113.1

153.6

282.4

175.3

142.5

n.a.

n.a.

281.1

759.5

702.4

177.7

214.4

228.2

918.4

Component Unknown (MS: 67, 95, 55, 41, 79, 107) Unknown (MS: 79, 93,67, 41, 55, 107) Unknown (MS: 69, 81, 41, 95, 109, 55)

2096 2119 2152

Terpenoids Terpenoid (MS: 67, 81, 95, 43, 123, 55) (E)-ß-Caryophyllene

1634 1753

Relative and absolute amounts of components of the flower scent of A. rotunda and volatiles trapped from freshly killed Heteroptera (Miridae: Capsus ater, Notostira elongata; and Lygaeidae: Peritrechus gracilicornis, Xanthochilus quadratus). Scent components are sorted by substance class and retention time. *, Acetone sample; ✝, Sample used for EAD measurements; tr, traces (relative amount < 0.05%). Content/Comments: 1–4, Number of EAD responses in 4 EAD-runs with the natural flower sample. a, EAD response using synthetic substances. b, EAD measurement employing synthetic substance elicited no response. l, small amount in leaf samples. s, component confirmed by synthetic substance.

7

Table S3 Components used as references in scent analyses and for bioassays Substance (E)-2-Hexenyl butyrate (E)-2-Hexenyl hexanoate (Z)-3-Hexenyl butyrate Acetone Butyl butyrate Decyl acetate Decyl butyrate Heptyl butyrate Hexyl (E)-2-butenoate Hexyl butyrate Hexyl hexanoate Hexyl isobutyrate Octyl butyrate Pentadecane Pentyl butyrate Tridecane Undecane

Origin SAFC Sigmal Aldrich Chemie GmbH Steinheim, Germany TCI Europe N.V., Zwijndrecht, Belgium SAFC Sigmal Aldrich Chemie GmbH Steinheim, Germany Merck, Schwalbach/Ts., Germany SAFC Sigmal Aldrich Chemie GmbH Steinheim, Germany SAFC Sigmal Aldrich Chemie GmbH Steinheim, Germany SAFC Sigmal Aldrich Chemie GmbH Steinheim, Germany SAFC Sigmal Aldrich Chemie GmbH Steinheim, Germany SAFC Sigmal Aldrich Chemie GmbH Steinheim, Germany SAFC Sigmal Aldrich Chemie GmbH Steinheim, Germany SAFC Sigmal Aldrich Chemie GmbH Steinheim, Germany SAFC Sigmal Aldrich Chemie GmbH Steinheim, Germany SAFC Sigmal Aldrich Chemie GmbH Steinheim, Germany Sigma Chemicals CO. St. Louis, MO, USA WAKO Pure Chemicals Industries, Ltd. Sigma Chemicals CO. St. Louis, MO, USA Sigma Chemicals CO. St. Louis, MO, USA

Purity ≥96% >95% ≥98% 99.8% 98% ≥95% 97% ≥98% ≥95% ≥98% ≥97% ≥98% ≥98% 99% ≥98% 99% 99%

Table S4 Amount of EAD-active components Miridae

Hexyl isobutyrate (Z)-3-Hexenyl butyrate (E)-3-Hexenyl butyrate Hexyl butyrate (E)-2-Hexenyl butyrate Heptyl butyrate Hexyl hexoate (E)-2-Hexenyl hexanoate Octyl butyrate

A. rotunda n=5 0.5–1.6 tr-0.1 tr-0.3 2.5-25.6 0.6-21.7 0-tr 1.1-5.9 0.5-6.6

C. ater n =3 tr-tr tr-tr 0.1-0.9 63.2-346.6 5.3-24.8 tr-0.1 0-tr 0-tr

N. elongata n =1 tr 0.2 3.4 1.5 74.2

0.2-1.6

0-tr

-

Lygaeidae P. X. gracilicornis quadratus n =2 n =1 0.1-0.2 0.5 tr-tr tr -

-

Absolute concentration range (in ng per flower per 20 min and ng per individual per 20 min, respectively) in dynamic headspace samples of EAD-active components found of both A. rotunda and true bugs. tr, absolute amount