New Phytologist Supporting Information Notes S1

Article title: Covariation and phenotypic integration in chemical communication displays: biosynthetic constraints and eco-evolutionary implications

Authors: Robert R. Junker, Jonas Kuppler1, Luisa Amo, James D. Blande, Renee M. Borges, Nicole M. van Dam, Marcel Dicke, Stefan Dötterl, Bodil K. Ehlers, Florian Etl, Jonathan Gershenzon, Robert Glinwood, Rieta Gols, Astrid T. Groot, Martin Heil, Mathias Hoffmeister, Jarmo K. Holopainen, Stefan Jarau, Lena John, Andre Kessler, Jette T. Knudsen, Christian Kost, Anne-Amélie C. Larue-Kontic, Sara Diana Leonhardt, Dani Lucas-Barbosa, Cassie J. Majetic, Florian Menzel, Amy L. Parachnowitsch, Rémy S. Pasquet, Erik H. Poelman, Robert A. Raguso, Joachim Ruther, Florian P. Schiestl, Thomas Schmitt, Dorothea Tholl, Sybille B. Unsicker, Niels Verhulst, Marcel E. Visser, Berhane T. Weldegergis and Tobias G. Köllner

Article acceptance date: 29 January 2017

1

Note S1 Scent samples and methods to identify chemical communication displays.

Species sampled, references to the original studies and a brief summary of the sampling methods and the analysis of scent bouquets are given.

2

# Author Species organism organ treatment # sample # compounds integration # modules # modules > 1 compound integration of modules 1 Larue Achillea millefolium plant flower NA 9 38 22.47 3 3 43.22 Tab. SI1-1 Species and 2 Tholl Arabidopsis thaliana plant flower NA 138 10 16.44 4 3 55.47 3 Hoffmeister Brassica juncea plant flower control 9 19 13.11 4 4 45.90 samples included in the 4 Hoffmeister Brassica juncea plant flower induced 9 18 15.30 2 2 17.15 5 Hoffmeister Brassica nigra plant flower control 9 20 21.37 5 4 56.92 study. For each sample, 6 Lusas-Barbosa Brassica nigra plant flower control 5 25 13.34 4 3 30.16 7 Hoffmeister Brassica nigra plant flower induced 9 20 18.89 3 3 44.48 8 Lusas-Barbosa Brassica nigra plant flower induced 5 26 7.93 4 4 29.94 the first author, the 9 Lusas-Barbosa Brassica nigra plant flower control 6 21 43.50 3 2 50.88 10 Lusas-Barbosa Brassica nigra plant flower induced 5 21 11.62 7 6 69.65 species, the organ as well 11 Larue Cirsium arvense plant flower NA 9 49 13.15 2 2 28.61 12 Knudsen Corydalis gotlandica plant flower NA 43 28 5.21 14 9 56.25 13 Etl Dieffenbachia aurantiaca plant flower NA 8 7 29.28 2 2 83.55 as the treatmemt are 14 Borges Ficus racemosa Phase a Stage D plant flower NA 10 33 26.31 2 2 30.04 15 Borges Ficus racemosa Phase a Stage N plant flower NA 10 13 23.55 2 2 23.16 given. Additionally, 16 Borges Ficus racemosa Phase b Stage D plant flower NA 10 31 24.96 2 2 21.45 17 Borges Ficus racemosa Phase b Stage N plant flower NA 10 19 12.58 2 2 50.56 sample size, the number 18 Majetic Ipomoea purpurea plant flower NA 51 25 11.87 7 4 31.58 19 Parachnowitsch Penstemon digitalis plant flower NA 88 23 10.77 6 4 26.57 20 Majetic Phlox bifida plant flower NA 8 8 21.65 2 2 49.90 of compounds detected, 21 Majetic Phlox carolina plant flower NA 8 26 18.54 3 3 40.05 22 Majetic Phlox drummondii plant flower NA 18 10 15.07 2 2 25.60 the phenotypic 23 Majetic Phlox drummondii wild plant flower NA 31 21 11.95 7 6 55.05 24 Majetic Phlox paniculata plant flower NA 9 22 18.48 3 3 35.03 integration, the number of 25 Majetic Phlox stolonifera plant flower NA 12 15 18.76 3 2 47.17 26 Majetic Phlox sublata plant flower NA 24 15 9.98 6 5 46.95 27 Majetic Polemonium caeruleum plant flower NA 9 6 12.30 2 2 23.86 modules, the number of 28 Hoffmeister Raphanus raphanistrum plant flower control 7 14 17.17 5 2 45.16 29 Hoffmeister Raphanus raphanistrum plant flower induced 7 14 6.70 5 4 44.18 modules with more than 30 Dötterl Silene latifolia plant flower NA 98 28 6.32 15 7 53.05 31 Schiestl Silene latifolia plant flower NA 737 28 7.74 6 3 23.93 one compound as well as 32 Glinwood Sinapis alba plant flower NA 10 10 8.52 2 1 10.44 33 Hoffmeister Sinapis arvensis plant flower control 7 6 24.91 2 1 22.50 34 Hoffmeister Sinapis arvensis plant flower control 8 14 35.10 2 2 72.85 the mean phenotypic 35 Hoffmeister Sinapis arvensis plant flower induced 7 6 12.96 2 2 24.25 36 Hoffmeister Sinapis arvensis plant flower induced 9 14 14.83 5 4 61.12 integration of modules are 37 Hoffmeister Sinapis arvensis plant flower induced 9 6 18.25 2 1 31.08 38 Hoffmeister Sinapis arvensis plant flower induced 8 6 23.78 2 2 42.13 listed. Methodological 39 Kuppler Sinapis arvensis plant flower NA 95 18 38.19 2 2 34.79 40 Hoffmeister Vicia faba plant flower control 41 24 20.42 5 4 64.52 41 Hoffmeister Vicia faba plant flower induced 14 24 9.01 7 6 51.86 details and additional 42 Hoffmeister Vicia faba plant flower induced 15 24 9.38 9 7 46.18 43 Hoffmeister Vicia faba plant flower induced 12 24 13.90 7 6 66.77 information are given 44 Hoffmeister Vicia faba plant flower induced 7 24 18.48 4 4 38.87 45 Knudsen Vigna unguiculata var. spontanea plant flower NA 177 39 2.99 23 11 36.73 below. NA, information 46 Borges Ficus racemosa Phase c1 Stage D plant fruit NA 10 44 17.01 2 2 28.56 47 Borges Ficus racemosa Phase c1 Stage N plant fruit NA 10 43 10.09 2 2 20.78 48 Borges Ficus racemosa Phase c2 Stage D plant fruit NA 10 45 18.56 3 2 28.07 not applicable. 49 Borges Ficus racemosa Phase c2 Stage N plant fruit NA 10 43 10.79 3 2 17.37 50 Plotto Fragaria x ananassa plant fruit NA 11 59 10.50 5 5 26.53 51 Plotto Fragaria x ananassa plant fruit NA 22 68 13.02 9 6 47.72 52 Plotto Fragaria x ananassa plant fruit NA 22 67 13.44 2 2 20.30 53 Plotto Fragaria x ananassa plant fruit NA 23 68 18.59 3 3 36.26 54 Plotto Fragaria x ananassa plant fruit NA 8 45 21.17 2 2 27.36 55 Plotto Fragaria x ananassa plant fruit NA 16 68 22.91 7 7 40.36 56 Plotto Fragaria x ananassa plant fruit NA 18 66 23.41 5 4 33.41 57 Plotto Fragaria x ananassa plant fruit NA 8 62 33.35 2 2 44.04 58 Plotto Tangerine plant fruit NA 25 168 6.74 23 16 51.47 59 Glinwood Beta vulgaris plant leaves control 19 13 34.57 2 2 47.47 60 Glinwood Beta vulgaris plant leaves induced 10 13 42.87 4 4 86.04 61 van Dam Brasscia oleracea plant leaves control 13 110 19.70 8 6 41.17 62 van Dam Brasscia oleracea plant leaves control 12 85 21.32 2 1 21.90 63 van Dam Brasscia oleracea plant leaves induced 13 106 17.79 8 7 49.67 64 van Dam Brasscia oleracea plant leaves induced 13 100 18.64 33 23 75.32 65 Lusas-Barbosa Brassica nigra plant leaves control 5 12 12.39 3 3 34.54 66 Lusas-Barbosa Brassica nigra plant leaves induced 5 13 17.42 2 2 12.90 67 Lusas-Barbosa Brassica nigra plant leaves control 9 19 9.94 5 5 56.66 68 Lusas-Barbosa Brassica nigra plant leaves control 8 14 35.05 3 3 48.15 69 Lusas-Barbosa Brassica nigra plant leaves induced 9 19 9.74 6 4 48.65 70 Lusas-Barbosa Brassica nigra plant leaves induced 8 14 33.36 6 4 85.30 71 Weldegergis Brassica oleracea plant leaves control 10 54 10.96 9 9 37.32 72 Weldegergis Brassica oleracea plant leaves induced 10 54 6.97 6 6 29.09 73 Weldegergis Brassica oleracea plant leaves induced 10 54 17.11 11 8 57.19 74 Glinwood Hordeum vulgare cv Alva plant leaves NA 8 21 21.64 5 3 53.93 75 Glinwood Hordeum vulgare cv Barke plant leaves NA 8 18 15.84 4 4 42.77 76 Glinwood Hordeum vulgare cv Frieda plant leaves NA 8 17 17.56 2 2 21.54 77 Amo de Paz Malus silvestris plant leaves control 16 75 10.92 18 15 50.78 78 Amo de Paz Malus silvestris plant leaves induced 16 75 3.61 15 11 42.70 79 Kost_Heil Phaseolus lunatus plant leaves control 9 10 64.09 6 4 85.16 80 Kost_Heil Phaseolus lunatus plant leaves induced 9 10 42.20 2 1 53.47 81 Blande Pinus sylvestris plant leaves control 16 25 20.06 7 5 58.83 82 Blande Pinus sylvestris plant leaves induced 17 25 15.12 2 2 20.67 83 Uniscker Populus niger plant leaves control 17 40 14.76 6 5 32.03 84 Uniscker Populus niger plant leaves control 17 40 29.16 2 1 30.88 85 Uniscker Populus niger plant leaves induced 17 53 9.20 24 14 74.41 86 Uniscker Populus niger plant leaves induced 17 75 21.84 2 2 11.13 87 Uniscker Populus niger plant leaves induced 17 77 22.12 6 6 41.20 88 Uniscker Populus niger plant leaves induced 17 54 31.93 5 5 47.21 89 Glinwood Salix viminalis plant leaves control 8 33 12.13 4 4 42.68 90 Glinwood Salix viminalis plant leaves induced 8 35 24.44 2 2 26.93 91 Leonhardt Austroplebeia australis animal NA NA 8 56 12.16 5 5 45.32 92 Schmitt Chrysis mediata animal NA NA 8 59 43.77 16 12 86.42 93 Menzel Crematogaster scutellaris animal NA NA 12 33 4.31 4 4 16.97 94 Menzel Crematogaster scutellaris animal NA NA 8 32 9.66 3 3 14.62 95 Ruther Dibrachys cavus animal NA NA 9 54 17.89 1 1 35.60 96 Ruther Dibrachys cavus animal NA NA 9 54 17.89 1 1 0.00 97 Ruther Dibrachys cavus animal NA NA 9 51 19.60 2 2 31.05 98 Ruther Dibrachys cavus animal NA NA 9 52 26.82 8 5 71.18 99 Groot Heliothis subflexa animal NA NA 203 11 23.59 5 4 62.17 100 Groot Heliothis virescens animal NA NA 397 7 38.09 3 2 64.55 101 Verhulst Homo sapiens animal NA NA 16 15 14.07 4 3 61.36 102 Groot Mamestra brassicae animal NA NA 93 8 29.40 2 1 39.37 103 Menzel Myrmica rubra animal NA NA 10 56 16.28 2 2 36.08 104 Menzel Myrmica rubra animal NA NA 8 54 16.34 3 3 32.53 105 Menzel Myrmica rubra animal NA NA 11 58 18.70 5 5 43.24 106 Menzel Myrmica rubra animal NA NA 10 54 19.42 11 8 61.15 107 Menzel Myrmica rubra animal NA NA 11 53 21.15 7 6 51.97 108 Menzel Myrmica rubra animal NA NA 140 58 22.27 8 5 47.61 109 Menzel Myrmica rubra animal NA NA 10 56 24.73 2 2 31.25 110 Menzel Myrmica rubra animal NA NA 8 51 26.29 5 3 60.02 111 Menzel Myrmica rubra animal NA NA 10 52 27.76 4 3 52.06 112 Menzel Myrmica rubra animal NA NA 6 51 28.08 3 3 44.40 113 Menzel Myrmica rubra animal NA NA 6 48 28.58 4 4 51.71 114 Menzel Myrmica rubra animal NA NA 11 54 30.81 3 3 52.98 115 Menzel Myrmica rubra animal NA NA 8 53 30.82 2 2 23.53 116 Menzel Myrmica rubra animal NA NA 11 55 37.30 2 2 65.89 117 Menzel Myrmica rubra animal NA NA 5 45 40.96 2 2 37.22 118 Menzel Myrmica rubra animal NA NA 9 53 41.78 2 2 42.89 119 Menzel Myrmica rubra animal NA NA 6 46 42.78 2 1 44.80 120 Schmitt Odynerus spinipes Chemotyp 1 animal NA NA 21 48 23.14 2 2 45.08 121 Schmitt Odynerus spinipes Chemotyp 2 animal NA NA 15 61 39.20 4 3 50.73 122 Leonhardt Partamona orizabaensis animal NA NA 9 32 10.37 4 4 33.49 123 Groot Plutella xylostella animal NA NA 69 3 30.85 2 1 93.32 124 Schmitt Pseudospinolia neglecta animal NA NA 17 50 40.07 2 2 36.84 125 Leonhardt Scaptotrigona pectoralis animal NA NA 10 49 14.00 25 11 82.37 126 Leonhardt Tetragonilla collina animal NA NA 52 72 37.08 2 2 60.08 127 Leonhardt Tetragonula carbonaria animal NA NA 15 87 18.45 16 11 63.76 128 Leonhardt Tetragonula melanocephala animal NA NA 41 52 36.66 4 3 70.05 129 Jarau Trigona corvina animal NA NA 117 7 7.73 2 2 2.09 Samples along with methodological details and additional information are given below, ordered as they appear in Table S1-1. Additional information can be found in original publications.

3

(1, 11) Species: Achillea millefolium, Asteraceae; Cirsium arvense, Asteraceae

Organism / organ: flowers

Authors / contributors: Anne-Amélie C. Larue-Kontic1, Robert A. Raguso2 & Robert R. Junker1

Affiliation: 1University of Salzburg, Department of Ecology and Evolution, Salzburg, Austria

2Department of Neurobiology and Behavior, Cornell University, Ithaca, USA

Reference: Larue A-AC, Raguso RA, Junker RR (2016) Experimental manipulation of floral scent bouquets restructures flower-visitor interactions in the field. Journal of Animal Ecology 85: 396-408

Location: fallow land, Salzburg, Austria

Method sketch / description of data:  Experimental setup: Flower-visitor observations and scent manipulation of living flowers from A. millefolium and C. arvense plants.

 Scent sampling / analyses: Volatiles were collected by using dynamic headspace sampling. Flowers during anthesis from living plants were enclosed within scentless polyethylene tetraphthalate (PET) oven bags (Toppits® Cofresco Fischhalteprodukte GmbH & Co. KG, Minden, Germany), headspace was enriched for 90 min and scented air was sucked through volatile traps for 2 min with a flow rate of 200 ml min-1. Volatile traps contained a mixture of 1.5 mg Tenax-TA (mesh60-80; Supelco, Germany) and 1.5 mg Carbotrap B (mesh 20-40; Supelco, Germany). Flower tissues were dried for at least 48 h at 40°C to obtain the dry mass. Volatiles were desorbed from traps using an automatic TD (thermal desorption) system (model TD-20, Shimadzu, Japan) coupled with a GC–MS (model QP2010 Ultra EI, Shimadzu, Japan). GC was equipped with a 60 m long column (Zebron ZB-5, Newport Beach, USA) with a diameter of 0.25 mm and a film thickness of 0.25 µm. The column flow (helium) had a rate of 1.5 ml min-1 and the oven temperature was kept constant for 1 min at 40°C before it increased with 6°C min-1 until the maximum of 250°C. The MS interface was set to 260°C and the ion source was at 200°C. For identification of the scent compounds, we used the GCMSsolutions Software (Version 2.72, Shimadzu Corporation) and mass spectra of authentic standards as well as spectral libraries (ADAMS, ESSENTIALOILS-23P, FFNSC 2, W9N11) and Kovat’s indices generated using n-alkanes.

 Funding: This work was funded by the Deutsche Forschungsgemeinschaft (DFG, JU2856/1-1) to RRJ and by NSF Grant DEB-0746106 to RAR.

4

(2) Species: Arabidopsis thaliana, Brassicaceae

Organism / organ: plant / flowers

Authors / contributors: Dorothea Tholl1

Affiliation: 1Department of Biological Sciences, Virginia Tech, USA

Reference: Dorothea Tholl, Feng Chen, Jana Petri, Jonathan Gershenzon and Eran Pichersky (2005) Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers, Plant Journal, 42, 757–771

Location: Max Planck Institute for Chemical Ecology, Jena, Germany

Method sketch / description of data:  Experimental setup: Seventy inflorescences were detached each (n = 3–6) from flowering plants of 37 different Arabidopsis accessions. Plants were grown in chambers under controlled temperature and light conditions (22°C, 160 µmol m-2 s-1 PAR, 16 h : 8 h, light : dark cycle). Inflorescences of most accessions had an average of four to five open flowers.

 Scent sampling / analyses: Detached inflorescences were placed in a small glass beaker filled with tap water and transferred to 1 l bell jars. Emitted volatiles were collected for 8 h on 25 mg Super Q (Supelco; Sigma-Aldrich, St Louis, MO, USA) traps in a closed loop stripping procedure according to Donath & Boland (1995). Volatiles were eluted from the traps with 100 µl CH2Cl2, and 120 ng of nonyl acetate were added as a standard. Samples from volatile collections were analyzed on a Hewlett-Packard 6890 gas chromatograph coupled to a Hewlett-Packard 5973 quadrupole mass selective detector. Separation was performed on (5%-phenyl)-methylpolysiloxane column (J&W Scientific, Folsom, CA, USA) of 30 m × 0.25 mm i.d. × 0.25 m thickness. Helium was the carrier gas (flow rate of 2 ml min-1), a splitless injection (injection volume of 2 µl) was used, and a temperature gradient of 5°C min-1 from 40°C (3-min hold) to 240°C was applied. Compounds were identified by comparison of retention times and mass spectra with those of authentic standards and with reference spectra in the NIST and Wiley libraries (Agilent Technologies, Palo Alto, CA, USA). Mass spectrometry was performed with a transfer line temperature of 230°C, source temperature of 230°C, quadrupole temperature of 150°C, ionization potential of 70 eV, and scan range of 50 to 400 atomic mass units. For quantification, primary ion peaks of each compound were integrated (single ion method) and the amounts were calculated in relation to the response of nonyl acetate at mass-to-charge ratio (m/z) 69. Response curves for the quantified compounds relative to the internal standard were generated by injecting a mixture of equal amounts of authentic standards and internal standard. Emissions were determined in ng h-1 per 70 inflorescences.

Donath, J. and Boland, W. (1995) Biosynthesis of acyclic homoterpenes – enzyme selectivity and absolute configuration of the nerolidol precursor. Phytochemistry, 39, 785–790.

Funding: National Science Foundation Grant IBN-0211697 to Eran Pichersky and funds of the Max Planck Society to Jonathan Gershenzon

5

(3–5, 7, 28, 29, 33-38) Species:

Sinapis arvensis L., Brassicaceae

Brassica nigra L., Brassicaceae

Brassica juncea L., Brassicaceae

Raphanus raphanistrum L., Brassicaceae

Organism / organ: plant / flowers

Authors / contributors: Mathias Hoffmeister1,2 & Robert R. Junker2

Affiliation: 1Heinrich-Heine-University, Institute of Sensory Ecology, Düsseldorf, Germany 2University of Salzburg, Department of Ecology and Evolution, Salzburg, Austria

Reference: Hoffmeister M, Wittköpper N, Junker RR (2015) Herbivore-induced changes in flower scent and morphology affect the structure of flower-visitor networks but not plant reproduction. OIKOS, in press. doi: 10.1111/oik.02988

Location: University of Düsseldorf, Germany

Method sketch / description of data:  Experimental setup: Experiment to test the effect of leaf herbivory on floral scent emission in different Brassicaceaes (n = 4). For herbivory treatment, larvae of the Brassicaeae specialist Phaedon cochleariae were allowed to feed inside of clip cages on single leafs of potted plants for 2 d 3–4 wk after germination; control plants were equipped with clip cages containing no larva. When flowering scent was sampled in

control and herbivore-treated plants (nplant = 7–9 per species and treatment).  Scent sampling / analyses: Scent was sampled from unpicked wrapped inflorescences (oven bags, Toppits, Minden, Germany) of control and herbivore-treated plants as well as from surrounding air (empty oven bags) to determine flower-specific compounds. After scent accumulation for 30 min, volatiles were collected in traps filled with a mix (1 : 1) of Tenax-TA (mesh 60-80) and Carbotrap B (mesh 20-40; Supelco, Bellafonte, USA), where air was pulled from flower bags (flow rate: 200 ml min-1, sampling: 2 min). Traps were analysed using an automatic thermal desorption system (TD-20, Shimadzu, Japan; ZB-5 fused silica column, Phenomenex, Aschaffenburg, Germany) coupled with a GC-MS (model QP2010 Ultra El, Shimadzu, Japan). Column flow was set to 1.5 ml min-1, GC oven temperature was increased to 250°C (6°C min-1) and held for 1 min and the MS interface worked at 220°C. We processed GC/MS data with the GCMSolution package, Version 2.72 (Shimadzu Corporation 2012). For compound identification we compared compounds to mass spectra and retention times of commercially available standard substances or to compounds listed in mass spectral libraries Wiley Registry 9th Edition, NIST 2011 and FFNSC2.

Funding: Deutsche Forschungsgemeinschaft (DFG JU 2856/2-2).

6

(6, 65-70) Species: Brassica nigra L., Brassicaceae

Organism / organ: plant / leaves and flowers

Authors / contributors: Dani Lucas-Barbosa / Marcel Dicke

Affiliation: Laboratory of Entomology, Wageningen University, Wageningen, The Netherlands.

Reference: Bruinsma, M., Lucas-Barbosa, D., ten Broeke, C. J. M., van Dam, N. M., van Beek, T. A., Dicke, M. & van Loon, J. J. A. Journal of Chemical Ecology 40, 39-49, (2014).

Location: Glasshouse of Wageningen University, Wageningen, The Netherlands

Method sketch / description of data: Experimental setup: Glasshouse experiment with potted Brassica nigra individuals (n = 20). Emission rates were quantified for compounds that were detected in at least 50% of the samples of one of the treatments.

Scent sampling and analyses: To investigate whether flowering B. nigra plants respond locally and systemically to herbivore infestation, we collected volatiles emmited by leaves and flowers of infested and non-infested control plants. Each B. nigra plant was infested with 100 second instar P. brassicae larvae, spread over two leaves, and allowed to feed for 48 h. To prevent caterpillars from moving to flowers, cotton wool was placed around the petiole leaf stalk or the caterpillars were confined to clip cages. When plants were tested, caterpillars had consumed about 60 % of the leaves where they were placed. Control plants were of the same batch of plants, of the same age and similar in height and number of flowers, but were not infested with herbivores. Either leaves or flowers of infested and control plants were enclosed in an oven bag (Toppits® Brat-Schlauch, polyester; 32 cm × 32 cm × 70 cm; Toppits, Minden, Germany). A strip of bag material wrapped around the stem and above or below the inflorescence was used to close the bag. Synthetic air was flushed through the bag at a flow rate of 300 ml min1 (224-PCMTX8, air-sampling pump Deluxe, Dorset, UK; equipped with an inlet protection filter) by inserting Teflon tubing through an opening in the upper part of the bag. Air was sucked out and headspace volatiles were collected in the glass tube filled with Tenax for 1.5 h at a flow rate of 250 ml min1 through a second Teflon tube at the opening of each bag, and connecting it to a glass tube filled with about 90 mg of Tenax-TA 25/30 mesh (Grace-Alltech; USA). Headspace samples were analysed in a gas chromatograph with a thermodesorption unit (GC) (6890 series, Agilent, Santa Clara, USA) connected to a mass spectrometer (MS) (5973 series, Agilent, Santa Clara, USA). Compounds were identified by comparison of mass spectra with those of NIST, Wiley libraries and the Wageningen Mass Spectral Database of Natural Products. Identity was confirmed by comparison of retention index described in the literature and the respective index calculated during this study.

Funding: The Earth and Life Sciences council of The Netherlands Organisation for Scientific Research (NWO-ALW).

7

(12) Species: Corydalis gotlandica Lidén, Papaveraceae

Organism / organ: plant / flowers

Authors / contributors: Jette T. Knudsen12

Affiliation: 1Dept. of Biology, Lund University, Lund, Sweden, 2Nattaro Labs AB, Medicon Village, 223 81 Lund, Sweden.

Acknowledgement: I acknowledge Lotta Hallenfur for help with collection of scent samples

Reference: NA

Location: Gotland and Stora and Lilla Karlsö

Method sketch / description of data:  Experimental setup: Inflorescences on rooted plants (N=43) in natural settings.

 Scent sampling / analyses: Dynamic headspace samples were collected in situ from one inflorescence per plant. Each inflorescence was covered with a polyacetate oven bag and an absorbent tube, filled with 20 mg Tenax Gr (mesh size 60-80) was inserted into the bag and the air within the bag was sucked through the absorbent tube with a battery driven membrane pump at a flow rate of 90–110 ml min-1. In parallel blank samples were collected from leaves or air. Samples were collected for 4-6 h between 10:00 - 16:00 h and were extracted with 250 l of hexane. The samples were analysed by coupled GC-MS on a HP 6890 GC connected to a HP 5973 mass selective detector. Emission was standardized to %/inflorescence. Most compounds were identified by comparison of mass spectra and retention times with standard compounds. Alternatively, compounds were identified using the mass spectral libraries NIST 2011 and Adams 2007.

Funding: Länsstyrelsen, Gotland

8

(13) Species: Dieffenbachia aurantiaca Engl., Araceae

Organism / organ: plant / flowers

Authors / contributors: Florian Etl1,2

Affiliation: 1University of Vienna, Department of Botany and Biodiversity Research, Vienna, Austria 2University of Salzburg, Department of Ecology and Evolution, Salzburg, Austria

Reference: FLORIAN ETL, ANDREAS BERGER, ANTON WEBER, JÜRG SCHÖNENBERGER, STEFAN DÖTTERL (2016), NOCTURNAL PLANT BUGS USE cis- JASMONE TO LOCATE INFLORESCENCES OF AN ARACEAE AS FEEDING AND MATING SITE (HETEROPTERA: MIRIDAE). Journal of Chemical Ecology, 42: 300

Journal of Chemical Ecology, accepted.

Location: Tropenstation la Gamba, Costa Rica

Method sketch / description of data:

Experimental setup: Dynamic head space collection with Dieffenbachia aurantiaca individuals (n = 8) in their natural habitat which is a tropical lowland rainforest in the southernmost part of Costa Rica. Collections were made during the period of strong scent emission (pistillate phase; ca. 18:30; as determined by human nose and indicated by attraction of large numbers of mirid bugs during preliminary observations).

Scent sampling / analyses: Dynamic headspace samples were collected from one inflorescence per plant. Inflorescences were bagged with polyethylene oven bags (10 × 30 cm; Toppits, Germany) and scent was trapped for 2 min (either directly after bagging (n = 6) or 10 min after bagging (n = 2)) on adsorbent tubes (quartz glass tube: length 25 mm; inner diameter 2 mm) filled with 1.5 mg each of Carbotrap B (mesh 20-40, Supelco, Germany) and Tenax TA (mesh 60-80; Supelco, Germany). For scent collection, a membrane pump (Gardner Denver, Germany) was used and the flow was set at 200 ml min-1. To obtain negative controls, we conducted the same procedure but with empty oven bags (n = 3).

The samples were analyzed by GC/MS (QP2010Ultra, Shimadzu Corporation, Japan) coupled to a thermal desorption unit (TD-20, Shimadzu, Japan) and equipped with a ZB-5 fused silica column (5% phenyl polysiloxane; 60 m long, inner diameter 0.25 mm, film thickness 0.25 µm, Phenomenex, USA). Samples were run at a column flow (carrier gas: helium) of 1.5 ml min-1. GC oven temperature started at 40°C, then increased by 6°C per min to 250°C and held for 1 min. The MS interface worked at 260°C and the ion source at 200°C. Mass spectra were taken at 70 eV (in EI mode) from m/z 30 to 350. The GC/MS data were processed using the GCMSolution Version 4.11 (Shimadzu Corporation, Japan). Compounds were identified by the NIST 11, Wiley 9, FFNSC 2, Essential Oils and Adams 2007 mass spectral data bases and confirmed by comparison of mass spectra and retention times with those of authentic standards.

Funding: FE was supported by a KWA scholarship of the University of Vienna and by a scholarship of the ‘Verein zur Förderung der Tropenstation La Gamba’.

9

(14–17, 46–49) Species: Ficus racemosa L. (Section Sycomorus), Moraceae

Organism / organ: plant / inflorescence

Authors / contributors: Renee M. Borges1, Jean-Marie Bessière2 & Yuvaraj Ranganathan1

Affiliation: 1Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560012, India 2 Ecole Nationale Supérieure de Chimie de Montpellier, Montpellier Cedex 5, France

Reference: Borges, R.M., Bessière, J.-M. and Ranganathan, Y., 2013. Diel variation in fig volatiles across syconium development: making sense of scents. Journal of Chemical Ecology 39:630–642.

Location: Indian Institute of Science Campus, Bangalore

Method sketch / description of data:  Trees were sampled in situ.

Scent sampling / analyses: Volatiles of F. racemosa were collected from A–C2 phases from 10 trees, one fig bunch per tree. Two samples per phase from the same syconia were collected (11:00 h and 23:00 h on the same day) by dynamic headspace adsorption in situ. Figs were enclosed in a polyethylene terephthalate (NalophanTM) bag (Kalle Nalo GmbH, Wursthullen, Germany) through which a constant airflow over an Alltech Super Q® volatile collection trap (ARS Inc., Gainesville, FL, USA) was maintained for 1 h per sample at incoming and outgoing flow rates of 111 ml min−1 and 94 ml min−1, respectively, using glass flowmeters (Aalborg Instruments and Controls, Orangeburg, NY, USA) controlled by a micropump (KNF Neuberger GmbH, Germany; model number NMP50KNDC 12VDC). Incoming air was cleaned using activated charcoal filters (Sigma-Aldrich). VOC traps were eluted with 150 μl of dichloromethane, and the eluate concentrated by solvent evaporation at room temperature to 10 μl, to which 0.5 μl of the internal standard cumene was added at a concentration of 200 ng ml−1.

Funding: Ministry of Environment and Forests, Department of Biotechnology, Government of India

10

(18) Species: Ipomoea purpurea L. Roth, Convolvulaceae:

Organism / organ: plant / flowers

Authors / contributors: Cassie Majetic1 & Robert A. Raguso2

Affiliation: 1Department of Biology, Saint Mary’s College, Notre Dame, IN, USA 2Department of Neurobiology and Behavior, Cornell University, Ithaca, NY, USA

Reference: Majetic CJ, Rausher MD, & Raguso RA (2010) The pigment-scent connection: Do mutations in regulatory vs. structural anthocyanin genes differentially alter floral scent production in Ipomoea purpurea? South African Journal of Botany, 76: 632-642

Location: Saint Mary’s College Glasshouse, Notre Dame, IN, USA

Method sketch / description of data:  Experimental setup: Common garden experiment with inbred mutant or wildtype genetic lines (n = 51).

 Scent sampling / analyses: Dynamic headspace samples were collected from two detached flowers per plant. The flowers were enclosed with an oven bag and the emitted volatiles were then trapped on 10 mg SuperQ between quartz wool plugs in a glass Pasteur pipette for 1 h using a pump (flowrate c. 250 ml min-1). Samples were collected between 7:30 - 10:30 h. Samples were eluted with 300 µl hexane immediately following collection and stored at -20°C until analysis. Before analysis, samples were blown down to a volume of 75 µl and an internal standard of 5 µl of 0.03% toluene was added. Samples were analyzed using a Shimadzu GC17A gas chromatograph (EC- wax column) with a QP mass spectrometer detector (Shimadzu Corporation, Kyoto, Japan). Compounds were identified by comparing the mass fragment signatures of peaks to NIST and Wiley electronic spectral libraries. We also compared our volatile compound retention times and m/z ratios to the compounds identified using authentic terpenoid standards or Kovat's indices. Emission rates of ng compound g-1 fresh flower mass/h were calculated using external sesquiterpene standard curves.

Funding: CJM was supported by the Eli Lilly/Saint Mary’s College New Faculty Scholars program; RAR was supported by US National Science Foundation grant DEB-0746106.

11

(19) Species: Penstemon digitalis Nutt. ex Sims, Plantaginaceae

Organism / organ: plant / flowers

Authors / contributors: Amy L. Parachnowitsch1 & André Kessler2

Affiliation: 1Plant Ecology and Evolution, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden. 2Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY, USA

Reference: Parachnowitsch AL, Raguso RA & Kessler A (2012) Natural selection to increase floral scent emission, but not flower size or colour in bee-pollinated Penstemon digitalis. New Phytologist 195:667–675

Location: Thompkins County, NY, USA (42º29’249’’N 77º25’671’’W)

Method sketch / description of data:  Experimental setup: Common garden experiment with Penstemon digitalis individuals from three source populations (n = 88).

 Scent sampling / analyses: Dynamic headspace samples were collected from the whole inflorescence. The inflorescence was enclosed with 500 ml polyethylene cup and continuously sampled for 8 h and the emitted volatiles were trapped on activated charcoal absorbent vials (ORBO-32, SIGMA-Aldrich) using a pump (flow rate 450-500 ml min-1). Samples were collected between 10:30 - 6:30 h and taken over 4 d. ORBO- 32 absorbent vials were each eluted with 350 ml of dimethylchloride (SIGMA®) with 430 ng tetralin added for an internal standard. Samples were analyzed using a Varian 2200 GC/MS equipped with an EC WAX-column (Alltech Associates, USA). Compounds were identified by comparison of mass spectra and retention times with standard compounds or using the mass spectral library Nist 2011.

Funding: Ecology and Evolutionary Biology Department at Cornell (ALP), NSF grants DEB 0746106 (RAR) and DEB 0717139 (AK)

12

(20–27) Species: All Phlox and Polemonuim species, Polemoniaceae: Polemonium caeruleum ‘Bressingham Purple’ and ‘Album’ (n=9) Phlox bifida ‘Betty Blake’ and ‘Alba’ (n=8) Phlox carolina ‘Bill Baker’ and ‘Miss Lingard’ (n=8) Phlox drummondii ‘21st Century Mix’ (n=18) Phlox drummondii Hook. Wild (n=31) Phlox paniculata ‘David’s Lavender’, ‘Tall Tenor’, and ‘David’ (n=9) Phlox stolonifera ‘Pink Ridge’ and ‘Bruce’s White’ (n=12)

Organism / organ: plant / flowers

Authors / contributors: Cassie Majetic1 & Robert A. Raguso2

Affiliation: 1Department of Biology, Saint Mary’s College, Notre Dame, IN, USA 2Department of Neurobiology and Behavior, Cornell University, Ithaca, NY, USA

Reference: Majetic CJ, Levin DA, & Raguso RA (2014) Divergence in floral scent profiles among and within cultivated species of Phlox. Scientia Horticulturae, 172: 285-291

Location: Saint Mary’s College Glasshouse, Notre Dame, IN, USA

Method sketch / description of data:  Experimental setup: Common garden experiment with inbred horticultural lines of Polemoniaceae species (see complete list of sample sizes above).

 Scent sampling / analyses: Dynamic headspace samples were collected from one inflorescence per plant. The inflorescence was enclosed with an oven bag and the emitted volatiles were then trapped on 10 mg SuperQ between quartz wool plugs in a glass Pasteur pipette for 1 h using a pump (flowrate c. 250 ml min-1). Samples were collected between 10:00 - 14:00 h. Samples were eluted with 300 µl hexane immediately following collection and stored at -20°C until analysis. Before analysis, samples blown down to a volume of 75 µl and an internal standard of 5 µl of 0.03% toluene was added. Samples were analysed using a Shimadzu GC17A gas chromatograph (EC-wax fused column) with a QP mass spectrometer detector (Shimadzu Corporation, Kyoto, Japan). Compounds were identified by comparing the mass fragment signatures of peaks to NIST and Wiley electronic spectral libraries. We also calculated Kovat’s indices for all compounds and used this information to further verify compound identity where possible. Emission rates of ng compound g-1 fresh flower mass h-1 were calculated using the internal standard.

Funding: CJM was supported by the Eli Lilly/Saint Mary’s College New Faculty Scholars program; RAR was supported by US National Science Foundation grant DEB-0746106.

13

(30) Species: Silene latifolia Poiret, Caryophyllaceae

Organism / organ: flowers

Authors / contributors: Stefan Dötterl

Affiliation: University of Salzburg, Department of Ecology and Evolution, Salzburg, Austria

Reference: Dötterl S, Wolfe LW, Jürgens A (2005) Qualitative and quantitative analyses of flower scent in Silene latifolia. Phytochemistry, 66:203-213

Location: University of Bayreuth, Germany

Method sketch / description of data:  Experimental setup: Common garden experiment with potted individuals (n = 98).

 Scent sampling / analyses: Dynamic head-space samples were collected from single, newly opened flowers. A flower was enclosed within a polyethylene oven bag (Toppits®) and the emitted volatiles were trapped for 2 min in an adsorbent tube through the use of a membrane pump (ASF Thomas, Inc.). We took ChromatoProbe quartz microvials of Varian Inc. (length: 15 mm; inner diameter: 2 mm), cut the closed end, filled them with a mixture (1 : 1) of 3 mg Tenax-TA (mesh 60-80) and Carbotrap (mesh 20-40), and used them as adsorbent tubes. The samples were analysed by thermal desorption GC/MS on a Varian Saturn 2000 System using a 1079 injector, that had been fitted with the ChromatoProbe kit. A ZB-5 column (5% phenyl polysiloxane) was used for the analyses (60 m long, inner diameter 0.25 mm, film thickness 0.25 µm, Phenomenex). Component identification was carried out using the NIST 02 mass spectral data base (NIST algorithm), or MassFinder 2.3, and confirmed by comparison of retention times with published data (Adams, 1995; Davies, 1990). Identification of individual components was confirmed by comparison of both mass spectrum and GC retention data with those of authentic standards. For quantification of compounds known amounts of lilac aldehydes, trans-β-ocimene, cis-3-hexenyl acetate, benzaldehyde, phenylacetaldehyde, and veratrole were injected, and the mean response of these compounds was used for quantification.

Adams, R.P. (1995) Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. Allured Publishing Corporation, Carol Stream, IL, USA. Davies, N.W. (1990) Gas-chromatographic retention indexes of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. Journal of Chromatography A, 503, 1–24.

Funding: German Research Foundation (Research Training Group 678)

14

(31) Species: Silene latifolia Poiret, Caryophyllaceae

Organism / organ: flowers

Authors / contributors: Florian Schiestl

Affiliation: Institute of Systematic Botany, University of Zürich, Zollikerstrasse 107, 8008 Zürich Reference: Waelti, M. O., Page, P. A. Widmer, A., Schiestl, F. P. (2009). How to be an attractive male: floral dimorphism and attractiveness to pollinators in a dioecious plant. BMC Evolutionary Biology 9: 190.

Location: Switzerland

Method sketch / description of data: s. reference

Funding: SNF Swiss national science foundation

15

(32) Species: Sinapis alba, Brassicacea

Organism / organ: inflorescence

Authors / contributors: Robert Glinwood

Affiliation: Dept Crop Production Ecology, Swedish University of Agricultural Sciences, Box 7043, S750 07 Uppsala, Sweden

Reference: unpublished

Location: Controlled environment chamber, SLU, Sweden

Method sketch / description of data:  Experimental setup: Laboratory experiment with potted plants. 10 replicates

Scent sampling / analyses: Inflorescences were enclosed in a glass vessel (450 ml), closed using an aluminum manifold with a hole for the stem. Charcoal filtered air was pumped into the vessel (push flow 600 ml min-1) through a Teflon tube. Volatiles were trapped in glass tubes containing Tenax TA (50 mg) by letting air flowing through it (pull flow 300 ml min-1). Volatiles were collected for 24 h.

After volatile collection, internal standards (nonane and dodecanal) were injected onto the Tenax tubes. Quantification was made by GC using internal standards and compounds tentatively identified by GC/MS using NIST (08) and commercial standards where available.

 Funding: The Swedish Foundation For Strategic Environmental Research (Mistra) and Carl Tryggers Stiftelsen.

16

(39) Species: Sinapis arvensis L., Brassicaceae

Organism / organ: plant / flowers

Authors / contributors: Jonas Kuppler1,2 & Robert R. Junker2

Affiliation: 1Heinrich-Heine-University, Institute of Sensory Ecology, Düsseldorf, Germany 2University of Salzburg, Department of Ecology and Evolution, Salzburg, Austria

Reference: Kuppler J, Höfers MK, Wiesmann L & Junker RR (2016) Time-invariant differences between plant individuals in interactions with arthropods correlate with intraspecific variation in plant phenology, morphology and floral scent. New Phytologist, in press. doi: 10.1111/nph.13858

Location: Botanical Garden of the University of Salzburg, Austria

Method sketch / description of data:  Experimental setup: Common garden experiment with potted Sinapis arvensis individuals (n = 94). Plants were covered with net at least c. 12 h before sampling.

 Scent sampling / analyses: Dynamic headspace samples were collected from one inflorescence per plant. The inflorescence was enclosed with a polyester oven bag for 10 min and the emitted volatiles were then trapped on 1.5 mg Tenax (mesh 60-80) and 1.5 mg Carbotrap B (mesh 20–40) in a quartz vial for 2 min using a pump (flowrate 200 ml min-1). Samples were collected between 8:00 - 12:00 h. They were analysed using an automatic thermal desorption system (TD-20, Shimadzu, Japan; ZB-5 fused silica column, Phenomenex, Aschaffenburg, Germany) coupled with a GC-MS (model QP2010 Ultra El, Shimadzu, Japan). Emission was standardized to ng/h/flower. Compounds were identified by comparison of mass spectra and retention times with standard compounds, which are commercially available. Alternatively, compounds were identified using the mass spectral libraries Wiley 9, Nist 2011, FFNSC 2, Essential oils and Adams 2007 as well as the database available in MassFinder 3.

Funding: Graduate School ‘Evolutionary Networks: Organisms, Reactions, Molecules’ (E- Norm) of the Heinrich-Heine-University, Düsseldorf, Germany, and the Deutsche Forschungsgemeinschaft (DFG JU 2856/2-2).

17

(40–44) Species: Vicia faba L., Fabaceae

Organism / organ: plant / inflorescences including flowers, leafs and EFNs

Authors / contributors: Mathias Hoffmeister1,2 & Robert R. Junker2

Affiliation: 1Heinrich-Heine-University, Institute of Sensory Ecology, Düsseldorf, Germany 2University of Salzburg, Department of Ecology and Evolution, Salzburg, Austria

Reference:

Location: University of Düsseldorf, Germany

Method sketch / description of data:  Experimental setup: Experiment to test the effect of different herbivory treatments on floral scent emission. We tested four herbivory treatments, plants were either subjected (1) to a single jasmonic acid treatment (spraying of leafs until drip-off, 1 d before scent

sampling, nplants = 7), (2) to multiple jasmonic acid treatments (7 d and 1 d before scent

sampling, nplants = 14), (3) to Spodoptera exigua (2 larvae feeding on leafs in clip cages

for 7 d until sampling, nplants = 12) or (4) to Aphis fabae (80 aphids feeding on leafs in

clip cages for 7 d until sampling, nplants = 15). Control plants (C) were either sprayed

with a solution containing no jasmonic acid for treatment 1 (nplants = 9) and 2 (nplants =

7), or equipped with empty clip cages for treatment 3 (nplants = 12) and 4 (nplants = 13).  Scent sampling / analyses: Scent was sampled from unpicked wrapped inflorescences (oven bags, Toppits, Minden, Germany) as well as from surrounding air (empty oven bags) to determine plant-specific compounds. Volatiles were collected into traps filled filled with Poropak-Q (ARS, Inc, Gainesville, USA, flow rate: 1000 ml min-1, sampling: 60 min). Subsequently volatiles were eluted with 150 µl Dichloromethane containing Nonylacetate (10 ng µl-1) as internal standard. By means of a AOC-20i autoinjector (Shimadzu, Tokyo, Japan) 1 µl of the eluate was injected at 220°C (split ratio 1 : 1) into a Shimadzu GC-MS-QP2010 Ultra (Shimadzu, Tokyo, Japan, ZB-5 fused silica column, Phenomenex, Aschaffenburg, Germany). Column flow (carrier gas: helium) was set to 3 ml min-1. The GC oven temperature started at 40°C, was then increased to 220°C (10°C min-1) and held for 2 min. The MS interface worked at 250°C and the ion source at 200°C. Mass spectra were taken at 70 eV (in EI mode) from m/z 30 to 530. We processed GC/MS data with the GCMSolution package, Version 2.72 (Shimadzu Corporation 2012). Compounds were identified, comparing them to mass spectra and retention indices of commercially available standard substances or to compounds listed in mass spectral libraries Wiley Registry 9th Edition, NIST 2011 and FFNSC2. Using the internal standard we determined the emission rates of the identified volatiles.

Funding: Deutsche Forschungsgemeinschaft (DFG JU 2856/2-2).

18

(45) Species: Vigna unguiculata (L.) Walp. ssp. unguiculata var. spontanea (Schweinf.) Pasquet, Fabaceae/Leguminosae

Organism / organ: plant / flowers

Authors / contributors: Jette T. Knudsen12 & Rémy S. Pasquet3.

Affiliation: 1Dept. of Biology, Lund University, Lund, Sweden, 2Nattaro Labs AB, Medicon Village, 223 81 Lund, Sweden, 3IRD, Dept ECOBIO, 44 Bd de Dunkerque, 13572 Marseille cedex 02, France.

We acknowledge Beatrice Elesani for growing the plants and help with floral scent collection

Reference: NA Location: Muhaka Field Station, 32 km S-SE of Mombasa, Kenya

Method sketch / description of data:  Experimental setup: Screen house grown cowpea plants from seeds collected at 19 different locations mainly in coastal Kenya (n = 177). Plants were screened off from pollinator visitation.

 Scent sampling / analyses: Dynamic headspace samples were collected from one flower per plant. Each flower was covered with a polyacetate oven bag and an absorbent tube, filled with 20 mg Tenax Gr (mesh size 60-80) was inserted into the bag and the air within the bag was sucked through the absorbent tube with a battery driven membrane pump at a flow rate of 90–110 ml min-1. In parallel blank samples were collected from leaves or air. Floral scent collection started within 10 min of flower opening (c. 05:45-06:30 h local time) and lasted for about 120 min. Samples were extracted with 250 l of hexane. The samples were analysed by coupled GC-MS on a HP 6890 GC connected to a HP 5973 mass selective detector. Emission was standardized to %/flower. Almost all compounds were identified by comparison of mass spectra and retention times with commercially available standard compounds. Alternatively, compounds were identified using the mass spectral libraries NIST 2011 and Adams 2007.

Funding: SIDA/SAREC Grant (SWE-2005-340) awarded to JTK

19

(50–57) Species: Fragaria × ananassa Duch., Rosaceae

Organism / organ: plant / fruit

Authors / contributors: Anne Plotto1 & Celine Jouquand2

Affiliation: 1USDA, ARS, US Horticultural Research Laboratory, Fort Pierce, Florida, U.S.A. 2Institut Polytechnique Lasalle, Beauvais, France

Reference: Jouquand C, Plotto A, Goodner KL & Chandler CK (2008) A sensory and chemical analysis of fresh strawberries over harvest dates and seasons reveals factors that affect eating quality. J Amer Soc Hort Sci 133:859-867.

Location: Plant City, Florida, U.S.A.

Method sketch / description of data:  Experimental setup: Fruit harvested at commercial maturity from commercial fields in Plant City, Florida, on multiple harvests at monthly intervals.

 Scent sampling / analyses: About ten fruit per replication were homogenized for 20 s

in a blender, and an equal volume of saturated aqueous CaCl2 was added to stop enzymatic activity. Five millilitres of the mix + 3-hexanone internal standard were placed in a 20 mL vial. Volatiles were extracted using a 2-cm SPME fiber (50/30 mm DVB/Carboxen/PDMS; Supelco, Bellefonte, PA). After a sample equilibration time of 10 min at 40°C, the fiber was exposed to the headspace for 30 min at 40°C, then introduced into the injector of the GC-MS for 5 min at 250°C for desorption of volatiles. Identification of volatile compounds was carried out using an Agilent 6890 GC coupled with a 5973N MS detector and equipped with a DB5ms capillary column (60 m · 250 m · 1.00 m) (Agilent Technologies, Santa Clara, CA). Data were collected using the Chemstation G1701 AA (Agilent Technologies). Compounds were first identified by matching mass spectra with library entries (NIST/EPA/NIH Mass Spectral Library, Version 2.0d; National Institute of Standards and Technology, Gaithersburg, MD), then confirmed using retention indices calculated using retention time data from a series of alkane standards (C5–C17) run under the same chromatographic conditions. The relative concentration of volatile compounds was determined by normalizing the peak area of each compound to the peak area of internal standard.

Funding: University of Florida and USDA, ARS internal funds.

20

(58) Species: Tangarine, Citrus reticulata Blanco, Rutaceae

Organism / organ: plant / fruit

Authors / contributors: Anne Plotto1, & Fred G Gmitter2

Affiliation: 1USDA, ARS, US Horticultural Research Laboratory, Fort Pierce, Florida, U.S.A. 2University of Florida, Citrus Research and Education Center, Lake Alfred, Florida, U.S.A.

Reference: Miyazaki T, Plotto A, Goodner K & Gmitter FGJr. (2011) Distribution of aroma volatile compound in tangerine hybrids and proposed inheritance. J Sci Food Agric 91(3):449- 460 DOI 10.1002/jsfa.4205

Location: Citrus Research and Education Center, Lake Alfred, Florida, U.S.A.

Method sketch / description of data:  Experimental setup: Fifty to sixty fruit harvested from the University of Florida breeding population (single tree per genotype) (n = 25).

 Scent sampling / analyses: Fruit were juiced and 2.5 ml juice + 2.5 ml saturated calcium chloride were introduced to 20 mL sample vials. Volatiles were extracted using a 2-cm SPME fiber (50/30 mm DVB/Carboxen/PDMS; Supelco, Bellefonte, PA). After a sample equilibration time of 30 min at 40°C, the fiber was exposed to the headspace for 60 min at 40°C, then introduced into the injector of the GC-MS for 3 min at 250°C for desorption of volatiles. Identification of volatile compounds was carried out using an Agilent 6890 GC coupled with a 5973N MS detector and equipped with a DB5ms capillary column (60 m · 250 m · 1.00 m) or a DB-Wax (60 m · 250 m · 0.50 m) (Agilent Technologies, Santa Clara, CA). Data were collected using the Chemstation G1701 AA (Agilent Technologies). Compounds were first identified by matching mass spectra with library entries (NIST/EPA/NIH Mass Spectral Library, Version 2.0d; National Institute of Standards and Technology, Gaithersburg, MD), then confirmed using retention indices calculated using retention time data from a series of alkane standards (C5– C15) run under the same chromatographic conditions. The relative concentration of volatile compounds was determined by normalizing the peak area of each compound to the peak area of internal standard

Funding: University of Florida and USDA, ARS Internal funds

21

(59, 60) Species: Beta vulgaris L., Amaranthaceae

Organism / organ: plant / whole, non-flowering plant

Authors / contributors: Robert Glinwood

Affiliation: Dept Crop Production Ecology, Swedish University of Agricultural Sciences, Box 7043, S750 07 Uppsala, Sweden

Reference: unpublished

Location: Controlled environment chamber, SLU, Sweden

Method sketch / description of data:  Experimental setup: Laboratory experiment with potted plants (4–5 wk old at vegetative growth stage, eight to 10 leaves per plant). Ten replicates induced (infested with aphids), 19 replicates uninfested

Scent sampling / analyses: The plant was covered by a polyester baking bag (Melitta Scandinavia AB, Toppits 60 × 55 cm). Charcoal filtered air was pumped into the bag (push flow 600 ml min-1) from the lower part of the bag through a Teflon tube. Volatiles were trapped in glass tubes containing Porapak Q (mesh 50/80) by letting air flowing through it (pull flow 450 ml min-1). Induced plants were infested with 300 Aphid fabae (Hemiptera: Aphididae) for 72 h before volatile collection. Control plants were uninfested.

After 72 h the collected volatiles compounds were rinsed out with 750 µl redistilled Dicloromethane and internal standard added (2-tridecanone). The sample was then reduced to 50 µl and analyzed on GC-MS. Quantification was made using internal standards and compounds tentatively identified using NIST (08) and commercial standards where available.

 Funding: The Swedish Research Council (Formas), Carl Tryggers Stiftelse för Vetenskaplig Forskning and the Faculty of Natural Resources and Agricultural Sciences, SLU.

22

(71–73) Species: Arabidopsis thaliana, Brassica oleracea & Brassica nigra. organ: plant

Organism: Brevicoryne brassicae, Rhizobacteria, Pieris rapae, Eggs of Pieris brassicae, Eggs of Mamestra brassicae, Cotesia glomerata, Cotesia rubecula.

Author / contributor: Berhane T. Weldegergis Affiliation: Laboratory of Entomology, Wageningen University, P.O. Box 8031, 6700 EH Wageningen, The Netherlands

Reference:

1. Ana Pineda, Roxina Soler, Berhane T. Weldegergis, Mpoki M. Shimwela, Joop J.A. van Loon & Marcel Dicke (2013) Non-pathogenic rhizobacteria interfere with the attraction of parasitoids to aphid-induced plant volatiles via jasmonic acid signalling. Plant, Cell and Environment 36 (2), 393-404. doi: 10.1111/j.1365-3040.2012.02581.x 2. Wouter Kegge, Berhane T. Weldegergis, Roxina Soler, Marleen Vergeer-Van Eijk, Marcel Dicke, Laurentius A. C. J. Voesenek & Ronald Pierik (2013) Canopy light cues affect emission of constitutive and methyl jasmonate-induced volatile organic compounds in Arabidopsis thaliana. New Phytologist 200(3), 861-874. doi: 10.1111/nph.12407 3. Erik H. Poelman, Maaike Bruinsma, Feng Zhu, BerhaneT. Weldegergis, Aline E. Boursault, Yde Jongema, Joop J.A. van Loon, Louise E.M. Vet, Jeffrey A. Harvey & Marcel Dicke (2012) Hyperparasitoids use herbivore-induced plant volatiles to locate their parasitoid host. PLoS Biology, 10(11): e1001435. doi:10.1371/journal.pbio.1001435 4. Nina E. Fatouros, Dani Lucas-Barbosa, Berhane T. Weldegergis, Foteini G. Pashalidou, Joop J.A. van Loon, Marcel Dicke, Jeffrey A. Harvey, Rieta Gols & Martinus E. Huigens (2012) Plant volatiles induced by herbivore egg deposition affect insects of different trophic levels. PLoS ONE, 7(8): e43607. doi:10.1371/journal.pone.0043607 5. Roxina Soler, Ana Pineda, Yhua Li, Camille Ponzio, Joop J.A. van Loon, Berhane T. Weldegergis & Marcel Dicke (2012) Neonates know better than their mothers when selecting a host-plant. Oikos 121, 1923–1934. doi: 10.1111/j.1600- 0706.2012.20415.x

Location: Laboratory of Entomolgy, Wageningen University, Wageningen, the Netherlands

Method sketch / description of data:  Experimental setup: Glasshouse experiment with potted individual plants ranging 5 – 10 replicates for each treatment group.

Plant volatiles were collected from the headspace of each sample. Before volatile collection, the pots were removed and the plant roots and soil were carefully wrapped with aluminium foil. The plants were then placed in glass jars and sealed with a Viton-lined glass lid with an inlet and outlet. Compressed air was filtered by passing through charcoal before entering the sampling container. Volatiles were trapped by drawing air out of the jars through a stainless steel tube filled with 200 mg Tenax TA (20/35 mesh).Thermo Trace Ultra GC coupled with Thermo Trace DSQ MS (Thermo Fisher Scientific Waltham, USA) were used for separation and detection of plant volatiles. The collected volatiles were released thermally from the Tenax

23

TA cartridges using thermal desorption unit (Ultra 50:50, Markes, Llantrisant, UK), while re- collecting them in a thermally cooled universal solvent trap (Unity,Markes). Once the desorption process was completed, the cold trap was ballistically heated, while the volatiles being transferred to a ZB-5MSi capillary column: 30 m L × 0.25 mm I.D. × 1.00 µm F.T. (Phenomenex, Torrance, CA, USA) for further separation in a programmed GC oven temperature. The DSQ MS was operated in a scan mode and ionisation was performed in EI at 70 eV. The MS transfer line and ion source were set at 275 and 250°C, respectively. Identification of compounds was based on comparison of mass spectra with the NIST 2005 and Wageningen Mass Spectral Database of Natural Products libraries as well as linear retention indices (LRI). Quantitation (peak area measurements) was performed using a single (target) ion in selected ion monitoring (SIM) mode. Individual peak areas were corrected for the aerial fresh weight of each plant sample (peak area unit g-1 plant FW). For details on the methodologies please refer to references 1 – 5 above.

Funding: The Netherlands Organization for Scientific Research (NWO).

24

(74–76) Species: Hordeum vulgare L., Poaceae

Organism / organ: plant / whole, non-flowering plant

Authors / contributors: Robert Glinwood

Affiliation: Dept Crop Production Ecology, Swedish University of Agricultural Sciences, Box 7043, S750 07 Uppsala, Sweden

Reference: unpublished

Location: Controlled environment chamber, SLU, Sweden

Method sketch / description of data:  Experimental setup: Laboratory experiment with potted plants. Eight pots of 20 plats (1 pot = 1 replicate) for each of three cultivars Alva, Barke and Frieda

Scent sampling / analyses: A pot of 20 barley plants (two leaf stage) was covered by a polyester baking bag (Melitta Scandinavia AB, Toppits 60 × 55 cm). Charcoal filtered air was pumped into the bag (push flow 600 ml min-1) from the lower part of the bag through a Teflon tube. Volatiles were trapped in glass tubes containing Porapak Q (mesh 50/80) by letting air flowing through it (pull flow 450 ml min-1).

After 72 h the collected volatiles compounds were rinsed out with 750 µl redistilled Dicloromethane and internal standard added (1-nonene). The sample was then reduced to 50 µl and analyzed on GC-MS. Quantification was made using internal standards and compounds tentatively identified using NIST (08) and commercial standards where available.

 Funding: The Swedish Research Council (Formas), The Swedish Foundation For Strategic Environmental Research (Mistra) and the Faculty of Natural Resources and Agricultural Sciences, SLU.

25

(77, 78) Species: Malus silvestris Miller (variety De Costa), Rosaceae

Organism / Organ: plant / leaves

Authors / contributors: Luisa Amo1,2 & Marcel Visser1

Affiliation: 1 Department of Animal Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, The Netherlands 2 Department of Evolutionary Ecology, Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain

Reference: Amo, L., Jansen, J.J., van Dam, N.M., Dicke, M. & Visser, M.E. 2013. Birds exploit herbivore-induced plant volatiles to locate herbivorous prey. Ecology Letters, 16: 1348–1355.

Location: Heteren, The Netherlands

Method sketch / description of data:

Experimental setup: Common garden experiment with potted Malus silvestris individuals (n = 32). Sixteen individuals were uninfested and 16 individuals were infested with 30 winter moth Operopthera brumata caterpillars (L5 stage) during 3 d. Caterpillars were located in clip-cages, so they could only eat one leave. The day of chemical measurements, we removed caterpillars and cut the damaged parts of the leaves around 3 h before the chemical measurements. In the control uninfested trees, we also cut a similar number of leaves 3 h before the chemical measurements.

Scent sampling / analyses: Dynamic headspace samples were collected from one branch per tree (16 infested and 16 uninfested) between 16:30 and 19:00 h. The branch was enclosed with a polyethylene oven bag s and the volatiles were trapped on 150 mg Tenax TA and 150 mg Carbopack B (Markes International Limited, Llantrisant, UK) tubes for 2 h using a vacuum pump (flowrate 200 ml min-1). Traps were stored at 4°C for 10–11 wk until analysis. Volatiles were desorbed from the traps using an automated thermodesorption unit (model Unity, Markes, Llantrisant, UK) coupled with a GC-MS (model Trace, ThermoFinnigan, Austin, Texas). The volatiles were detected by the MS operating at 70 eV in EI mode. Mass spectra were acquired in full scan mode (33–300 amu. 0.4 scan s-1). Compounds were identified by their mass spectra using deconvolution software (AMDIS; NIST (International Institute of Standards and Technology, MD, USA) and US DOD (Department of Defense, DC, USA)) in combination with Nist 98 and Wiley seventh edition spectral libraries and by comparing their linear retention indices. In addition, mass spectra and/or linear retention indices of chromatographic peaks were compared with values reported in the literature. Additional confirmation for compound identification was obtained by interpolating retention indices of homologous series, or by comparing analytical data with those of reference substances. The integrated signals generated by the AMDIS software from the MSchromatograms were used for comparison between the treatments. Peak areas in each sample were divided by the total volume in ml that was sampled over the trap, to correct for small differences in flow rates over individual traps.

Funding: MEC postdoctoral programme to L.A. and NWO-VICI grant.

26

(79, 80) Species: Phaseolus lunatus, Fabaceae

Organism / organ: leaves

Authors / contributors: Christian Kost1,2 & Martin Heil2

Affiliation: 1Max Planck Institute for Chemical Ecology, Research Group Experimental Ecology and Evolution, Jena, Germany, 2University of Osnabrück, Department of Ecology and Evolution, School of Biology/ Chemistry, Osnabrück, Germany, 3CINVESTAV – Irapuato, Departamento de Ingeniería Genética, Irapuato, México.

Reference: Kost, C, Heil, M (2006). Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants. Journal of Ecology, 94(3), 619-628.

Location: coastal area, dirt roads leading to extensively used pastures or plantations, Puerto Escondido, Mexico.

Method sketch / description of data:  Experimental setup: Use of laboratory-grown plants to test the effect of feeding of a specialised leaf herbivore on the emission of volatile organic compounds. The five youngest leaves of two potted Lima bean plants were exposed to five adult Epilachna varivestis (Mexican bean beetle) for 2 d at room temperature, while two control plants were left undamaged. Volatiles were collected from the five youngest leaves of all four plants for the next 24 h. This experiment was replicated nine times.

 Scent sampling / analyses: Tendrrils were bagged in a PET foil (‘Bratenschlauch’, Toppits, Minden, Germany) that does not itself emit detectable amounts of volatiles. The emitted VOCs were collected continuously over 24 h on charcoal traps (1.5 mg char- coal, CLSA-Filters, Le Ruissaeu de Montbrun, France) using air circulation. After 24 h, leaves were dried for dry mass determination and volatiles were eluted from the carbon trap with dichloromethane (40 μl) containing 1-bromodecane (200 ng μl−1) as an internal standard. Samples were analysed on a GC-Trace mass spectrometer (Thermo Finnigan, www.thermofinnigan.com). The extracts were directly analyzed by GC-MS using fused-silica capillary tubes (15 m, 0.25 mm; Alltech, Unterhaching, Germany) coated with DB 1 (0.1 m). Helium at 60 kPa served as the carrier gas. Separation of the compounds was achieved under programmed conditions (40°C for 2 min, then at 10°C min 1 to 200°C). MS analysis was performed (model MD800, Fisons, Bellevue, WA) with the GC interface at 260°C and the scan range at 35 to 300 D. Individual compounds (peak areas) were quantified with respect to the peak area of the internal standard and related to the dry weight of the measured tendril.

 Funding: This study was financially supported by the German Research Foundation (DFG grants HE 3169/2–1,2,3,4) to MH.

27

(81, 82) Species: Pinus sylvestris L., Pinaceae

Organism / organ: plant / leaves (needles)

Authors / contributors: James D. Blande

Affiliation: Department of Environmental and Biological Sciences, University of Eastern Finland, Kuopio, Finland.

Reference: Heijari J, Blande JD & Holopainen JK (2011) Feeding of large pine weevil on Scots pine stem triggers localised bark and systemic shoot emissions of volatile organic compounds. Environmental and Experimental Botany, 71, 390-398.

Location: Research garden of the Kuopio campus of the University of Eastern Finland, Finland.

Method sketch / description of data:  Experimental setup: Field study with potted 2-yr-old Scots pine saplings. A total of 17 saplings were each infested with two large pine weevils, which were enclosed in mesh bags around the stem; 16 saplings were fitted with an empty mesh bag and were used as controls.

 Samples were collected from the foliage of the saplings by dynamic headspace analysis after 3 d of herbivore-feeding. The main shoot and side branches above the site of damage were enclosed in a polyethylene terephthalate (PET) bag (size 45 cm × 55 cm). Clean filtered air was pumped into the bag and the headspace was drawn out through a stainless steel tube filled with Tenax TA adsorbent (150 mg) (Supelco, mesh 60/80) at a flow rate of 200 ml min-1. Samples were analysed by GC-MS (Hewlett- Packard GC 6890, MSD 5973) with trapped compounds thermally desorbed with an automated thermal desorption unit (Perkin-Elmer ATD400). Emissions were calculated in units of ng g (DW)-1 h-1. Compounds were identified by comparing mass spectra and retention times with a series of commercially available terpene and green-leaf volatile standards. Compounds for which there was not an available standard were identified by comparison of mass spectra with the Wiley library.

Funding: Academy of Finland (decision numbers 111543, 256050, 251898 and 141053).

28

(83–88) Species: Populus nigra, Salicaceae

Organism / organ: Leaves

Authors / contributors: Andrea Clavijo McCormick1, Sandra Irmisch1, Andreas Reinecke2,4, G. Andreas Boeckler1, Daniel Veit3, Michael Reichelt1, Bill Hansson2, Jonathan Gershenzon1, Tobias G. Köllner1 & Sybille B. Unsicker1*

1 Max Planck Institute for Chemical Ecology, Department of Biochemistry 2 Max Planck Institute for Chemical Ecology, Department of Neuroethology 3 Max Planck Institute for Chemical Ecology, Department of Scientific Instrumentation and Utilities Management 4Current address: Max Planck Institute for Ornithology, Department of Behavioural Ecology and Evolutionary Genetics

Reference: Clavijo McCormick A., Irmisch S., Reinecke A., Boeckler G.A., Veit D., Reichelt M., Hansson B.S., Gershenzon J., Köllner T.G. & Unsicker S.B. (2014) Herbivore- induced volatile emission in black poplar: regulation and role in attracting herbivore enemies. Plant Cell and Environment, 37, 1909-1923.

Location: Glasshouse, climate chamber, Jena, Germany

Method sketch / description of data: To investigate the emission of volatiles from young P. nigra after Lymantria dispar caterpillar feeding, two individual trees (c. 1.20 m in height) of each of 20 different genotypes were selected. Twenty trees were infested with gypsy moth caterpillars and the other 20 functioned as controls. Thus each treatment contained 20 tree genotypes as replicates. During the experiment, trees were kept in a climate chamber. The foliage of each tree enclosed with PET foil (Toppits® Bratschlauch, Minden, Germany) Seven fourth instar L. dispar caterpillars were released in the PET bags covering the basal section of the black poplar foliage and allowed to feed for 41 h.

Volatiles were collected using a dynamic push-pull system. Charcoal filtered air was pumped into the PET bags at a flow rate of 1.5 l min-1. At the same time a portion of the air was pumped out of the bags with a second pump at a flow of 1 l min-1. The outgoing air passed through a trap packed with 20 mg Super Q adsorbent (ARS, Inc., Gainesville, USA) to trap the volatile compounds. Volatiles were collected for 4 h the second day after herbivore release on the plants. Volatile compounds were desorbed by eluting the filter twice with 200 µl dichloromethane containing nonyl acetate as an internal standard (10 ng µl-1).

Analysis of black poplar volatiles was carried out using GC-MS/FID analysis an Agilent 6890 series gas chromatograph (Agilent, Santa Clara, CA, USA) coupled either to an Agilent 5973 quadrupole mass selective detector (interface temp. 270 °C, quadrupole temp. 150°C, source temp. 230°C; electron energy 70 eV) or a flame ionization detector (FID, temp. 300°C). The constituents were separated with a DB-5MS column (30 m × 0.25 mm × 0.25 μm) and He

29

(mass detector) or H2 (FID) as carrier gases. One microliter of the sample was injected without splitting at an initial oven temperature of 40°C (2 min hold) followed by a ramp to 155°C (1°C min-1), a ramp to 300°C (60°C min-1), and a hold for 3 min. To confirm identification of coeluting compounds, samples were also run on an HP Innowax column (Agilent, 30 m × 0.25 mm × 0.25 μm) under the same conditions, except the final temperature gradient ended at 260°C.

Compounds were identified by comparison of retention times and mass spectra to those of authentic standards obtained from Fluka (Seelze, Germany), Roth (Karlsruhe, Germany), Sigma (St. Louis, MO, USA) or Bedoukian (Danbury, CT, USA) or to reference spectra in the Wiley and National Institute of Standards and Technology libraries and in the literature (Joulain & König, 1998). Standards not commercially available were provided by Wilfried A. König, University of Hamburg (essential oils of Oreodaphne porosa and Aloysia sellowii) and DMNT was kindly synthesized by Stefan Bartram (MPI-ICE). The quantity of each compound was determined from its peak area in the FID trace in relation to the area of the internal standard using the effective carbon number concept (Scanion & Wills, 1985). Chiral analysis was performed using a RtTM-ßDEXsm column (Restek, Bad Homburg, Germany) with a temperature program from 50°C (2 min hold) to 220°C (1 min hold) with a gradient of 2°C min-1.

Joulain, D. and König, W.A. (1998) The Atlas of Spectral Data of Sesquiterpene Hydrocarbons. EB-Verlag, Hamburg, Germany.

Scanlon, J.T. and Willis D.E. (1985) Calculation of Flame Ionization Detector Relative Response Factors Using the Effective Carbon Number Concept. Journal of Chromatographic Science, 23, 333–340.

Funding: This project was funded by the Max Planck Society

30

(89, 90) Species: Salix viminalis L., Salicaceae

Organism / organ: plant / whole, non-flowering plant

Authors / contributors: Robert Glinwood

Affiliation: Dept Crop Production Ecology, Swedish University of Agricultural Sciences, Box 7043, S750 07 Uppsala, Sweden

Reference: unpublished

Location: Controlled environment chamber, SLU, Sweden

Method sketch / description of data:  Experimental setup: Laboratory experiment with potted plants. Eight induced (infested) and eight control (uninfested) plants

Scent sampling / analyses: The plant (c. 30–40 cm high with 2–3 ‘twigs’) was covered by a polyester baking bag (Melitta Scandinavia AB, Toppits 60 × 55 cm). Induced plants were infested by 10 Anthocoris nemorum (Hemiptera: Anthocoridae) for 24 h. Charcoal filtered air was pumped into the bag (push flow 600 ml min-1) from the lower part of the bag through a Teflon tube. Volatiles were trapped in glass tubes containing Porapak Q (mesh 50/80) by letting air flowing through it (pull flow 450 ml min-1).

After 24 h the collected volatiles compounds were rinsed out with 750 µl redistilled Dicloromethane and internal standards added (1-nonene and Dodecanal). The sample was then reduced to 50 µl and analyzed on GC-MS. Quantification was made using internal standards and compounds tentatively identified using NIST (08) and commercial standards where available.

 Funding: The Swedish Research Council (Formas) and the Faculty of Natural Resources and Agricultural Sciences, SLU.

31

(91) Species: Austroplebeia australis F., Apidae

Organism / organ: animal / cuticular surface

Authors / contributors: Sara D. Leonhardt1 & Thomas Schmitt1 Affiliation: 1Würzburg University, Department of Animal Ecology and Tropical Biology, Würzburg, Germany

Reference: Leonhardt SD, Wallace HM & Schmitt T (2011) The cuticular profiles of Australian stingless bees are shaped by resin of the eucalypt tree Corymbia torelliana. Austral Ecology, 36: 537-543

Locations: Australia: Sydney, Elonora, Dalby

Method sketch / description of data:  Specimen sampling / analyses: Five to 10 specimens were obtained from the entrance of a bee colony by capturing leaving foragers in a clean clear plastic bag, killed by quick freezing and extracted in hexane for 5 min. Cuticular extracts were analysed by a Hewlett Packard HP 6890 Series gas chromatograph coupled to a Hewlett Packard HP 5973 Mass Selective Detector (Agilent Technologies, Böblingen, Germany) on a DB-5 fused silica capillary column (30 m _ 0.25 mm ID; d.f. ¼ 0.25 mm; J & W, Folsom, CA, USA) with helium used as carrier gas (constant flow of 1 ml/ min). Injection was carried out at 250°C in the splitless mode for 1 min. Temperature was raised from 60°C to 300°C with a 5°C min-1 heating rate and held at 300°C for 10 min. Electron impact mass spectra were recorded at 70 eV. Compounds were identified by comparison of mass spectra and retention times with standard compounds, which are commercially available. Alternatively, compounds were identified using the mass spectral libraries Wiley 9, Nist 98 and Adams EO Library 2205.

Funding: Deutsche Forschungs-Gemeinschaft (DFG project: LE 2750/1-1) and a grant of the German Excellence Initiative to the Graduate School of Life Science, University of Würzburg.

32

(93, 94) Species: Crematogaster scutellaris, Formicidae, Hymenoptera

Organism / organ: animal / body surface

Authors / contributors: Florian Menzel

Affiliation: University of Mainz, Institute of Zoology, Mainz, Germany

Reference: Menzel F, Woywod M, Blüthgen N, Schmitt T (2010): Behavioural and chemical mechanisms behind a Mediterranean ant-ant association. Ecological Entomology 35: 711-720

Location: Esterel massif, (Côte d’Azur, France) and Dolceacqua, Liguria, Italy.

Method sketch / description of data:

 Cuticular extracts were obtained by immersing between four and 12 freeze-killed ants in hexane for 10 min (as a pooled sample), and analysed using GC-MS with a Hewlett Packard 6890 series gas chromatograph coupled to a HP 5973 Mass Selective Detector. The GC was equipped with a J&W Scientific DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 μm). Temperature was kept at 60°C for 2 min, then increased by 60°C min−1 up to 200°C and subsequently by 4°C min−1 to 320°C, where it remained constant for 10 min. Helium was used as carrier gas with a constant flow of 1 ml min−1. The samples were injected in splitless mode for 30 s at 250°C. The electron impact mass spectra (EI-MS) were recorded with an ionisation voltage of 70 eV, a source temperature of 230°C, and an interface temperature of 325°C. The cuticular hydrocarbons were identified based on diagnostic ions and the Kovat’s retention index.

Funding: none

33

(95–98) Species: Dibrachys cavus, Hymenoptera: Pteromalidae

Organism / organ: whole body extract

Authors / contributors: Joachim Ruther

Affiliation: University of Regensburg, Institute of Zoology, Regensburg, Germany

Reference: Ruther J, Döring M, Steiner S (2011) Cuticular hydrocarbons as contact sex pheromone in the parasitoid Dibrachys cavus. Entomologia Experimentalis et Applicata 140: 59-68.

Location: Lab strain, originally collected from a bird’s nest near Hamburg, Germany.

Method sketch / description of data:  Experimental setup: Parasitic wasps were reared on freeze-killed pupae of the green bottle fly Lucilia Caesar L. (Diptera: Calliphoridae) at 25°C and 55% r.h. at a L16 : D8 photoperiod. Male and female wasps were excised from the host 1-2 d before the expected eclosion date and extracted immediately or 1-2 d after emergence.

 Scent sampling / analyses: Newly emerged and 1- to 2-d-old males and females were extracted for 30 min with 10 µl dichloromethane containing 10 ng µl-1 of n-tetracosane as an internal standard (n = 10 for each sex and age class). Extracts (1 µl per sample) were analysed by coupled gas chromatography–mass spectrometry (GC/MS) on a Fisons 8060 GC coupled to a Fisons MD 800 quadrupole MS (Thermo Finnigan, Egelsbach, Germany). Analytical conditions were as follows. Injector temperature: 280°C, column: 30 m × 0.32 mm inner diameter DB-5ms, film thickness 0.25 lm (J & W, Scientific, Folsom, CA, USA), carrier gas: helium, inlet pressure 10 kPa. The temperature program started at 150°C and increased at 3°C per min to 280°C. Methyl- branched hydrocarbons were identified using diagnostic ions resulting from the favoured fragmentation at the branching points and by comparing linear retention indices with literature data.

 Funding: Deutsche Forschungsgemeinschaft (DFG Ru 717/8-2).

34

(99, 100) Species: Heliothis virescens Fabricius and Heliothis subflexa Guenée, Noctuidae

Organism /organ: moth, female sex pheromone gland

Authors / contributors: Astrid T. Groot1,2 Affiliation: 1University of Amsterdam, Institute for Biodiversity and Ecosystem Dynamics (IBED), Amsterdam, the Netherlands 2Max Planck Institute for Chemical Ecology, Department of Entomology, Jena, Germany

Reference: Groot AT, Inglis O, Bowdrigde S, Santangelo RG, Blanco C, et al. 2009. Geographic and temporal variation in moth chemical communication. Evolution 63:1987-2003

Location: North Carolina State University

Method sketch / description of data:  (Copied from Groot et al. 2009): Eggs of Hv and larvae of Hv and Hs were collected in the field, as described in Groot et al. 2009 (reference above). Were reared to adults on artificial diet at North Carolina State University (NCSU). Hs larvae were given Physalis angulata fruits in addition to the artificial diet; pupae were separated by sex and checked daily for emergence. Pheromone glands were extracted from 2 to 5 d old virgin females that emerged from the field-collected larvae and/or from their female offspring (i.e. females that were reared in the lab for one generation). Glands were extracted and analyzed as described for M. brassicae.

Funding: National Research Initiative of the USDA Cooperative State Research, Education and Extension Service (CSREES), grant # 2005–00896, the W.M. Keck Center for Behavioral Biology, and the Blanton J. Whitmire Endowment at North Carolina State University.

35

(101) Species: Homo sapiens

Organism / organ: NA

Authors / contributors: Niels O. Verhulst1

Affiliation: 1Laboratory of Entomology, Wageningen University, P.O. Box 8031, 6700 EH Wageningen, the Netherlands

Reference: Verhulst NO, Beijleveld H,Qiu YT, Maliepaard C, Verduyn W, Haasnoot GW, Claas FHJ, Mumm R, Bouwmeester HJ, Takken W, van Loon JJA, & Smallegange RC 2013 Relation between HLA genes, human skin volatiles and attractiveness of humans to malaria mosquitoes. Infection Genetics and Evolution. 18: 87-93. dx.doi.org/10.1016/j.meegid.2013.05.009

Location: Wageningen University, The Netherlands

Method sketch / description of data:  Experimental setup: Samples were collected from nine individuals that were highly attractive to malaria mosquitoes and seven individuals that were poorly attractive to these malaria mosquitoes. Individuals were asked to refrain from eating spicy food, taking a shower and using perfumed cosmetics for 24 h before sampling.

 Scent sampling / analyses: Volatiles were collected by rubbing the sole of the foot over 100 glass beads for 10 min. The beads were transferred to steel cartridges and emanations analyzed by thermodesorption followed by GC–MS (Trace GC Ultra, quadruple mass detector, DSQ, Thermo Scientific, USA). Analytes were split to 1/6 of the total amount and separated on a RTX-5ms GC column (Restek, USA). Compounds were identified by comparing their mass spectra and retention times with those of authentic reference compounds. Relative quantification of the compounds was done based on characteristic mass ions for each compound using the software package Xcalibur (Version 2.07, Thermo Scientific, USA). Integration settings for each compound were inserted in a processing setup, batch processed and evaluated in the Quan browser.

Funding: The Foundation for the National Institutes of Health through the Grand Challenges in Global Health Initiative (GCGH#121), and a grant from the Earth and Life Science Foundation of the Netherlands Organization for Scientific Research (820.01.019).

36

(102) Species: Mamestra brassicae L., Noctuidae

Organism /organ: moth, female sex pheromone gland

Authors / contributors: Astrid T. Groot1,2 Affiliation: 1University of Amsterdam, Institute for Biodiversity and Ecosystem Dynamics (IBED), Amsterdam, the Netherlands 2Max Planck Institute for Chemical Ecology, Department of Entomology, Jena, Germany

Reference: Van Geffen K, Groot AT, Van Grunsven RHA, Donners M, Berendse F, Veenendaal E. 2015. Artificial night lighting disrupts sex pheromone in a noctuid moth. Ecological Entomology 40: 401-408

Location: Wageningen University and University of Amsterdam

Method sketch / description of data:  (see Van Geffen et al. 2015; summary:) In a glasshouse, 20 open-top compartments of 50 × 50 × 60 cm3 were constructed. Each compartment was randomly assigned to one of four artificial light treatments: green-rich, warm white, red-rich or no artificial light at night. Treatments were divided over five randomised blocks. Each each compartment contained five pupae, resulting in a nested design. Pupae were checked for emergence every day and freshly emerged moths were provided with sugar water. Each night, all three-night-old females were collected for gland extraction. Glands of 24, 22, 22, and 24 females from green, white, red and dark control treatments, respectively, were successfully extracted.

 Scent sampling / analyses: (full description in Groot et al. 2010): Each gland was extracted separately in a glass conical vial in 50 µl of n-hexane (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) containing 25 ng of the internal standard pentadecane. After 30–40 min, the glands were removed and the extract stored at -20 °C until GC analysis. For GC analysis, the extracts were reduced to 1–2 µl under a gentle stream of nitrogen and taken up with a 10 µl syringe (701SN 26S GA 2 needle; Hamilton, Reno, NV, USA) together with 1 µl of octane (Fluka, St Louis, MO, USA) to avoid evaporation. The sample was injected with a 7683 automatic injector into the splitless inlet of an HP7890 GC, which was coupled with a high resolution polar capillary column (DB-WAXetr (extended temperature range); 30 m ・ 0.25 mm ・ 0.5 lm) and a flame-ionization detector (FID) using the following program: 60°C (hold for 2 min) to 180°C (30°C min), followed by an increase of temperature to 230°C (5°C min). The column was then heated to 245°C at 20°C min) for 15 min. The FID was kept at 250°C. To identify the particular compounds, a multicomponent blend (compounds from Pherobank, Wageningen) containing all the pheromone compounds was injected into the GC before or after each daily series of injections. For quantitative analysis, the GC signal was integrated with the software CHEMSTATION (Agilent, Technologies Deutschland GmbH, Böblingen, Germany).

Groot AT, Classen A, Staudacher H, Schal C, Heckel DG. 2010. Phenotypic plasticity in sexual communication signal of a noctuid moth. Journal of Evolutionary Biology 23: 2731- 2738

Funding: NWO-STW grant 11110, Philips Lighting, and the Nederlandse Aardolie Maatschappij

37

(103–119) Species: Myrmica rubra, Formicidae, Hymenoptera

Organism / organ: animal / body surface

Authors / contributors: Florian Menzel

Affiliation: University of Mainz, Institute of Zoology, Mainz, Germany

Reference: Pamminger T, Foitzik S, Kaufmann KC, Schützler N, Menzel F (2014): Worker personality and its association with spatially structured division of labor. PLoS One 9: e79616

Location: Ober-Olmer forest near Mainz, Rhineland-Palatinate, Germany

Method sketch / description of data:

 Cuticular extracts were obtained by immersing individual freeze-killed ants in hexane for 10 min, and analysed using GC-MS with a Hewlett Packard 7890A series gas chromatograph coupled to a HP 5975C Mass Selective Detector. The GC was equipped with an Agilent HP-5MS fused silica capillary column (30 m × 0.25 mm × 0.25 μm). Temperature was kept at 60°C for 2 min, then increased by 60°C min−1 up to 200°C and subsequently by 4°C min−1 to 320°C, where it remained constant for 10 min. Helium was used as carrier gas with a constant flow of 1 ml min−1. The samples were injected in splitless mode for 30 s at 250°C. The electron impact mass spectra (EI-MS) were recorded with an ionisation voltage of 70 eV, a source temperature of 230°C, and an interface temperature of 325°C. The cuticular hydrocarbons were identified based on diagnostic ions and the Kovat’s retention index.

Funding: none

38

(120, 121, 124) Species: Odynerus spinipes (Vespidae), Pseudospinolia neglecta, Chrysis mediata (Chrysididae)

Organism / organ: insects

Authors / contributors: Thomas Schmitt

Affiliation: University of Würzburg, Department of Animal Ecology and Tropical Biology, Würzburg, Germany

Reference: Wurdack M, Herbertz S, Dowling D, Kroiss J, Strohm E, Baur H, Niehuis O, Schmitt T (2016) Striking cuticular hydrocarbon dimorphism in the mason wasp Odynerus spinipes and its possible evolutionary cause (Hymenoptera: Chrysididae, Vespidae). Proceedings of the Royal Sociecty B, London 282: 20151777

Location: Kaiserstuhl, Baden-Württemberg, Germany

Method sketch / description of data:  Experimental setup: Comparative study on chemical mimicry of a vespid host and its chrysidid brood parasitoids.

 Scent sampling / analyses: CHCs were extracted by submerging the freeze-killed wasps for 10 min in n-pentane. In all extracts, the volume of n-pentane was adjusted to 200 µl, and all extracts were stored at -20°C before chemical analysis. The CHC extracts were analyzed with a gas chromatograph (GC) coupled with a Mass Selective (MS) Detector. The CHC extracts were analyzed with a HP 6890 gas chromatograph (GC) coupled with a HP 5973 Mass Selective (MS) Detector (Hewlett Packard, Waldbronn, Germany). The GC (split/splitless injector in splitless mode for 1 min, injected volume: 1 µl at 250°C) was equipped with a DB-5 Fused Silica capillary column (30 m × 0.25 mm ID, 0.25 m³; J&W Scientific, Folsom, USA). Helium served as a carrier gas at a constant flow of 1 ml min-1. The following temperature program was used: start temperature 60°C, temperature increase by 5°C min-1 up to 300°C, isotherm at 300°C for 10 min. The electron ionization mass spectra (EI-MS) were acquired at an ionization voltage of 70 eV (source temperature: 230°C). Chromatograms and mass spectra were recorded and quantified (via integrated peak areas; initial threshold: 16, manually corrected) with the software HP Enhanced ChemStation G1701AA (version A.03.00; Hewlett Packard). Individual CHC compounds were chemically identified by considering the MS data base Wiley275 (John Wiley & Sons, New York, USA), the compound-specific retention time, Kovat’s indices, and the detected diagnostic ions (Carlson et al., 1998). Given that some substances could not be accurately separated with the above instrument and settings, we calculated the combined quantity by integrating over all substances within a peak in these cases. We additionally applied dimethyldisulfide (DMDS) derivatization before the gas chromatography-mass spectrometry (GC-MS) to identify the position of double bonds within unsaturated hydrocarbons. Note that it was not possible to reliably identify the position of double bonds in some alkenes and in most alkadienes due to low concentration of these compounds.

Carlson, D.A., Bernier, U.R. and Sutton, D.B. (1998) Elution Patterns from Capillary GC for Methyl-Branched Alkanes. Journal of Chemical Ecology, 24, 1845-1865.

 Funding: This work was funded by the Deutsche Forschungsgemeinschaft (DFG, SCHM 2645/2-1 and SCHM2645/3-1).

39

(123) Species: Plutella xylostella L., Plutellidae

Organism /organ: moth, female sex pheromone gland

Authors / contributors: Astrid T. Groot1,2 Affiliation: 1University of Amsterdam, Institute for Biodiversity and Ecosystem Dynamics (IBED), Amsterdam, the Netherlands 2Max Planck Institute for Chemical Ecology, Department of Entomology, Jena, Germany

Reference: Dekker O. 2012. The recent host expansion of the diamondback moth, Plutella xylostella and its effect on the female pheromone production – male sensitivity system. MSc thesis IBED, UvA. Supervisors: A.T. Groot & P. Roessingh.

Location: University of Amsterdam

Method sketch / description of data:  The moths were collected from the cages in their late instar/pupae stage and reared individually in 5 cm × 1.5 cm glass tubes and checked for emergence every day. The sex of the insects was determined after emergence by checking the abdomen of the adult moths. All adults were fed with honey-agar (50 ml water, 5 ml honey + 1 g agar- agar). One- to five-days-old females were used for the pheromone analysis. Glands were extracted and analysed as described for M. brassicae.

Funding: University of Amsterdam

40

(125) Species: Scaptotrigona pectoralis D., Apidae

Organism / organ: animal / cuticular surface

Authors / contributors: Sara D. Leonhardt1 & Thomas Schmitt1 Affiliation: 1Würzburg University, Department of Animal Ecology and Tropical Biology, Würzburg, Germany

Reference: Leonhardt SD, Rasmussen C & Schmitt T (2013) Genes versus environment: geography and phylogenetic relationships shape the chemical profiles of stingless bees in a global scale. Proceedings of the Royal Society - B, 280: 20130680, doi: 10.1098/rspb.2013.0680

Locations: Costa Rica: Monte Alto, Hojancha, Santa Cruz, Atenas

Method sketch / description of data:  Specimen sampling / analyses: 5–10 specimens were obtained from the entrance of a bee colony by capturing leaving foragers in a clean clear plastic bag, killed by quick freezing and extracted in hexane for 5 min. Cuticular extracts were analysed by a Hewlett Packard HP 6890 Series gas chromatograph coupled to a Hewlett Packard HP 5973 Mass Selective Detector (Agilent Technologies, Böblingen, Germany) on a DB-5 fused silica capillary column (30 m _ 0.25 mm ID; d.f. ¼ 0.25 mm; J & W, Folsom, CA, USA) with helium used as carrier gas (constant flow of 1 ml min-1). Injection was carried out at 250°C in the splitless mode for 1 min. Temperature was raised from 60°C to 300°C with a 5°C min-1 heating rate and held at 300°C for 10 min. Electron impact mass spectra were recorded at 70 eV. Compounds were identified by comparison of mass spectra and retention times with standard compounds, which are commercially available. Alternatively, compounds were identified using the mass spectral libraries Wiley 9, Nist 98 and Adams EO Library 2205.

Funding: Carlsberg foundation, Denmark; Deutsche Forschungs-Gemeinschaft (DFG project: LE 2750/1-1) and a grant of the German Excellence Initiative to the Graduate School of Life Science, University of Würzburg.

41

(126) Species: Tetragonilla collina S., Apidae

Organism / organ: animal / cuticular surface

Authors / contributors: Sara D. Leonhardt1 & Thomas Schmitt1 Affiliation: 1Würzburg University, Department of Animal Ecology and Tropical Biology, Würzburg, Germany

Reference: Leonhardt SD, Blüthgen N & Schmitt T (2019) Smelling like resin: terpenoids account for species-specific profiles in Southeast-Asian stingless bees. Insectes Sociaux, 56 (2): 157-170

Locations: Malaysia/Borneo: Sepilok, Danum Valley

Method sketch / description of data:  Specimen sampling / analyses: 5–10 specimens were obtained from the entrance of a bee colony by capturing leaving foragers in a clean clear plastic bag, killed by quick freezing and extracted in hexane for 5 min. Cuticular extracts were analysed by a Hewlett Packard HP 6890 Series gas chromatograph coupled to a Hewlett Packard HP 5973 Mass Selective Detector (Agilent Technologies, Böblingen, Germany) on a DB-5 fused silica capillary column (30 m _ 0.25 mm ID; d.f. ¼ 0.25 mm; J & W, Folsom, CA, USA) with helium used as carrier gas (constant flow of 1 ml/ min). Injection was carried out at 250°C in the splitless mode for 1 min. Temperature was raised from 60°C to 300°C with a 5°C min-1 heating rate and held at 300°C for 10 min. Electron impact mass spectra were recorded at 70 eV. Compounds were identified by comparison of mass spectra and retention times with standard compounds, which are commercially available. Alternatively, compounds were identified using the mass spectral libraries Wiley 9, Nist 98 and Adams EO Library 2205.

Funding: Carlsberg foundation, Denmark; Deutsche Forschungs-Gemeinschaft (DFG project: LE 2750/1-1) and a grant of the German Excellence Initiative to the Graduate School of Life Science, University of Würzburg.

42

(127) Species: Tetragonula carbonaria S., Apidae

Organism / organ: animal / cuticular surface

Authors / contributors: Sara D. Leonhardt1 & Thomas Schmitt1 Affiliation: 1Würzburg University, Department of Animal Ecology and Tropical Biology, Würzburg, Germany

Reference: Leonhardt SD, Wallace HM & Schmitt T (2011) The cuticular profiles of Australian stingless bees are shaped by resin of the eucalypt tree Corymbia torelliana. Austral Ecology, 36: 537-543

Locations: Australia: Elonora, Dalby, Brisbane, Shiptonsflat

Method sketch / description of data:  Specimen sampling / analyses: 5–10 specimens were obtained from the entrance of a bee colony by capturing leaving foragers in a clean clear plastic bag, killed by quick freezing and extracted in hexane for 5 min. Cuticular extracts were analysed by a Hewlett Packard HP 6890 Series gas chromatograph coupled to a Hewlett Packard HP 5973 Mass Selective Detector (Agilent Technologies, Böblingen, Germany) on a DB-5 fused silica capillary column (30 m _ 0.25 mm ID; d.f. ¼ 0.25 mm; J & W, Folsom, CA, USA) with helium used as carrier gas (constant flow of 1 ml min-1). Injection was carried out at 250°C in the splitless mode for 1 min. Temperature was raised from 60°C to 300°C with a 5°C min-1 heating rate and held at 300°C for 10 min. Electron impact mass spectra were recorded at 70 eV. Compounds were identified by comparison of mass spectra and retention times with standard compounds, which are commercially available. Alternatively, compounds were identified using the mass spectral libraries Wiley 9, Nist 98 and Adams EO Library 2205.

Funding: Carlsberg foundation, Denmark; Deutsche Forschungs-Gemeinschaft (DFG project: LE 2750/1-1) and a grant of the German Excellence Initiative to the Graduate School of Life Science, University of Würzburg.

43

(128) Species: Tetragonula melanocephala G., Apidae

Organism / organ: animal / cuticular surface

Authors / contributors: Sara D. Leonhardt1 & Thomas Schmitt1 Affiliation: 1Würzburg University, Department of Animal Ecology and Tropical Biology, Würzburg, Germany

Reference: Leonhardt SD, Blüthgen N & Schmitt T (2019) Smelling like resin: terpenoids account for species-specific profiles in Southeast-Asian stingless bees. Insectes Sociaux, 56 (2): 157-170

Locations: Malaysia/Borneo: Sepilok, Danum Valley

Method sketch / description of data:  Specimen sampling / analyses: 5–10 specimens were obtained from the entrance of a bee colony by capturing leaving foragers in a clean clear plastic bag, killed by quick freezing and extracted in hexane for 5 min. Cuticular extracts were analysed by a Hewlett Packard HP 6890 Series gas chromatograph coupled to a Hewlett Packard HP 5973 Mass Selective Detector (Agilent Technologies, Böblingen, Germany) on a DB-5 fused silica capillary column (30 m _ 0.25 mm ID; d.f. ¼ 0.25 mm; J & W, Folsom, CA, USA) with helium used as carrier gas (constant flow of 1 ml/ min). Injection was carried out at 250°C in the splitless mode for 1 min. Temperature was raised from 60°C to 300°C with a 5°C min-1 heating rate and held at 300°C for 10 min. Electron impact mass spectra were recorded at 70 eV. Compounds were identified by comparison of mass spectra and retention times with standard compounds, which are commercially available. Alternatively, compounds were identified using the mass spectral libraries Wiley 9, Nist 98 and Adams EO Library 2205.

Funding: Carlsberg foundation, Denmark; Deutsche Forschungs-Gemeinschaft (DFG project: LE 2750/1-1) and a grant of the German Excellence Initiative to the Graduate School of Life Science, University of Würzburg.

44

(129) Species: Trigona corvina Cockerell, 1913 (Hymenoptera, Apidae, Meliponini)

Organism / organ: animal / cephalic labial glands

Authors / contributors: Stefan Jarau & Lena John

Affiliation: Ulm University, Institute for Neurobiology, Helmholtzstr. 10/1, 89081 Ulm, Germany

References: Jarau S, Dambacher J, Twele R, Aguilar I, Francke W, Ayasse M (2010) The trail pheromone of a stingless bee, Trigona corvina (Hymenoptera, Apidae, Meliponini), varies between populations. Chemical Senses 35: 593-601 John L, Aguilar I, Ayasse M, Jarau S (2012) Nest specific composition of the trail pheromone of the stingless bee Trigona corvina (Apidae, Meliponini) within populations. Insectes Sociaux 59: 527-532

Location: Costa Rica; nests from three different populations: five nests from Heredia (Heredia Province), three nests from La Gamba (Puntarenas Province), one nest from Pozo Azul de Abangares (Guanacaste Province).

Method sketch / description of data:  Experimental setup: Cephalic labial gland extracts prepared from foraging bees collected from nine different colonies at three different locations were chemically analysed. Comparisons of the composition from these glands were carried out in order to test whether the trail pheromones produced by them show any nest-specific signal and whether there are differences between colonies within a certain population or between colonies from different populations (for details see John et al., 2012).

 Scent sampling / analyses: Cephalic labial glands were dissected from the heads of 117 foragers and individually placed in 100 µl hexane for 24 h at room temperature. The resulting extracts were reduced to volumes of 50 µl before gas chromatographic analyses. One microliter per sample was injected into a HP 5890 GC equipped with a 30 m DB-5MS column and with hydrogen (2 ml min-1 constant linear flow rate) as carrier gas. The GC was operated splitless at 50°C for 1 min, followed by a temperature increase to 310°C at a rate of 10°C min-1. The resulting gas chromatograms were used for quantitative analyses of the single compounds detected in the individual extracts by calculating the relative proportion of each compound within the total extract. Identification of the single compounds in the extracts was done by comparing their retention times with the retention times of pure reference compounds. Structure elucidation of these compounds by means of GC-MS analyses was carried out in an earlier study published by Jarau et al. (2010).

Funding: Deutscher Akademischer Austauschdienst (DAAD) and Deutsche Forschungs- gemeinschaft (DFG, JA 1715/3-1).

45