Journal of Chemical Ecology, Vol. 23, No. 12, 1997

VARIATION OF FLORAL SCENT EMISSION ANDPOSTPOLLINATION CHANGES IN INDIVIDUALFLOWERS OF Ophrys sphegodes subsp. sphegodes

FLORIAN P. SCHIESTL,1,* MANFRED AYASSE,1HANNES F. PAULUS,1 DIRK ERDMANN,2 and WITTKO FRANCKE2

1institute of Zoology, Department of Evolutionary BiologyUniversity of Vienna

Althanstrasse 14, A-1090 Vienna, Austria

2Institute of Organic Chemistry and BiochemistryUniversity of Hamburg, Hamburg, Germany

(Received March 11, 1997; accepted August 12, 1997)

Abstract—We investigated the scent composition of individual flowers ofOphrys sphegodes, its alteration following pollination, and of picked flowersby day and at night. Odor samples were collected by headspace sorption andanalyzed by gas chromatography and mass spectrometry. To evaluate thefunction of postpollination odor changes, we carried out behavioral tests onthe pollinator Andrena nigroaenea with pollinated and unpollinated flowers.We identified 27 volatiles in the flower scents. Aldehydes and alkanes weremost frequently found. Aldehydes were the most abundant class of compounds(40-50%). When flowers were picked, they emitted significantly lower totalamounts of volatiles than unpicked flowers, and their odor bouquets weresignificantly different. Comparison of scents released by day and at nightshowed no decrease in scent emission during nighttime, but the odor bouquetswere significantly different. Pollinated flowers produced significantly differentodor bouquets, and the total amount of scent emitted two to four days afterpollination was significantly lower compared with unpollinated flowers. Inaddition, behavioral tests with A. nigroaenea males showed that flowers weresignificantly less attractive three days after pollination. This reduced attrac-tiveness is hypothesized to guide pollinators to the unpollinated flowers withinan inflorescence, and thus increase the reproductive success of the plant.

Key Words—Ophrys sphegodes subsp. sphegodes, Andrena nigroaenea, flo-ral scent, floral semiochemicals, headspace sorption, postpollination changes,nocturnal scent emission.

*To whom correspondence should be addressed.

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0098-0331/97/1200-2881$l2.50/0 © 1997 Plenum Publishing Corporation

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INTRODUCTION

The Mediterranean orchid genus Ophrys is pollinated by means of sexual decep-tion, mostly by male bees and wasps. Lured by the odor and insectlike shapeof the flower, the pollinator alights on the labellum and attempts to copulatewith it (Kullenberg, 1961). During these so called “pseudocopulations,” themale contacts the pollinia, which become attached to its head or abdomen.Numerous behavioral tests have shown that the Ophrys-pollinator relationshipis highly specific: each Ophrys species is pollinated by males of usually onlyone or a few pollinator species (Kullenberg, 1961, 1973; Paulus and Gack,1986, 1994). These studies also showed that the flower’s odor is the mostimportant cue for eliciting male mating behavior and for ensuring the pollinatorspecificity in the male attracting communication signal.

In behavioral experiments it was shown that males of the sweat bee Lasio-glossum malachurum (Kirby) use odor signals to learn the identity of femalebees with which they have attempted to copulate, thereby avoiding these femalesin further encounters (Smith and Ayasse, 1987). In Ophrys flowers, pollinatingmale bees also learn the odor bouquet of a visited flower and subsequentlymodify their response to it. Andrena nigroaenea (Kirby) males learn the odorsof individual O. sphegodes Miller flowers and avoid copulatory attempts infurther visits (Ayasse et al., 1996). Although the flower odors of numerousOphrys species have been chemically analyzed (Borg-Karlson, 1987, 1990; Borg-Karlson et al., 1985, 1987), none of the studies have addressed the variation ofodor bouquets emitted by single flowers. Our experiments (Ayasse et al., 1996)suggest that there is some inter- and intraindividual variation within floral odorbouquets of O. sphegodes that may influence the behavior of the male pollinator.

In bees, postmating changes of the females’ mate-attracting odor signalshave been shown, which direct males to virgin females (Ayasse et al., 1993;Engels et al., 1993). Similarly, in Ophrys orchids, a decrease and alteration offloral odor bouquets after pollination might lead to a lower attractiveness ofthese flowers. Besides the advantage of reduced costs, this may be of selectivevalue if one plant bears more than one flower, since the plants’ overall repro-ductive success can be increased by directing pollinators to the unpollinatedflowers of an inflorescence (Tollsten, 1993). Although some studies have doc-umented postpollination changes in scent emission of orchids (e.g., Tollstenand Bergstrom, 1989; Tollsten, 1993), none dealt with Ophrys.

Few studies proved an alteration in floral volatile production of orchidsafter injury. In orchids of the genus Cycnoches, excised labella emit considerablyaltered fragrance profiles more than 8 hr after excision (Gregg, 1983). In Ophrysorchids, alteration of odor emission of picked flowers has not yet been inves-tigated. However, behavioral tests showed that picked plants are generally still

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attractive for pollinators over several days (Paulus and Gack, 1990). This firststudy dealing with changes of scent emission in picked Ophrys plants is a basefor further scent investigations and behavioral experiments.

In orchids pollinated by nocturnal animals, an increased scent productionduring the night was reported (e.g., Nilsson, 1978; Kaiser, 1993). In contrast,apple flowers, which are primarily bee pollinated during the daytime, producelower amounts of scent during the night (Loughrin et al., 1990). Since orchidsof the genus Ophrys are also pollinated by day-active insects, we expect flowersto emit lower amounts of volatiles during the night versus the day. In the absenceof pollinators, the production of biologically active fragrances can be regardedas redundant. This study is a first approach to understand changes in odor profilesemitted by Ophrys orchids during the night.

In order to obtain information about intraspecific variation of Ophrys flowerodor emission under various conditions, the following aspects are addressed:(1) quality of odors—none of the recent studies dealing with odor emission ofOphrys flowers includes O. sphegodes subsp. sphegodes; (2) postpollinationchanges in flowers with respect to their odor and color and in terms of theirattractiveness for pollinators; (3) effect of picking on odor emission; and (4)comparison of odor emission during day and night.

METHODS AND MATERIALS

Plant Material. Ophrys sphegodes subsp. sphegodes grows in the Med-iterranean region and Central Europe. It is pollinated by Andrena nigroaeneamales (Godfery, 1925; Kullenberg and Bergstrom, 1973; Paulus and Gack,1986, 1990), a solitary bee species that is common in the area of our study. Weinvestigated individual plants of O. sphegodes growing in the “Bisamberg”area in Lower Austria. Plants were dug up, numbered, and transferred to potsin the laboratory. The scent of 36 single flowers was collected over two weeks.In addition, 25 plants were picked and kept in glass tubes filled with water.These freshly picked plants were investigated over three subsequent days. Thescent of 17 unpicked flowers that were hand pollinated immediately after anthesiswas collected during the following four days. Furthermore, 17 picked flowerswere left unpollinated and their scent was collected during the night (21:00-05:00hr) in a completely dark room.

Flower Odor Headspace Sampling. For the collection of volatiles, singleflowers were placed in glass tubes (approx. 5 ml) open at both ends, which wereplugged with cotton wool. Air was pulled over the flowers (30 ml/min for 6-8hr) by a battery-operated membrane pump. Air coming into the flower chamberwas cleaned by an adsorbent tube filled with 120 mg activated charcoal (SKC).The air exited through a cartridge that contained 1.5 mg activated charcoal

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(CLSA Filter) and onto which the volatiles were trapped. Contamination fromthe environment was checked by collecting volatiles from the laboratory air(control sample). The fragrance was eluted with 20 pl carbon disulfide (MerckUvasol) according to the technique described in Habich and Grob (1984).

Chemical Analysis. Analyses of volatiles were carried out by combinedcapillary gas chromatography-mass spectrometry. A mass spectrometer VG 70-250 SE (Vacuum Generators) was linked to a gas chromatograph HP 5970(Hewlett-Packard) equipped with a DB5 capillary column (30 m X 0.32 mm)operating splitless at 50°C for 30 sec, followed by opening the split and pro-gramming to 220°C at a rate of 4°C/min. Helium served as the carrier gas. Acombination of a quadrupole mass spectrometer (Fisons MD 800) and a gaschromatograph (Fisons 8060) was also used. Compound identifications are basedon comparison of mass spectra and gas chromatographic retention times ofnatural products with authentic reference samples. To determine the enantio-meric composition of 2-nonanol and endo-brevicomin, a capillary column(25 m X 0.25 mm) coated with a 1:1 mixture of octakis (6-0-methyl-2.3-di-0-pentyl)-7-cyclodextrin and OV-1701 was used while hydrogen served as car-rier gas. For determination of absolute amounts collected, n-octadecane wasadded as an internal standard to all samples. GC analyses were performed witha Hewlett-Packard 5890 Series II gas chromatograph under the conditionsdescribed above.

Behavioral Tests. In seven O. sphegodes plants from Bisamberg, one flowerof an inflorescence was hand pollinated in the field, and plants were picked afterthree days. In Ophrys orchids, it can be easily determined if a flower is pollinatedby checking the stigma for deposited pollen massulae. Behavioral experimentswere then carried out in Oberweiden/Lower Austria, where A. nigroaenea malesregularly patrol. In a dual-choice experiment, one pollinated and one unpolli-nated flower were offered together on the same stem for 3 min to A. nigroaeneamales in a patrolling area. On three plants the pollinated flower was older thanthe unpollinated one, and on four plants it was reverse. Copulation attemptswith the flower were recorded.

Postpollination Flower Color Change. Twenty flowers were pollinated inthe field and their color was compared with 20 unpollinated flowers on the sameplants. The color change of 10 flowers was observed after three days and thatof another 10 flowers after seven and 10 days. Flowers with dark-brown colorwere regarded as unchanged, and flowers with a yellow-whitish appearance wereregarded as wilted.

Statistics. Relative and total amounts of single compounds and of chemicalclasses of compounds were tested for significant differences between unpolli-nated and pollinated flowers, picked and unpicked flowers, and day and nightscent emission of picked flowers by means of Mann-Whitney U test (nonnormaldistributed data), t tests (normal distributed data), and stepwise discriminant

SCENT EMISSION AND POSTPOLLINATION CHANGES IN FLOWERS 2885

function analysis (Norusis, 1993). To approach normality and reduce hetero-geneity of variances, all data for discriminant analysis were transformed byln(1 + x). The standardized discriminant function coefficient was used to assessthe importance of variables within the discriminant functions.

RESULTS

Odor of Unpicked Flowers. We identified 27 compounds in the headspacesamples of O. sphegodes (Table 1, Figure 1). The volatiles consisted of a blendof nine straight-chain aldehydes, nine straight-chain alkanes, six terpenoids, andthree nonane derivatives [nonanone, nonanol, and (–)-endo-brevicomin]. Alde-hydes were the most abundant class of compounds, having a relative proportionof 40-50% in all samples. Among the aldehydes, nonanal and decanal weremost prominent. 2-Nonanol was detected as a mixture of enantiomers in a ratioof R:S = 85:15. endo-Brevicomin was found to be represented by the almostpure (–) enantiomer. GC analysis used in this study was not able to separatethe following substances: heptanal + nonane, octanal + decane, or 2-nonanol+ undecane.

FIG. 1. Gas chromatogram of the headspace sample of an unpollinated, unpicked Ophryssphegodes flower. For names of compounds, see Table 1.

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Postpollination Changes in Floral Scents. Three groups of flowers werecompared: unpollinated flowers, flowers one day after pollination, and flowerstwo to four days after pollination. Two to four days after pollination, the relativeamounts of some hydrocarbons and 6-methyl-5-hepten-2-one decreased signifi-cantly, while those of most aldehydes and a-pinene increased (Mann-WhitneyU test, P < 0.05, Table 1). A discriminant analysis revealed that there was asignificant difference between the odor bouquets of unpollinated and pollinatedflowers (discriminant function 1: x2 = 46.6; df = 8; P < 0.001; discriminantfunction 2: x2 = 6.2; df = 3; P > 0.05, Figure 2). According to the stan-dardized discriminant function coefficients, dodecane, tridecane, undecanal, and6-methyl-5-hepten-2-one contributed most to the discriminant function (i.e.,they showed the largest values for the standardized canonical discriminant func-tion coefficient).

The mean total amount of scent released by single flowers of unpicked,unpollinated plants was 66 ± 7 ng/hr/flower (Figure 3). Flowers 24 hr afterpollination showed a slight decrease in the total amount of scent and flowerstwo to four days after pollination emitted significantly lower total amounts ofvolatiles compared to unpollinated flowers (t test, t = 2.04, P < 0.05, Figure3). Almost all compounds were affected by this decrease, but the reductions

FIG. 2. Comparison of odor bouquets of unpollinated and pollinated O. sphegodes flow-ers by means of a discriminant analysis. Odor bouquets of unpollinated flowers weresignificantly different from those of pollinated flowers as discussed in the text.

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FIG. 3. Mean (±SE) of the total amounts of volatiles emitted by unpollinated and pol-linated O. sphegodes flowers. Flowers two to four days after pollination produced sig-nificantly lower amounts of volatiles compared with unpollinated flowers (t test, t =2.04, P < 0.05).

were significant only for 6-methyl-5-hepten-2-one and 2-nonanol+undecane(Mann-Whitney U test, P < 0.05).

Odors of Picked Versus Unpicked Flowers. Picked flowers released smallerrelative amounts of some hydrocarbons and 2-nonanone and higher relativeamounts of some aldehydes (Mann-Whitney U test, P < 0.05, Table 1). Mul-tivariate analysis showed significant differences between the odor bouquets ofpicked and unpicked flowers (x2 = 45.5; df = 7; P < 0.001). Dodecane,tridecane, heptanal, and 2-nonanone contributed most to the differences. Fur-thermore, the total amount of scent released by picked flowers was significantlylower compared with unpicked flowers (Mann-Whitney U test, P < 0.05, Figure4). All components were affected by this decrease in volatile emission.

Odor Emitted During Night Versus Day. Floral odor collected during thenight contained significantly lower relative amounts of most aldehydes and6-methyl-5-hepten-2-one and significantly higher relative amounts of mosthydrocarbons, a-pinene, limonene, 1,8-cineole, and 2-nonanol than samplescollected during the day (Mann-Whitney U test, P < 0.05, Table 1). A dis-

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FIG. 4. Mean (±SE) of the total quantity of volatiles emitted by flowers of unpickedand picked O. sphegodes flowers. Flowers of picked plants released significantly lessvolatiles (Mann-Whitney U test, P < 0.05).

criminant analysis showed a significant difference between the composition ofodor bouquets emitted during night versus day (x2 = 9 1 . 1 ; d f = 5 ; P < 0.001).Tetradecane, heptadecane, hexanal, and 1,8-cineole contributed most to thediscriminant function. Furthermore, the total amount of scent emitted increasedduring the night (Mann-Whitney U test, P > 0.05).

Bee Behavioral Tests. In the behavioral tests, males of A. nigroaenea madesignificantly fewer copulation attempts on O. sphegodes flowers three days afterpollination than on unpollinated flowers (Mann-Whitney U test, P < 0.001,Figure 5).

Postpollination Change in Flower Color, In terms of color change, 80%of flowers had wilted three days after pollination compared to only 50% ofunpollinated flowers (Mann-Whitney U test, P > 0.05). Within the other sam-ple, flowers were checked seven and 10 days after pollination. After seven days,100% of the pollinated flowers had wilted compared to only 20% of the unpol-linated controls (Mann-Whitney U test, P < 0.05). After 10 days, 60% ofunpollinated flowers had wilted.

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FIG. 5. Mean (±SE) number of copulation attempts by Andrena nigroaenea males onunpollinated and pollinated O. sphegodes flowers. Pollinated flowers were significantlyless attractive than unpollinated ones (Mann-Whitney U test, P < 0.001).

DISCUSSION

Among Ophrys orchids, which are usually interfertile, floral odor is themost important factor for ensuring the reproductive success and isolation ofspecies in sympatry (Kullenberg, 1961, 1973; Paulus and Gack, 1986, 1994).The pollinator male insect apparently perceives the floral odor as the sex pher-omone of the female insect since both elicit the same behavioral response. Theodor triggers searching behavior in the male and releases male copulatory move-ments on the flower labellum, which result in the attachment of the pollinia tothe bee (Kullenberg, 1961; Kullenberg and Bergstrom, 1975, 1976a; Bergstrom,1978). Ophrys flowers usually produce complex bouquets of volatiles (Borg-Karlson, 1990), although presumably not all components are important for theattraction of male pollinators. Behavioral experiments have shown that onlycertain compounds are active in stimulating mating behavior (Kullenberg andBergstrom, 1976b). Besides the odor, the size and pilosity of the labellum alsocontribute to the species specificity of pollinator attraction (Kullenberg, 1961).

Depending on the conditions, the quantitative compositions of the odorpatterns of the flowers show characteristic alterations (Table 1). Changes can

SCENT EMISSION AND POSTPOLLINATION CHANGES IN FLOWERS 2891

not be generally attributed to any specific class of compounds, neither withrespect to carbon skeletons (acetogenins vs. mevalogenins) or to functionalgroups (hydrocarbons vs. alcohols vs. carbonyl compounds). The concentrationof a-pinene is lowest in intact flowers, of which the concentration of the non-terpenoids 6-methyl-5-hepten-2-one and geranylacetone are highest. Changesamong the acetogenins seem to be slightly more obvious.

2-Nonanol is widespread in the plant and animal kingdoms, and a definedmixture of enantiomers serves as part of the queen pheromone in the stinglessbee, Scaptotrigona postica Latr. (Engels et al., 1990, 1993). Both the exo- andendo- isomers of brevicomin are well known pheromones of bark beetles of thegenus Dendroctonus (Byers, 1989). However, endo-brevicomin was also foundin the odor of Ophrys speculum Link (Francke et al., 1995), and it may play arole in the pollination of O. sphegodes by A. nigroaenea.

Scent production and maintenance by flowers is expensive in terms ofenergy resources (Vogel, 1962; Arditti, 1979). Therefore, many orchid specieshave evolved various mechanisms for energy conservation, such as cessation ofscent production and fast wilting immediately after pollination (Arditti, 1979).Few investigations have examined the changes of floral odor following polli-nation. In Platanthera bifolia (L.) Rich., Tollsten and Bergstrom (1989) reporteda decrease in scent production 24 hr after pollination, and Tollsten (1993) alsofound changes in the floral odor bouquets of flowers two and five days afterpollination. These changes were interpreted as causing a reduced attractivenessin pollinated flowers. Neiland and Wilcock (1995) studied Ophrys lutea Cav.flowers and found them to be visibly unchanged for at least three days afterpollination. They assumed that the flowers remained attractive for several daysafter pollination and thus promoted additional insect visitation; however, theydid not investigate the changes in floral odors.

In contrast to these results, we found the color of pollinated O. sphegodesflowers to fade three days after pollination, whereas many of the unpollinatedflowers remained unchanged. The changes in scent involved a decrease of totalamount of scent produced as well as an alteration of the odor bouquet. Besidesreduced costs for scent production in pollinated flowers, a second advantage ofaltered odor bouquets is the directing of pollinators to unpollinated flowers ofthe plant. Behavioral tests with male A. nigroaenea confirmed that pollinatedflowers have a reduced attractiveness compared to unpollinated ones. The advan-tage of reducing floral attractiveness after pollination (Faegri and van der Pijl,1979) may be enhanced by the generally low visitation rates by pollinators thatcharacterize Ophrys orchids (Paulus, personal communication). Moreover, inorchids with pollinia, such as Ophrys, most or all of the pollen of one flowercan be removed by a single pollinator. A pollinated flower, therefore, no longerfunctions as a pollen donor and benefits only little by a visit of a second polli-nator (Casper and La Pine, 1984). Therefore, if a plant bears more than one

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flower, as is usual in O. sphegodes (Hutchings, 1987), its reproductive successcan be enhanced by guiding the few pollinators to the unpollinated flowers ofan inflorescence. This can be achieved by reducing the attractiveness of polli-nated flowers. Our findings very clearly confirm these theoretical assumptionsfor O. sphegodes. Pollinated flowers change their odor bouquets and also theircolor, and are thus less attractive to potential pollinators.

Little attention has been paid to floral scent emission of orchids after pick-ing. Gregg (1983) found some differences in odor profiles of intact and excisedlabella of Cycnoches spp. investigated after more than 8 hr. However, as longas they were analyzed within 8 hr after excision, no great changes were found.Picked Ophrys flowers often have been used for both floral odor and pollinatorbehavioral studies, and our findings here show that odor can be significantlyaltered following picking. Picked O. sphegodes flowers showed a great decreasein total amount of volatiles and odor bouquets differed considerably from intactflowers. Nevertheless, these picked flowers are still attractive to pollinators overseveral days (Ayasse, unpublished data), which suggests that behaviorallyimportant volatiles are not immediately affected by picking.

Nocturnal scent emission has been investigated mostly in orchids pollinatedby nocturnally active animals. Nilsson (1978) found great differences in scentproduction during day and night of Platanthera chlorantha (Cluster) Rchb.flowers, which are pollinated predominantly by night-active moths. Malus Xdomestica Borkh. and Nicotiana otophora Gresebach flowers are pollinated pri-marily by day-active insects and emit greater amounts of volatiles during theday (Loughrin et al., 1990). Pollinators of O. sphegodes are also active onlyduring the day and one could expect the scent production to decrease or evento cease during the night. In our samples, scent bouquets collected during dayand night were significantly different. However, we found no decrease in noc-turnal scent production. Methodological reasons might in part account for thesefindings. Under natural conditions, lower temperatures during the night maycause a reduced fragrance emission. We collected our samples under laboratoryconditions, where no or a smaller temperature decrease occurred. Neverthelessour findings prove that scent emission does not completely cease during thenight due to diurnal rhythms. Since odor bouquets are significantly differentduring day and night, a reduced production of biologically active compoundsduring the night may be possible. In future studies on nocturnal scent emissionof Ophrys flowers, samples should be collected under constant light and tem-perature conditions or under natural conditions in the field.

This first study dealing with odor variability of individual flowers in Ophrysconfirms that odor variability is an important mechanism for influencing thebehavior of the pollinators. Its alteration after pollination reduces the attractive-ness of these flowers, and pollinators are guided to unpollinated flowers. Thismechanism of maximization of the reproductive success of a plant appears to

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be especially important in the genus Ophrys, whihh is characterized by a lowvisitation rate. Our present investigations focus on the identification of the male-attracting compounds within the odor bouquets by conducting electrophysiolog-ical recordings and behavioral tests with synthetic compounds. Knowledge aboutthese biologically active compounds would allow much more complete inter-pretation of changes in scent emission and its functional significance.

Acknowledgments—We thank Roman Kaiser (Givaudan-Roure Research Ltd.) for his inval-uable advice concerning odor sampling and for reference substances, Heidi E. M. Dobson andRobert J. Paxton for many helpful comments on the manuscript, and the district governments ofKorneuburg and Ganserndorf and the Biological Research Institute Illmitz/National Park Neusied-lersee-Seewinkel for issuing collecting licenses. Financial support to F.P.S. was provided by theNiederosterreichische Landesregierung. Supported by a grant of the FWF Austria (P12275-B10).

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