Form-specific fragances fromOphrys insectifera L. (Orchidaceae) attract species of different pollinator genera. Evidence of sympatric speciation?

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Chemoecotogy 4/I:39-45 (1993) 0937-7409/93/010039 07 S1.50 + 0.20 1993 Birkhuser Verlag, Basel Form-specific fragances from Ophrys insectifera L. (Orehidaceae) attract species of different pollinator genera. Evidenee of sympatric speciation? Anna-Karin Borg-Karlson 1"3, Inga Groth 2'3, Lennart gren 3, and Bertil Kullenberg 3 Department of Organic Chemistry, The Royal Institute of Technology, S-100 44 Stockholm, Sweden 2 Department of Chemical Ecology, Gothenburg University, Reutersgatan 2c, S-413 20 Gteborg, Sweden 3 Ecological Research Station of Uppsala University, lands Skogsby 6280, S-386 93 Frjestaden, Sweden Abstraet. Two closely related forms of Ophrys insec- tifera were observed in the field to attract different pollinator species selectively. O. i. ssp. insectifera at- tracted males of two species of Argogorytes (Sphecidae, Hymenoptera Aculeata) and O. i. ssp. aymoninii at- tracted Andrena combinata males (Andrenidae, Apoidea, Hymenoptera Aculeata). A third form, O. aff. i. ssp. insectifera, attracted none of these three species. Volatile compounds from flowers and inflorescences of the three forms (originating from land, Sweden, and Aveyron, France) were collected, using entrainment, enfleurage, and solvent extraction techniques, and iden- tified by gas chromatography-mass spectrometry. Scent differences between the three forms were con- firmed in the amounts of aliphatic hydrocarbons (Cll- C19), methyl esters (C~4 C~8), short chain aliphatic 1-alcohols (C6 Cl2), and monoterpene alcohols (C~o). Key words, pollination semiochemicals attractants - aliphatic alcohols - aliphatic hydrocarbons - aliphatic methyl esters - linalool - Hymenoptera Aculeata- Andrena - Argogorytes - Orchidaceae- Ophrys - Osmia Introduction At least on the Baltic island of land, the orchid Ophrys insectifera L. ssp. insectifera (IS) (Fig. 10-15) is pollinated by sexually excited males of the two sphecid wasps Argogorytes rnystaceus (L.) and A. fargei (Shuck- ard) (Sphecidae, superfamily Sphecoidea, Hymenoptera Aculeata) (Kullenberg 1950, 1952, 1956, 1961). The morphologically very similar form O. insec- tifera ssp. aymoninii Breistroffer 1981 (Fig. 19 and 20) (AF) was recently observed being pollinated by another far-related vector, namely males of the solitary bee Andrena (Simandrena) combinata (Christ) (Andrenidae, superfamily Apoidea, Hymenoptera Aculeata) in Valle de Cernon, Aveyron, France (Kullenberg unpublished observations). These findings are not in agreement with the original "pollination systems" by Kullenberg (1961), Kullenberg & Bergstrm (1976), and Borg- Kartson (1990). Therefore, we decided to make a chem- ical analysis of the flower volatiles from this and yet another form from Southern France, O. aff. insectifera ssp. insectifera (IF) (Fig. 16 and 17),- which pollina- tion was not clarified in detail during the field studies (Kullenberg unpublished observations) -and compare them with the flower volatiles of the IS form from land, which is carefully analyzed earlier (gren & Borg-Karlson 1984; Borg-Karlson et al. 1987). Furthermore, scanning electron micrographs (SEM) were made of flower labella and some superficial integu- mental surfaces of the corresponding pollinators (both sexes). The aim was to visualize any possible differences in those labellar epidermal structures that communicate the tactile and proprioceptive stimuli influencing the behavior of the attracted male (Kullenberg 1956, 1961). The chief purpose of the study was to search for chemical differences in the floral odors that can explain the attraction of two far-related pollinator species to the very near-related orchid forms IS and AF of O. insectifera. Materials and methods Collection of biologica] materials Flowers of AF and IF were collected for chemical and morphological investigations mainly in the terrain of Causse Noir and Causse du Larzac, Aveyron, in the southern part of the French Massif Central. Rather rich populations of AF and IF were found about 14 15 km from Millau close to the road D 110, of AF also in the rocky landscape of Montpellier le Vieux connected to this road. The flowers were prepared immediately in the field for the chemical and morpho- logical investigations. Andrena combinata, Argogorytes mystaceus, and A. fargei were collected in the same areas as the orchids. Insect behavior The attractivity of AF was tested in the field, in France on maies of Andrena combinata, in Sweden on males of Argogorytes fargei, A. mustacens, A. vaga Panzer, A. haemorrhoa (Fabricius), A. nigroaenea (Kirby), and Osmia rufa (L.) (common hymenopteran species that fly in the same season as Argogorytes fargei and A. mystaceus, pollina- tors of IS on land). The following test materials were presented to the insects in their mating llight areas, on land, on males of Andrena haemorrhoa and 40 A.-K. Borg-Karlson et al. A. nigroaenea, and in Uppland on males of A. haemorrhoa and A. vaga: a) Whole plants (the three individuals tested in Sweden has been bred in a greenhouse) with exposed flowers b) Same as a but with the inflorescences hidden by green tulle permitting the evaporation of flower odors c) Hexane extracts of flower labella on pieces of black velvet, uncov- ered or covered by green tulle d) A mixture of aliphatic l-alcohols on pieces of black velvet (100 ng/5 min test occasion). The mixture consisted of equal amounts of 1-heptanol, 1-octanol, 1-nonanol, 1-decanol, 1-undecanol, and 1-dodecanol, each diluted 1:100 (v:v) in hexane. The attractivity of IF on A. eombinata was tested in the same manner as that of AF in France, using whole plants in pots. On land, hexane extracts (5 flowers/ml hexane) were tested on Argogo- rytes fargei and A. mystaceus. The various types of behavior exhibited by the insects attracted to the flowers or the dummies were classified according to standard methods (Kullenberg 1956, 1961, 1973; Teng 1979; Borg-Karlson 1990). The test methods and results were compared with those of earlier studies of IS (Kullenberg 1956, 1961; Borg-Karlson et al. 1987) and the old and new results were compiled in Table 3. Chemical methods The volatiles emitted from the inflorescences of IF, AF, and IS were collected on PorapakQ via entrainment (Borg-Karlson et al. 1987) or enfleurage techniques (100 mg of PorapakQ was added to 4 -5 flower labella for 24 la) or via extraction of flower labella (5 labella/ml solvent) with hexane for 3 d (Bergstrm et al. I980; Borg-Karlson et al. 1987; Borg-Karlson 1990). The investigations were first made in I986 and then repeated in 1987 and 1988. The odoriferous com- pounds adsorbed via entrainment on PorapakQ were eluted with 4 ml of redistilled pentane (Merck p.a. grade) and the eluate was carefully concentrated at 42C prior to the chemical analyses. The labellar extracts in hexane were concentrated at 70C. The chemical identifications were made on a Finnigan 4500 GC-MS instrument, using a fused silica column, coated with Super- oxFA (25 m, i.d. 0.15 mm) with temperature programming (60C for 4 min, followed by an increase of 5/min up to 220C). The mass spectra and retention values were compared with those of available authentic samples and with reference spectra. Scanning electron microscopy Three to five flowers from different individuals of each O. insectifera form were chosen for the morphological studies. They were fixated in 3% glutaraldehyde in 0.1 M phosphate buffer solution, dehydrated in an ascending ethanol series, and dried in a critical point apparatus after being gradually transferred into Freon TF 133. The insects were air-dried on a pin, and then sectioned into body parts, which were glued to specimen holders with conductive glue. Before examination in the electron microscope, the preparations were coated with gold/ palladium to a thickness of about 350 by means of a sputter. Micrographs were taken at 15-20 kV, using a Jeol JSM-35 scanning electron microscope. Resu l ts Chemist ry The three fo rms o f O. insectifera showed character i s t i c d i f fe rences in the i r pat te rn o f emi t ted vapors f rom f resh in f lo rescences (Tab le 1 and 2). Sorpt ion and so lvent ext rac t techn iques gare complementary resu l ts : Wi th the ent ra inment and enf leurage methods , the vo la t i l es ' spect rum o f AF showed the presence o f a ser ies o f a l iphat i c 1 -a lcoho ls (C6-C~2) and a few CHEMOECOLOGY Table 1 Volatile compounds identified by GC-MS in three forms of Ophrys insectifera Substances Ophrys i. ssp O. aff i. ssp. O. i . ssp. aymoninii insectifera insectifera France France Sweden (AF) (IF) (IS) Aliphatic hydrocarbons undecane + + + tridecane + + + + tridecene - - + tetradecane + + + + tetradecene - - + + pentadecane + + + + + pentadecene (a) - - + pentadecene (b) - - + hexadecane + + + hexadecene - - + heptadecane + + + + heptadecene + + + + + nonadecane + + + + nonadecene - + + Aliphatie aleohols 1-pentanol + + - 2-pentanol + - + 1-hexanoI + + - 2-hexanol + + 1-heptanol + + - 1-octanol + + + + 1-nonanol + + + + - 2-nonanol - - + 1-dodecanol + + + - 1-tetradecanol + - - 1-hexadecanol + + + + 1-octadecanol + + - Aliphatic esters methyl tetradecanoate + + methyl pentadecanoate + - - methyl hexadecanoate + tr + methyl hexadecenoate + - tr methyl octadecanoate + - - Terpenoids 6-methyl-5-heptene-2-one + + + linalool + - trans-furanoid linalool oxide - - + bornyl acetate + + + geranylacetone + - + oxygenated monoterpene + - - trans-thujanol + -- - cyclosativene + + + + + + + c~-farnesene + + +++ large amounts (>20% of total amounts); ++ minor amounts (2 20% of total); + small amounts (Vol. 4, [993 Form-specific fragrances from Ophrys insectifera 4l Table 2 Principal compound classes present in the flower of three forms of Ophrys insectifera according to GC-MS (SuperoxFA) Ophrys i. ssp O. aff i. ssp. O.i. ssp. aymoninii insectifera insecti~bra France France Sweden (AF) (IF) (IS) aliphatic hydrocarbons + + + + + Cll-Cl9 aliphatic hydrocarbons + + + + + + + + + C20 C27 aliphatic alcohols + + + + C 6 el 2 aliphatic methyl esters + + Ci4-Ct5 aliphatic methyl esters + + + + + + + C16 CI8 oxygenated monoterpenes + + + Clo sesquiterpenes + + + C15 +++ large amounts (>20% of total amounts); ++ minor amounts (between 2 and 20% of total); + small amounts (42 A.-K. Borg-Karlson et al. CHEMOECOLOGY Fig. 1 Labellum of Ophrys insectifera ssp. insectifera from land, seen slightly from above. Bar 2 mm, Magnification 7.2 x Fig. 2 Argogorytes fargei female from land, head and thorax without wings and other appendages, lateral view from right. Bar 2 mm, Magnification 10 x Fig. 3 Argogorytes fargei from land, female abdomen, distalmost segments, dorsal view. Bar 1 mm, Magnification 22 x Fig. 4 Ophrys insectifera ssp. aymoninii from Aveyron, Causse Noir. Same view as in Fig. 1. Bar 2 mm, Magnification 7.0 x Fig. 5 Andrena combinata female from Aveyron, Vaile de Cernon, Causse du Larzac. Thorax without wings and other appendages (no head), lateral view from leit. Bar I mm, Magnification 13 x Fig. 6 Same animal as in Fig. 5. Abdomen, dorsal view. Bar 1 mm, Magnification ll x Fig. 7 Same animal as in Fig. 5. Terminal tergites. Bar 250 gm, Magnification 35 x Fig. 8 Same animal as in Fig. 5. Right wing, dorsal view. Bar 1 mm, Magnification 11 x Fig. 9 Ophrys aff. insectifera ssp. insect!fera from Aveyron, Causse Noir. Same view as in Fig. 1. Bar 2 mm, Magnification 7.0 x Kar lson unpubl ished results). The hydrocarbons with uneven carbon numbers release strong EAG responses when tested one by one on Argogorytes male antennae; the synergistic effects are not known (gren & Borg- Kar lson 1984). The biological signif icance of these hydro- carbons in Argogorytes might lie in the construct ion of nest cells, in increasing the penetrat ion of venom into the prey, and/or in the attract ion o f males to females. The clear but weak att ract ion o f Andrena ovatula males to IS (Ku l lenberg 1961) is most probab ly explained by the presence o f a!iphatic 1-alcohols (C6-C~2) in a very low concentrat ion in the flower. These are, as ment ioned above, identical with those found in the mand ibu la r gland secretion in a number o f Andrena species (Teng 1979; Borg -Kar l son et al. 1985; Borg -Kar l son 1990). In IF, the hydrocarbons C1~-C19 are rauch less abundant . Males of Swedish Argogorytes species did not respond to hexane extracts of this Ophrys form in field Fig. 10-15 Ophrys insectifera ssp. insectifera (IS), flowers seen from the front. Central part of land, Southwestern Sweden. Note the var:ation of the shape of the labella and of the shape and color of the labdlar patches Fig. 16-17 O. aff. insectifera ssp. inseetifera (1F), Aveyron, South- ern France. Note the resemblance of the shape of the labella to that of the flower labella of the flowers from land Fig. 18 O. insectifera forma (IS). Northern part of land. Note the resemblance regarding shape and coloration of the flowers of O. insect(fera ssp. aymoninii (Figs. 19 and 20) Fig. 19-20 O. insectifera ssp. aymoninii (AF). Aveyron, Causse Noir, Southern France Fig. 21 Andrena combinata c~, head with pollinia of O. insectifera ssp. ayrnoninii, seen from the fiont. Drawing by I. Thomasson Vol. 4, 1993 Form-specific fragrances from Ophrys insectifera 43 10, 11 12 13, 14 15 16, 17 18 19, 20 21 44 A.-K. Borg-Karlson et al. experiments on land (Table 3) and its pollinator is still unknown. The chemical attraction mechanisms in Andrena- Ophrys pollination relationships are not yet fully under- stood. In all hitherto investigated Ophrys forms pollinated by Andrena males, analyses by GC-MS indi- cate the presence of large amounts of aliphatic straight- chain alcohols, carrying their hydroxyl group in position 1 or 2, and in some forms also oxygenated terpenes, such as geraniol, linalool, citronellol, and E,E-farnesol (Borg-Karlson et al. 1985). The amounts of these compounds are low in taxa that are not polli- nated by Andrena species. The way, in which they need to be combined to act as the field-observable species- selective attractants, is unknown. A parallel, concerning chemical and morphological diversification, is found in the form complex Ophrys fusca Link-O. murbecki (Fleichm.) So, in which the amouns of primary and secondary aliphatic alcolaols in the fragrances vary between the forms (Borg-Karlson et al. 1985). Within this form complex, several pollination syndromes are developed with corresponding differ- ences in size of both pollinators and flower labella (Kullenberg 1961; Paulus & Gack 1981, 1983; Warncke & Kullenberg 1984). Kullenberg proposed (1956), that the hairy bulbous field in the proximal part of the Ophrys labellum mim- ics the thorax of the female of the pollinator, the central field mimics the wings, and the labellar tip mimics the abdominal tip. Agren et al. (1984) found congruences in the pilosity patterns between the plant mimic IS and the hymenopteran model Argogorytes females. Such a clear morphological parallelism was not observed here between AF and Andrena cornbinata females. In fact, the Ophrys forms investigated differed very little in pilosity. The study was based on only a few individuals, and the normal variation due to age, water status, etc. remains to be shown, but seems at present to be as large as the variation between forms. The slightly greater disorder of the hairs of AF than of those of IF and IS may be understood as a developing adaptation to the more hairy pollinator, A. combinata. It is probable, that a combination of stimuli of several modalities, not the touch feeling of individual hairs per se, acts as the releaser of the copulation behavior. Then there is no necessity of an exact congru- ence of the single hairs between model and mimic to achieve the adequate (from the flower's point of view) pollinator posture (Kullenberg 1961). Moreover, the mechanical properties of the hairs are unknown. The stiffness and lengths of the hairs are factors that com- bine with the overall bulkiness and solidity of the labellum to optimize the function of the flower as a female dummy and thus to optimize its chances of getting pollinated. During the visits of Andrena combinata and Argogo- rytes in the flower, the frons of the male is directed against the gynostemium. The pollinia then attach to his head (Fig. 21). Ophrys species of the Arachni- tiformes and Araneiferae groups guide their Andrena pollinators similarly, those of the Fusci-Luteae group CHEMOECOLOGY inversely with the head downwards and the distal seg- ments against the basal structures of the labella. This study shows that the traditional biosystematic way of classification through measuring the macro- morphological characters is not sufficient for a correct description and analysis of the variation in biolog- ical forms. With the help of chemical analyses of the variation in odor composition, separate biolog- ical forms may be indicated which are not easily visual- ized by the human eye in the morphological flower structure. It may be legitimate to see the studies of chemical structures alluded to as biological morphology on a molecular level. In such research, the studies of form and function are intimately brought together. The conclusion is, that the variation in chemical composition of the flower fragrances has led to different pollination syndromes to the extent, that the three forms of O. insectifera are in reality genetically isolated from each other and are in the process of evolving into separate species. This may be regarded as an example of sympatric speciation. Acknowledgements We thank Dr Heidi Dobson for valuable comments on earlier drafts of the manuscript and Mr Gary Wife for skilful assistance at the SEM for some of the pictures. Financial support from the Swedish Natural Science Research Council, the Bank of Sweden Tercentenary Foundation, and the Axel and Margaret Ax:son John- son Foundation is gratefully acknowledged. References gren L, Borg-Karlson A-K (1984) Responses of Argogorytes (Hymenoptera: Sphecidae) males to odor signals from Ophrys insectifera (Orchidaceae). Preliminary EAG and chemical investi- gation. 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