THE POLLINATION ECOLOGY OF ORCHIS GALILAEA (BORNM. ET SCHULZE) SCHLTR. (ORCHIDACEAE)

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1 New Phytol. (1982) 90, ^ I 5 THE POLLINATION ECOLOGY OF ORCHIS GALILAEA (BORNM. ET SCHULZE) SCHLTR. (ORCHIDACEAE) BY R. J. BINO,* A. DAFNIf AND A. D. J. MEEUSE* *Hugo de Vries Laboratory, University of Amsterdam, Plantage Middenlaan 2", 1018 DD Amsterdam, The Netherlands f Institute of Evolution,, Israel (Received 30 June 1981) SUMMARY The flower of Orchisgalilaea (Bornm. et Schulze) Schltr. does not contain nectar. Only the males of Halictus marginatus are attracted and pollinate the orchid; females visit the flowers of other plant families. We suggest that O. galilaea has evolved a pollination mechanism based on a sexual attraction specific for the males of H. marginatus. This sexual attraction is the result of the strong, musk-like scent. INTRODUCTION Little is known about the pollination of the 23 Orchis species recognized by de Soo (1980). Most of them are considered to be adapted to pollination by Hymenoptera (Van der Pijl and 'Dodson, 1966), but only two Orchis species are known to be nectariferous, viz.. Orchis coriophora L. (Eberle, 1974) and O. sancta L. (Dafni and Ivri, 1979). Most of the species of Orchis rely on an attraction based on deceit (Daumann, 1941). Experiments with the nectarless flowers of O. israelitica Baumann et Dafni showed that this Orchis mimics the nectar-producing, liliaceous Bellevalia fiexuosa Bioss. (Dafni and Ivri, 1981). Vogel (1972) observed that pollination in O. papilionacea is positively influenced by the repetitive flight pattern performed by males of solitary bees. Orchis galilaea is an east Mediterranean plant common in Israel. It grows on terra rossa and calcareous soil types in both sunny and shady areas. The flowers of O. galilaea are small (10 to 12 mm), numerous (15 to 90 flowers per plant), and the height of the plant (30 to 70 cm) is quite tall in comparison with other species of Orchis. The flowering period is normally from March to April. Flowering is basipetalous and there is great variation in the colour of the flower (Dafni, 1979). Type 1: Flower white, centre of the labellum spotted deep purple; sepals and petals with purple veins. Type 2: Flower pinkish-purple; the labellum has a pale centre with purple spots; sepals and petals with purple veins. Type 3: Flower yellowish-green; the sepals and petals purple-veined and -spotted. The purpose of the present study was to examine the interaction between pollinators and O. galilaea, a species of the nectarless Orchis type. In addition we studied the influence of colour differences on the rate of fertilization X/82/O2O315 + O The New Phytologist

2 3i6 R. J. BiNO, A. D A F N I AND A. D. J. MEEUSE M A T E R I A L S AND M E T H O D S Experiments were mainly carried out on (Ein Alon), Kfar Shamay and near (March to April 1980). Inflorescences of 10 to 15 plants were cut off and arranged in water-filled beakers placed between stands of O. galilaea, except at Kfar Shamay, where we transferred inflorescences of O. italica Poiret, O. anatolica Boiss., O. caspia Trautv. and O. galilaea to a natural stand of Trifolium resupinatum L., approximately 200 m away from the original site of O. galilaea. Insects visiting the flowers of O. galilaea were collected with a net, killed with carbon tetrachloride and kept for identification. The possible significance of olfactory cues was investigated using a box in which intact flowers were placed. Scent emanated through holes covered by fine cotton gauze, yet visual cues were eliminated. The fruiting percentages of the different colour types were obtained by marking each plant in full flower and by counting the capsules. Swelling of the capsule, 3 to 6 days after the flowers had withered, indicates fruit setting and is easily detectable. The possible incidence of autogamy was checked by enclosing each of 22 inflorescences at the bud stage within gauze to exclude pollinators. We could not establish the presence of floral nectar in any of the collected spurs: tests with Fehling's reagent showed the absence of reducing sugars in the whole tissue. RESULTS Pollinators Only males of Halictus {Evylaeus) marginatus Bralle = Lasioglossum {Evylaeus) marginatum (Bralle) visited the flowers and carried pollinia on their heads (Table 1). In most cases two pollinia were attached to the head of a bee, but in three cases four pollinia were found. We tried to catch the bees before they could make contact with the flowers, in order to ascertain that all attached pollinia had been collected during previous visits to other O. galilaea plants. Behaviour of pollinators Insect visits started at a.m., increasing in frequency during the next 2 h. The frequency of visiting was low in overcast weather, so that the observations were made only on sunny days (at temperatures of 20 to 25 C). All the bees approached the flowers against the wind. Several patterns were recorded in the behaviour of the Halictus bees (Table 2). Most of the bees made small turns, passed by the inflorescence, returning to approach the plant again and flying away without landing. This behaviour we considered as an 'approach'. Some bees approached the flowers, hovered for several seconds around the inflorescence, touched and even landed on the labellum for a few seconds before flying away. This behaviour we considered as a 'visit'. Relatively few insects flew more directly to the flowers. On landing a bee remained in the same position on the labeilum for several to 30 s and was not easily disturbed at this stage. Subsequently it started to move over the labellum and with a quick movement put its head into the flower and obtained the pollinia on the clypeus and frons part of its head. Afterwards the bee flew away, circled around the same plant and returned sometimes to the same inflorescence, but might also approach the flowers of a different O. galilaea plant. This behaviour we considered as a' pollinia attachment'.

3 Pollination ecology of Orchis galilaea 317 The highest number of bees present on one infiorescence at the same time was four; no flocking of males was noticed. The transfer experiment carried out in Kfar Shamay showed that the bees did not stay at the same location all the time. Thus five males of H. marginatus (Table 1) carried pollinia of O. galilaea from plants growing at least 200 m away. The pollinia of O. galilaea are distinct in colour and shape from those of other orchid species growing in the area. Neither H. marginatus nor any other insect was seen to carry pollinia of O. italica, O. anatolica or caspia Trautv. which were also growing nearby. Table 1. Numbers of Halictus marginatus which visited Orchis galilaea Location Date Habitat msects caught Percentage carrying pollinia (Ein Alon) Kfar Shamay Sarcopoterietum spinosii Disturbed Quercetum caliprinii Sarcopoterietum spinosii spinosii 6-4 Trifolium resupinatum Reid Table 2. Behaviour of Halictus marginatus visiting Orchis galilaea Location Pollinia Date Approaches h~^ Visits h * attachment h ' Hours of observation (closed biotope) Kfar Shamay Mass samples Mass collections of insects visiting flowers in the vicinity of O. galilaea showed that H. marginatus is one of the most common halictids. The two sexes may easily be distinguished as the females have a clearly visible groove on the abdomen which is absent in the males. Most were females (Table 3), which can possibly be explained by the fact that H. marginatus is a social bee and the females are the workers. Although we could not identify all captured bees, it was evident that related taxa of the genera Halictus and Andrena and Apis mellifera L. were present in the areas studied. Scent The flowers emit a musk-like scent easily perceived by man. The smell is kept for months even in dried plants. There is no observed periodicity in the scent emission in living material. Experiments with the covered flowers showed that the scent is very important for the attraction of the bees, for five males oih. marginatus

4 3i8 R. J. BiNO, A. DAFNI AND A. D. J. MEEUSE landed on the box and stayed there for several seconds. The bees behaved in this way, both in experiments placed in natural vegetation, and in places some distance from plants of O. galilaea. Fruit setting and polymorphism As no fertilized capsules were found in the bagged inflorescences, it was concluded that autogamy had not occurred. Bees showed no preference for either green (Type 3) or white (Type 1) flowers. An experiment was carried out in the area by putting the inflorescences of 10 to 15 plants of these colour Table 3. Number o/halictus marginatus in mass samples. Mass sampling was carried out by sweeping the net through the vegetation stand near Orchis galilaea Location Date No. of males females Kfar Shamay (of which 2 with pollinia) Table 4. Fruit-set in flowers of different colour. Fruiting percentages were obtained by marking each plant in full flower. The swelling of the capsule 3 to 6 days after fertilization indicate fruit-set and was easily detectable Location green-flowered plants green flowers white-flowered plants white flowers No. and percentage of capsules of green type No. and percentage of capsules of white type (closed biotope) (21 %) 128(35%) 207(25%) 113(42%) X^ () = 16; between the two colour types. Carmel) = 15: at 5% level of significance no significant difference types in different containers. We placed the beakers about 0 5 m from each other. Many times the bees flew from the green to the white inflorescences (or vice versa). As might be expected, in view of the behaviour of the bees, no statistical difference in fruit setting between colour types was discovered (Table 4). The preference of Halictus for predominantly open vegetation was reflected by the fact that a higher percentage of fruit setting was recorded at the relatively open site of compared with the site in forest vegetation at. DISCUSSION Only the males of H. marginatus pollinate O. galilaea, although females and related insects visit the flowers of other plant families found close by. This indicates that O. galilaea with its strong musk-like smell has evolved a pollination mechanism based on a sexual attraction speciflc for the males of H. marginatus. As has been shown by Barrows (1975) in experiments with Lasioglossum zephyrum {Halictini), a specific scent is likely to be important in bringing about

5 Pollination ecology o/orchis galilaea 319 a normal sexual meeting between halictine bees. According to Barrows, the specific olfactory cue is so dominant that other sense-based inputs (such as visual or tactile ones) are less important. Thus in laboratory studies copulatory behaviour of the male could be misdirected towards black ink dots if the aphrodisiac was present. The sequence of behaviour of a male H. marginatus interacting with the flower of O. galilaea is the same as described by Barrows for a male L. zephyrum tempted by a female of the same species. The possible conclusion is that the flowers of the orchid emit a scent similar to the pheromone of the female H. marginatus. The field experiments did not show any appreciable importance of visual cues in the attraction of the bees, because the colour types did not evoke any difference in the behaviour of the pollinators. Orchis galilaea seems to deceive its pollinators mainly by olfactory cues. But once attracted, bees focus their pouncing behaviour on the dark spots in the middle of the labellum, thus releasing the pollinia from the flower. Orchis galilaea still attracts the males in the presence of large numbers of females. The strong scent of the flowers, and the persistent resistance of the females to the mating attempts of the males (Barrows, 1975) may explain this phenomenon. The pollination mechanism in Orchis galilaea may perhaps represent a less advanced stage on the path of evolution towards the highly adapted Ophrys flower with its bizarre pseudocopulation mechanism (Kullenberg, 1961). ACKNOWLEDGEMENTS We wish to express our gratitude to Drs H. Wiering (Amsterdam) for the identification of the Halictus species and to Ms M. Edelbroek (Nigtevecht) for her valuable suggestions and comments on the manuscript. REFERENCES BARROWS, E. M. (1975). Mating behaviour in Halictine bees. III. Copulatory behaviour and olfactory communication. Insectes Sociaux, 22, DAFNI, A. (1979). Orchids of Israel. Arbeitskreis Heimische Orchideen, 3, DAFNI, A. & IVRI, I. (1979). Pollination ecology of, and hybridization between. Orchis coriophora L. and O. collina Sol. ex Russ. Nevi Phytologist, 83, DAFNI, A. & IVRI, I. (1981). Pollination ecology of Orchis israelitica. Oecologia, 49, DAUMANN, E. (1941). Die anbohrbaren Gewebe und rudimentaren Nektarien in der Blutenregion. Beihefte zum Botanischen Zentralblatt, 61, DE Soo, R. (1980). Orchidaceae. In: Elora Europea, 5, Cambridge University Press, Cambridge. EBERLE, G. (1974). Nektarausscheidung im Sporn des Wanzenknabenkrautes. Die Orchidee, 25, KULLENBERG, B. (1961). Studies in Ophrys pollination. Zoologiska Bidrag Fran Uppsala, 34, 34. PIJL, L. VAN DER & DODSON, C. H. (1966). Orchid flowers: their pollination and evolution. Miami Press, Florida. VOGEL, S. (1972). Pollination von Orchis papilionacea in den Schwarmbahnen von Eucera tuberculata. Die Orchidee, Sonderheft,

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