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Choice Experiments Using Artificial Flowers Reveal Colour Preferences of Long-proboscid Flies (Diptera, Nemestrinidae) Under Field Conditions Wahlversuche mit Blütenattrappen zeigen Farbpräferenzen langrüsseliger Fliegen (Diptera, Nemestrinidae) unter Feldbedingungen SANDRA VALENTIN, KLAUS LUNAU & STEVE JOHNSON Summary: Long-proboscid flies belonging to the families Nemestrinidae and Tabanidae are important pollinators in southern Africa. However, no experiments have been conducted on the role of flower colour and nectar guide patterning for attraction of these flies. Model flowers varying in colour and nectar guide patterning were presented to Prosoeca ganglbaueri and Prosoeca sp. (Nemestrinidae) under field conditions in South Africa. It was established that both fly species show strong colour preferences. In particular, they avoid colours from the long wavelength sector (human yellow and orange). Prosoeca sp. showed a significant preference for model flowers with guide lines in contrast to flower dummies without lines. Furthermore, models with black lines were preferred over models with blue lines. Although we were not able to distinguish between innate and conditioned preferences, these experiments reveal that long-proboscid flies are able to discriminate strongly among flowers that differ in colour and nectar guide patterning. Preferences by longproboscid flies for certain colours probably account for the distinct patterns of convergent evolution in the visual advertising of plants pollinated by these flies. Keywords: long-proboscid flies, Nemestrinidae, Prosoeca ganglbaueri, Prosoeca sp., choice experiments Zusammenfassung: Langrüsselige Fliegen aus den Familien der Nemestrinidae und Tabanidae sind wichtige Bestäuber im südlichen Afrika. Bisher lagen keine Experimente über die Funktion bestimmter Blütenfarben und Blütenfarbmuster für die Anlockung dieser Fliegen vor. Unter Freilandbedingungen wurden in Südafrika Blütenattrappen, welche in Farbe und Farbmustern variierten, Prosoeca ganglbaueri und Prosoeca sp. (Nemestrinidae) präsentiert. Dabei stellte sich heraus, dass beide Fliegenarten starke Farbpräferenzen besitzen und besonders Farben aus dem langwelligen Sektor (gelb und orange für den Menschen) meiden. Prosoeca sp. zeigte eine signifikante Präferenz für Blütenattrappen mit Führungslinien gegenüber Blütenattrappen ohne Linien. Ebenfalls auffällig war, dass Prosoeca sp. auch zwischen verschiedenen Linienfarben unterschied, wobei sie schwarze Linien gegenüber blauen Linien bevorzugte. Obwohl es nicht möglich war, zwischen angeborenem und erlerntem Verhalten zu unterscheiden, machen diese Experimente deutlich, dass die Fliegen in hohem Maße fähig sind, zwischen Blütenfarben und Farbmustern zu diskriminieren. Die Farbpräferenzen der langrüsseligen Fliegen sind möglicherweise dafür verantwortlich, dass die von diesen bestäubten Blüten konvergent bestimmte Eigenschaften des Schauapparates evolvierten. Schlüsselwörter: langrüsselige Fliegen, Nemestrinidae, Prosoeca ganglbaueri, Prosoeca sp., Spontanwahlexperimente
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1. Introduction In contrast to other countries, South Africa has a remarkable high representation of plants with long and narrow corolla tubes. These correspond to pollinators such as butterflies, moths, bees, sunbirds and flies which feed on nectar in these flowers with their elongated mouthparts. The long-proboscid flies are an important feature of the pollinator fauna of South Africa. Two fly families, the Tabanidae and Nemestrinidae contain many South African representatives with remarkable, needle-like proboscises, which are often longer than their own body (GOLDBLATT & MANNING 2000). In Moegistorhynchus longirostris (Wiedemann, 1819) the proboscis can be 80 mm in length which is up to three times longer than the body. There are three different genera of long-proboscid Nemestrinidae in South Africa: Prosoeca, Stenobasipteron and Moegistorhynchus. They occur between South Namibia and the Cape Peninsula, around the mountains of Lesotho, as well as in KwaZulu-Natal, Mpumalanga, the Northern Province, and Swaziland (GOLDBLATT & MANNING 2000). The genus Philoliche (Tabanidae) also contains many species with elongated proboscis (up to 40 mm) used for feeding on nectar. Whereas nemestrinids feed only on nectar, Philoliche females also feed on blood, like other tabanids. Therefore the mouthparts of Philoliche have an additional portion for blood-sucking which works autonomously from their proboscis (VOGEL 1954). The actual proboscis length of longproboscid species of the Nemestrinidae and Tabanidae can vary between the different species as well as in a single species and also vary between the regions where they occur. The proboscis of the Nemestrinidae is tucked under the body when flying or sitting, whereas the proboscis of tabanids is carried in the front all time. Pollination by long-proboscid flies in South Africa was first documented by MARLOTH (1908) and later more detailed by VOGEL
(1954). Although a pollination system based on long-proboscid flies and long-spurred flowers has been described also for India (FLETCHER & SON 1931) and California (GRANT & GRANT 1965), it seems particularly well developed in South Africa (GOLDBLATT & MANNING 1996). In general most of these pollination systems are located in the southern hemisphere and there are no known cases from the tropics. The life cycles of South African species of Nemestrinidae are still unknown. However, during the last 20 years much attention has been devoted to the study of pollination by long-proboscid flies. REBELO et al. (1985) described that some flowers of the South African Ericaceae have been specialized on pollination by long-proboscid flies. This flower syndrome, termed rhinomyiophily, has also been described for a variety of Iridaceae (GOLDBLATT et al. 1995, 2000; GOLDBLATT & MANNING 1996, 1999), Geraniaceae (STRUCK 1997) and Orchidaceae (JOHNSON & STEINER 1995, 1997). Among rhinomyiophilous plants there are several endemic species, which underlines the importance of long-proboscid flies for conservation of plant biodiversity in South Africa. According to the flower syndrome of rhinomyiophily some general features are as follows: The flowers are mostly zygomorphic, odourless and the petals often have a characteristic flower colour pattern. Spurs are quite narrow, a bit curved or sigmoid and mostly between 35 mm and 70 mm long. Except from a few Orchidaceae they offer small amounts of nectar with a moderately high sugar concentration (mostly 24-29 %), and a high sucrose : hexose ratio (GOLDBLATT & MANNING 1996). GOLDBLATT & MANNING (1996) have described that even within one plant family, the Iridaceae, different guilds have developed which are adapted to different species of long-tongued flies. For example, flowers of the Lapeirousia silenoides group are pollinated by Prosoeca peringueyi (GOLDBLATT et al. 1995; GOLDBLATT & MANNING 1996), whereas flowers of the Lapeirousia fabricii group are only pollinated by Moe-
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gistorhynchus longirostris (GOLDBLATT et al. 1995). POTGIETER et al. (1999) have further shown that some nemestrinid flies also visit flowers of Lamiaceae. Flowers visited by long-proboscid flies vary markedly in colour and particular colours seem to be associated with particular flowers. For example, flowers visited by the tabanid fly Philoliche aethiopica tend to have a pink colour (JOHNSON 2000). The aim of this study, therefore, was to establish whether long-proboscid flies exhibit preferences among flowers according to colour and pattern (nectar guides). 2. Material and Methods 2.1. Study sites Different species of long-proboscid flies were observed in the Province of KwaZulu-Natal (South Africa) and the adjacent region of Lesotho. The study site for spontaneous choice experiments on an undescribed species of the genus Prosoeca was located in the Karkloof Region on top of Mount Gilboa, whereas experiments on Prosoeca ganglbaueri have been carried out on both sides of the border region between South Africa and Lesotho at Ramatselisos Gate (28°55.782’ E; 30°03.244’ S; 2275 m a.s.l.) close to Sehlabatebe National Park. Both sites are characterized by rocky grassland habitats and are located in summer-rainfall areas. The different study sites are shown in figure 1, including spots for the observation of flies and the collection of flowers visited by long-proboscid flies.
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coloured flower dummies were carried out. The flower dummies were presented among real flowers that had been visited by the flies. A record was made every time a fly visited one of the flower dummies. The arrangement of the artificial flowers was randomised and also changed at regular intervals to avoid site effects. To test whether the flies have preferences for flowers with colour patterns, flower dummies were used. Flower dummies without patterns and with line patterns were tested simultaneously. The basic construction of all artificial flowers is shown in figure 2. In this experiment the number of flights towards the different flower dummies was counted. The results of the choice experiments were analysed with the Chi-square-test. 2.3. Material It was recorded, which long-proboscid fly species were feeding on which plant species and in which region. To identify plants, field guides (KILLICK 1990; POOLEY 2003) were used. To identify some insects the field guide of PICKER et al. (2002) was used. Flowers which were visited by long-proboscid flies were taken back to the lab in order to measure their spectral reflection. The spectral reflection of the flower dummies was also measured (fig. 3). The measurements were carried out with a S 2000 Ocean Optics photometer in the range of wavelengths between 300 nm and 700 nm. 3. Results
2.2. Choice experiments
3.1. Choice experiments with Prosoeca sp.
All experiments have been carried out between November 2004 and March 2005, between 8.00 a.m. and 3.00 p.m. Observations were possible only during sunny, warm and windless periods as the activity of the flies decreased markedly when it was cloudy or windy. To test whether the flies exhibit colour preferences, choice experiments with different
On Mount Gilboa the long-proboscid fly Prosoeca sp. visited the blue flowers of Agapanthus campanulatus (Agapanthaceae). These were the only insects observed to visit this plant species during the whole observation period. When offered a choice of several flower dummies differing in colour, Prosoeca sp. flew only towards the blue ones. After 50 flights towards the blue
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Fig. 1: The map shows the complete region of the Drakensberg and the surrounding area (Province of KwaZulu-Natal in South Africa and the adjacent region of Lesotho). The five study sites are marked with bright dots. Abb. 1: Die Karte zeigt das gesamte Gebiet der Drakensberge und Umgebung (Provinz KwaZuluNatal in Südafrika und benachbarte Region von Lesotho). Die Standorte, an denen Beobachtungen und Experimente durchgeführt wurden, sind als helle Punkte eingezeichnet.
flower dummies and none to the other colours the experiment was terminated and further tests concerning flower pattern were carried out. Figure 4 shows the flower petals of A. campanulatus, which have flower markings in the same colour as the petals, just a bit more dull. Blue
flower dummies with dark blue lines were prepared to simulate these flowers. Additional blue flower dummies with black lines and without lines have been prepared. The three different types have been presented to the flies simultaneously (fig. 5).
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Fig. 2: Artificial flower. The stem consists of wood and the side-branches of toothpicks. The ten flowers were made from coloured cardboard. All flowers of one artificial flower were of the same colour. Flower dummies for the choice experiments were made in nine different colours. Abb. 2: Blütenattrappe. Der Stängel besteht aus Holz und die Seitenäste bestehen aus Zahnstochern. Die zehn Blüten wurden aus farbigen Pappen zusammengeklebt. Alle Blüten einer Blütenattrappe haben dieselbe Farbe. Attrappen für Wahlexperimente wurden in neun verschiedenen Farben erstellt.
Fig. 3: Spectral reflection properties of the different flower dummies, which have been used for the choice experiments. Abb. 3: Spektrale Reflexionseigenschaften der verschiedenen für die Wahlversuche verwendeten Blütenattrappen.
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Fig. 4: Reflexion of Agapanthus campanulatus and the blue flower dummies. Note that the flowers of A. campanulatus possess a dark-blue line in every petal. Abb. 4: Reflexionsspektrum von Agapanthus campanulatus und der blauen Blütenattrappen. Man beachte, dass die A. campanulatus Blüten jeweils pro Blütenblatt eine dunkelblaue Linie besitzen.
In the preference test, blue flower dummies with dark blue lines, with black lines and without lines were tested. The flies have been able to discriminate between the different flower dummy types. The flower dummies with black lines have been preferred significantly over flower dummies with blue lines, and the flies strongly preferred flower dummies with lines over flower dummies without lines. 3.2. Choice experiments with Prosoeca ganglbaueri The large long-proboscid fly Prosoeca ganglbaueri was feeding on flowers of Zalusianskya microsiphon (Scrophulariaceae) (fig. 6). This fly species was the only insect visiting and pollinating flowers of Z. microsiphon as well as
flowers of Disa nivea (Orchidaceae) (see JOHNSON et al. 2003; ANDERSON et al. 2005). While the flowers of Z. microsiphon offer nectar, the flowers of D. nivea do not (ANDERSON et al. 2005). In contrast to A. campanulatus, both flowering plant species mentioned above do not flower in clusters but are scattered separately and occur together with many other flowers. There were several other insects, such as bombylid flies, butterflies and syrphid flies, visiting other flowers in the community. Table 1 shows how often Prosoeca ganglbaueri visited the coloured flower dummies. While the colours beige, white, mauve, blue and lilac have been visited frequently, the colours pink, purple, red, orange, and yellow have been ignored completely. It was obvious that not all colours have been visited from the
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Fig. 5: Flight frequency of Prosoeca sp. towards the blue flower dummies with different colours of lines and dummies without lines. Abb. 5: Anflughäufigkeiten von Prosoeca sp. an blaue Blütenattrappen mit blauen Linien, mit schwarzen Linien und ohne Linien. Table 1: Number of approaches of Prosoeca ganglbaueri towards the different flower dummies. The last line indicates how many plants of Zalusianskya microsiphon were still in flower on each observation day. The flower-density refers to a study site which covered approximately 50 m². Tab. 1: Anzahl der Anflüge von Prosoeca ganglbaueri auf die verschiedenen Blütenattrappen. In der letzten Zeile ist angegeben, wie viele Zalusianskya microsiphon-Pflanzen am jeweiligen Beobachtungstag auf der gesamten Untersuchungsfläche von etwa 50 m² noch in Blüte standen.
very beginning of the experiment, but that during the orientation close to the target corresponding to the low spatial resolution of the range increased with time. compound eyes (WEHNER 1981). The results 4. Discussion of the spontaneous choice experiments with Prosoeca sp. established that these flies defiFlower colour is an important signal for nitely have colour preferences and use them detection and recognition of flowers during for flower detection, because the visited blue the distant orientation of insect flower-visi- flower dummies had the most similar reflectors (KEVAN 1983; CHITTKA & MENZEL 1992), tion to the flowers of Agapanthus campanulawhereas patterns on flowers only play a role tus. Although the spectral reflections of Entomologie heute 18 (2006)
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Fig. 6: Prosoeca ganglbaueri visiting the flowers of Zalusianskya microsiphon (Scrophulariaceae). Abb. 6: Prosoeca ganglbaueri beim Blütenbesuch von Zalusianskya microsiphon (Scrophulariaceae).
mauve and lilac flower dummies have also been quite similar to the blue ones, they have never been visited. The colour preferences of the flies are finely tuned and closely match the corolla colour of the single nectar food plant. Although flower dummies with blue lines were more similar to A. campanulatus flowers, the flower dummies with black lines have been preferred. Obviously the preference to guide line colours is less precise than that to flower colours. However, the similarity of the real flower colour and the colour of the artificial flower is only one aspect. Some insects tend to fly towards areas which have a high chromatic contrast against their background. This background can be soil, vegetation (KEVAN 1983; MENZEL & SHMIDA 1993; CHITTKA et al. 1994) or other parts of the flower (KEVAN 1983; LUNAU 1990). Therefore, for guide lines it is possible that the contrast between guide line colour and corolla colour is more important than the colour similarity between guide lines of natural flowers and flower dummies. Experiments on artificial flowers with guide lines have also been done with
the syrphid fly Eristalis tenax (LUNAU et al. 2005). It was shown that the flies found the reward on a central yellow dot quicker when guided by black lines than without guide lines. As Prosoeca sp. did not try to probe the flower dummies it could not be tested whether the lines actually help the fly in finding the nectar source. It is also still unclear whether the colour preference for blue is an innate or learned behaviour. As the lifecycle of this fly is still unknown it was not possible to get naïve flies in order to test innate behaviour. Also cage experiments with captured flies failed, because the flies died within a few hours, or permanently tried to escape from the cage when they lived longer than a day. The flowers of A. campanulatus grew in clusters and other plants did not flower nearby. Thus, the flies probably had no experience with other flowers. As Prosoeca sp. is a species discovered quite recently it is unknown whether this fly visits other plants in other areas. In contrast to Prosoeca sp. the choice experiments with Prosoeca ganglbaueri showed no
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colour preference for a single colour. In the first days the fly visited white, beige and mauve flower dummies. These colours were similar to the cream flowers of Zalusianskya microsiphon and Disa nivea. After a lapse of a few days the flies additionally visited the blue coloured flower dummies, and in the last two observation days they also visited the lilac ones. So the range of visited colours increased within the observation period. This can obviously be attributed to the simultaneous reduction of the flower density of Z. microsiphon and D. nivea. As the offer of the actual nectar sources was running shorter, new food sources were needed and searched for. In the first observation days it was not necessary for the flies to look for other flowers and one could always see them flying in straight direction to their flowers. But in the last observation days it was possible to see them investigating other flowers like Lobelia flaccida (blue flowering), Dianthus basuticus (pink flowering) or Helichrysum eckloniis (white-silver flowering). It was, however, remarkable that the flies never visited yellow flowers, even though there had been a lot of them in flower. Indeed, the flies also ignored yellow, orange and red flower dummies. Therefore, Prosoeca ganglbaueri exhibits clear colour preferences when foraging in the field. For Prosoeca sp. the offer of food sources also was running short at the end of the observation period, but in contrast to P. ganglbaueri these flies never tried to fly towards other coloured flower dummies. This could be a hint that the preference for blue is an innate preference. Observations on Nemestrinidae and Tabanidae in general indicate that flies are attracted by yellow and orange flowers, but, instead, exhibit a preference for cream, pink or blue colours. This matches the flower colour patterns of flowers known to be pollinated by long-proboscid flies (GOLDBLATT & MANNING 2000). As the long-proboscid flies of South Africa represent two different families (VOGEL 1954), it would be fruitful to determine whether Nemestrinidae and Tabanidae differ in their flower colour preferences. Entomologie heute 18 (2006)
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Sandra Valentin Prof. Dr. Klaus Lunau AG Sinnesökologie Institut für Neurobiologie Heinrich Heine University Düsseldorf Universitätsstr. 1 D-40225 Düsseldorf E-Mail:
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Prof. Steve Johnson University of KwaZulu-Natal P. Bag X01 Scottsville Pietermaritzburg South Africa E-Mail:
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