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Ecology |
Department of Plant Systematics, University of Bayreuth, D-95440 Bayreuth, Germany
Received for publication January 13, 2004. Accepted for publication August 24, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-farnesene, 2-phenyl ethanol, pentadecane, -muurolene, phenyl acetaldehyde, and linalool oxide (pyranoid) were either present only in specific taxa or varied greatly in their relative amount between the taxa. An analysis of the scent data using the chord-normalized expected species shared (CNESS) distances of chemical profiles of the species, followed by visualization of the data with nonmetric multidimensional scaling (NMDS) showed that most species belonging to the same genus have similar chemical compositions. The differences in the chemical composition of anther volatiles are discussed with respect to the taxonomy and pollination biology of the species.
Key Words: anther volatiles • GC-MS • Ranunculaceae • thermal desorption
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Floral odor differences between species have often been interpreted as a cue for attracting distinct pollinators (Raguso and Pichersky, 1995
; Schiestl and Ayasse, 2002
). In a number of studies correlations between the fragrance composition of flowers and the type of flower visitor have been found (Knudsen and Tollsten, 1993
, 1995
; Miyake et al., 1998
; Andersson et al., 2002
). Even in species with the same pollination syndrome, it has been shown that floral odors may also function as isolating mechanisms (Hills et al., 1972
; Williams and Dodson, 1972
). In addition to their ecological significance, floral fragrances have been recognized as chemotaxonomic markers in some plant taxa (Gerlach and Schill, 1989
; Dahl et al., 1990
; Harborne, 1993
; Knudsen, 1994
; Knudsen and Mori, 1996
). Floral odor compounds are somewhat limited markers however, due to homoplasy and high intraspecific variation (see Williams and Whitten, 1999
; Azuma et al., 2001
; Barkman, 2001
; Levin et al., 2001
).
Floral scents are most often emitted by the petals, but other floral parts may also play a role in scent production and scent emission (Dobson, 1994
). Pollen odors are considered in evolutionary terms to be the oldest olfactory attractants in flowers (Faegri and van der Pijl, 1979
). They probably evolved as defense compounds against pollen-feeding animals and pathogens (Pellmyr and Thien, 1986
). However, when plants became dependent on animals as pollen vectors, some attractive compounds were included among pollen volatiles (Dobson and Bergström, 2000
). Odors of the androecium and in particular of pollen have shown to be species specific and to differ quantitatively and/or qualitatively from other floral parts (von Frisch, 1923
; von Aufsess, 1960
; Buchmann, 1983
; Dobson, 1987
; Dobson et al., 1990
, 1996
; Knudsen and Tollsten, 1991
; Pichersky et al., 1994
; Bergström et al., 1995
; Dobson and Bergström, 2000
). Pollen volatiles have been of special interest in relation to pollen-collecting bees as important cues for flower discrimination (Dobson, 1987
, 1991
), for example, generalist honeybees having the ability to distinguish between pollen odors of different plant taxa (Doull, 1966
), and pollen specialist bees using floral and pollen fragrances to locate their specific host plant (Dobson, 1987
).
The Ranunculaceae family consists of approximately 59 genera and 2500 species worldwide, centered in temperate and boreal habitats (Tamura, 1993
). Phylogenetic analysis indicates that the family is monophyletic (Hoot, 1995
; Hoot et al., 1999
) and placed within the Ranunculales among "basal tricolpates." The family shows a wide variation in flower structures, and a high diversity of pollination modes (Heywood, 1993
). Most species are entomophilous, although some species of Thalictrum are anemophilous. Self-pollination also occurs. Usually floral nectaries are present, however, in Anemone, Clematis, Thalictrum, and many species of Pulsatilla nectaries are absent and these species rely on pollen as only food resource for attracting pollinators. In most other genera we find flowers with numerous stamens and nectaries, which from the functional point of view are nectar-producing pollen-flowers (Kugler, 1970
). Flowers are more or less wide open as in Ranunculus and Caltha, more cup- or bowl-shaped as in Pulsatilla and Trollius. Further, although the family is insect-pollinated in the main, we find hummingbird-pollinated species in the genus Aquilegia due to the evolution of deep nectar spurs (Hodges, 1997a
, b
; Endress, 2001
). Due to the observed variability in their pollination biology Ranunculaceae are especially interesting for ecological and taxonomical approaches to floral scent chemistry. However, little data on floral scent chemistry of Ranunculaceae is available (e.g., Cimicifuga simplex in Groth et al., 1987
; several Actaea species in Pellmyr et al., 1987
). To our knowledge, with the exception of Ranunculus acris (Bergström et al., 1995
), no data on floral scent of either of the species we studied has been published. Pollen and stamen odor chemicals of Ranunculaceae have been investigated by Bergström et al. (1995)
and Dobson and Bergström (2000)
. Unfortunately, the study of pollen volatiles with adapting headspace techniques used for flowers has been of limited success due to the low rate of volatile release from pollen compared with other scent-producing floral parts (Dobson and Bergström, 2000
). Flamini et al. (2002)
showed that the Solid Phase Microextraction (SPME) method dramatically can lower the threshold for odor sampling and detection by GC-MS. In the present study we used an alternative method to investigate pollen volatiles, namely thermal desorption of mature anthers in quartz microvials inserted into a modified gas chromatograph (GC) injector (Amirav and Dagan, 1997
; Wilkinson and Ladd, Undated). Thus, we analyzed the anther scent chemistry of 12 species from six genera (Anemone, Aquilegia, Caltha, Pulsatilla, Ranunculus, and Trollius) of the Ranunculaceae. The objectives of this paper are to provide chemical descriptions of pollen and anther odors in Ranunculaceae species and to relate scent composition to their taxonomy and pollination biology. Of special interest in the latter case are oligolectic bee species, which collect pollen for their larvae only at a specific plant family or plant genus, and it is assumed that such specialized bee species use pollen odors for host plant finding (Dobson and Bergström, 2000
). Further, we compare our results with the data obtained by other authors (Knudsen and Tollsten, 1991
; Bergström et al., 1995
; Dobson and Bergström, 2000
; Flamini et al., 2002
) that have used headspace techniques to analyze pollen volatiles.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Gas chromatography (GC)/mass spectrometry (MS)
The samples were analyzed on a Varian Saturn 2000 System using a 1079 injector that had been fitted with the ChromatoProbe kit. This kit allows the thermal desorption of small amounts of solids or liquids contained in quartz microvials (Amirav and Dagan, 1997
; Wilkinson and Ladd, Undated). The ChromatoProbe microvial was loaded into the probe, which was then inserted into the modified GC injector. The injector split vent was opened (1/20) and the injector heated to 40°C to flush any air from the system. The split vent was closed after 2 min and the injector was heated at 200°C/min, then held at 150°C for 2 min, after which the split vent was opened (1/20) and the injector cooled down.
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). Electronic flow control (EFC) was used to maintain a constant helium carrier gas flow of 1.8 mL/min. The GC oven temperature was held for 4.6 min at 40°C, then increased by 6°C per min to 260°C and held for 1 min. After each run the column was cleaned by heating at 100°C/min to 300°C. The MS interface was 260°C and the ion trap worked at 175°C. The mass spectra were taken at 70eV (in EI mode) with a scanning speed of 1 scan/s from m/z 40 to 650. The GC-MS data were processed using the Saturn Software package 5.2.1. Component identification was carried out using the NIST 98 mass spectral data base (NIST algorithm) and confirmed by comparison of retention times with published data (Davies, 1990
; Adams, 1995
). Identification of individual components was confirmed by comparison of both mass spectrum and GC retention data (Kovats Index) with those of authentic standards.
Statistical analyses
Using the thermal desorption technique we got volatile compounds, and also compounds that are nonvolatile under natural conditions. These nonvolatile compounds belong to the pollenkitt lipids and were studied in detail, e.g., by Schulz et al. (2000)
in Helianthus annuus. Both pollen volatiles and pollenkitt lipids play important roles in the interaction with pollinators (Singh et al., 1999
; Dobson and Bergström, 2000
). However, pollenkitt lipids do not act as volatiles and therefore all lipids were excluded from further analysis.
We used the CNESS (chord-normalized expected species shared) distance index, ranging between 0 and the square root of 2, to determine the differences between the single samples. CNESS is a metric version of Grassle and Smith's (1976)
NESS similarity index, which was originally built to compare faunal or floral samples. NESS and CNESS can be regarded as more generalized forms of the Morisita index (Morisita, 1959
). Wolda (1981)
investigated many quantitative similarity indices and found that all but one, the Morisita index, were strongly influenced by sample size and diversity. The only disadvantage of the Morisita index is the high sensitivity to changes in the abundance of the most abundant species (Wolda, 1981
), or in our case, the most abundant compounds. However, this problem can be solved by using NESS or CNESS, which can be adjusted, by altering the m-parameter to emphasize the importance of minor compounds in the data (Wolda, 1983
). We calculated CNESS indices using the updated version of the COMPAH (Combinatorial Polythetic Agglomerative Hierarchical Clustering) program (Boesch, 1977
), provided by Gallagher at UMASS/Boston (http://www.es.umb.edu/edgwebp.htm). We determined the best CNESS-m using the method described in Trueblood et al. (1994)
.
We used nonmetric multidimensional scaling (NMDS) in the STATISTICA 5.1 package to detect meaningful underlying dimensions and to visualize similarities between samples (see Borg and Lingoes, 1987
). To evaluate how well (or poorly) the particular configuration produces the observed distance matrix the stress value is given. The smaller the stress value, the better is the fit of the reproduced ordination to the observed distance matrix (Clarke, 1993
). We used the Mann-Whitney U test to compare intraspecific variation in the chemical profiles to interspecific variation. We therefore compared the pairwise dissimilarities (CNESS, explained above) between individual samples within a species/genus to those between samples among species/genera. A variance component analysis was used to estimate the contribution of single compounds to the obtained total variation (presence/absence and/or amount) between the taxa.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). We detected 116, and identified 103 volatiles using the thermal desorption technique. Dominant compound classes were fatty acid derivatives, isoprenoids and benzenoids, whereas phenyl propanoids and nitrogen-containing compounds were rarely found. The most commonly occurring compounds, found for each of the species, were fatty acid derivatives (e.g., protoanemonin, octanal, nonanal, decanal) and benzenoids (phenyl acetaldehyde, 2-methoxy-4-vinylphenol). Numbers ranged from 28 components in A. canadensis to 70 components in P. rubra. However, the scent profiles in all species were dominated only by few components (1–6). Dominant compounds reaching at least a content of 20% were protoanemonin in Pulsatilla and Ranunculus (48–98%), octanal in Aquilegia (28–42%), pentadecane in P. rubra (26%), nonanal in T. europaeus (22%), (E,E)--farnesene in T. europaeus (35%), -muurolene in C. palustris (22,4%), 2-phenyl ethanol in A. sylvestris (39%), and phenyl acetaldehyde in A. canadensis (22,4%).
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-farnesene explains a variation of 5.0% and is the main compound in T. europaeus. 2-phenyl ethanol explains a variation of 4.6% and is the most abundant compound in A. sylvestris. Phenyl acetaldehyde and pentadecane combined explain 4.3% of total variation. Phenyl acetaldehyde can be found in higher amounts in A. canadensis and A. sylvestris, whereas pentadecane is typical for Pulsatilla and A. sylvestris. -muurolene and linalool oxide (pyranoid) combined are responsible for 3.6% of total variation and can be found especially in C. palustris (Table 2).
Chemical composition of genera
Anemone sylvestris is the only species where benzenoids are the dominant compound class (49.2%) with 2-phenyl ethanol and phenyl acetaldehyde as main components. Other abundant compound classes in A. sylvestris were fatty acid derivatives (41.8%), especially pentadecane and nonanal, and sesquiterpenoids (8.0%), e.g., (E,E)--farnesene.
All samples of four studied Aquilegia species were very similar in their scent in comparison to samples of the other investigated taxa. The dominant compound class of these Aquilegia species were fatty acid derivatives (59.0–73.2%), with octanal as the main component in all studied species. Additional abundant fatty acid derivatives in this genus are protoanemonin and nonanal. Benzenoids, including dimethoxytoluene, 2-phenyl ethanol and phenyl acetaldehyde, reached an amount of 18.4–28.7%.
In C. palustris sesquiterpenoids (47.7%), fatty acid derivatives (27.1%), and monoterpenoids (23.0%) were dominating the scent. Typical compounds were -muurolene, protoanemonin, and linalool oxide (pyranoid).
Fatty acid derivatives are the dominant compound class in the Pulsatilla (78.2–96.5%) and Ranunculus (86.4–99.5%) species too. The dominant compound in these taxa is protoanemonin, reaching 48.4% in P. rubra and 98.0% in R. platanifolius. The Pulsatilla species are furthermore characterized by pentadecane as an abundant compound (e.g., P. rubra with 25.5%), Important compounds in P. rubra were also monoterpenoids with 28 compounds (12.1%).
Trollius europaeus is dominated by sesquiterpenoids (46.2%), especially (E,E)--farnesene, and fatty acid derivatives (42.9%) with nonanal and cis-jasmone as main compounds. In this species we also found relatively high amounts of nitrogen-containing compounds (4.7%), with indole most abundant.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Dobson and Bergström, 2000
), beetles (Bartlet et al., 1993
, 1997
; Cook et al., 2002
), and syrphids (Golding et al., 1999
) use pollen odor cues in resource location. However, the chemical composition of pollen scent in most plant taxa has been largely ignored because of the difficulty in sampling. Furthermore, exploring the role of pollen scents in pollination systems is hindered by difficulties in testing the behavioral responses to the odor. Using headspace techniques pollen odor samples have been found to differ from whole flower odor in chemical composition (Flamini et al., 2002
), and the number of compounds identified is often lower (Knudsen and Tollsten, 1991
; Dobson et al., 1996
). Bergström et al. (1995)
found differing fragrance profiles when comparing pollen samples with whole stamens (anthers and filaments). In contrast to the studies that used dynamic headspace methods we used only the anthers, not whole stamens, and the fragrance profiles of pollen samples and whole anthers in R. acris and A. californica gave similar results in terms of quality and quantity of volatile compounds (S. Dötterl, unpublished data). Therefore, we assume that in Ranunculaceae the small amount of tissue of the normally very thin anther walls does not contain any particular volatiles that would interfere with the analysis of pollen volatiles in whole anthers. Nevertheless, this might be different in other plant taxa and further investigations are needed to find putative differences between anther and pollen odors. Another aspect we should bear in mind is that scent emitted by both, anthers or pollen, is probably adsorbed by each other in flowers offering pollen in their opened anthers. Due to the spatial closeness of anthers and pollen, the origin of the odors is of minor importance for their function as olfactory cues for pollen-searching flower visitors. Even for the taxonomical approach, the existence of scent differences between anthers and pollen and the origin of volatiles would be neglectable, as long as we compare scent profiles of similar structures, e.g., whole stamens with whole stamens, and anthers with anthers.
Altogether, our results indicate that the lower number of volatiles found in pollen samples by other authors is more likely to reflect the much lower concentration of volatiles in pollen compared to that of whole flowers rather than the absence of such compounds. For example, Bergström et al. (1995)
studied the spatial fragrance patterns within the flowers of Ranunculus acris, using the dynamic headspace method with Porapak Q as adsorbent and pentane to elute the volatiles, and found only six pollen volatiles in comparison to 30 total flower volatiles. The most abundant compounds were protoanemonin and -farnesene, followed by 2-phenyl ethanol, heptanol, octanol and linalool oxide (furanoid). In contrast, thermal desorption allowed us to detect a total of 30 compounds from mature anthers of R. acris (and the same number in pure pollen samples; S. Dötterl, unpublished data). Ten of these 30 compounds were found by Bergström et al. (1995)
in the scent of whole flowers. Therefore, it can be assumed that the lower number of compounds found by Bergström et al. (1995)
might be due to detection limits. The results of Flamini et al. (2002)
lead to similar conclusions. They performed a headspace analysis of pollen and different floral parts of Laurus nobilis using the solid-phase microextraction (SPME) method. They found 58 pollen volatiles and explained the high number of compounds in comparison to previous publications (Dobson et al., 1987
, 1990
; Bergström et al., 1995
) with the higher concentration capability of the SPME technique. The advantage of the SPME technique, and the ChromatoProbe technique compared to dynamic headspace methods with solvent extraction, is the loss of the elution step with a solvent, and therefore the loss of a dilution effect (see also Raguso and Pellmyr, 1998
). Using ChromatoProbe, it is furthermore not necessary to trap the volatiles on a selective adsorbent or a selective SPME fiber.
Anther odor composition in relation to pollination biology and taxonomy
Generally, our data show that the differences in the anther fragrances between the investigated Ranunculaceae genera reflect to some degree the taxonomy, but there are also correlations to the pollination biology of the species. It is evident from our results that differences in the volatiles between genera were greater than differences within genera and that it is possible to differentiate Ranunculaceae genera on the basis of their anther volatiles. Additionally, we show for the first time that CNESS is very useful to analyze floral scent data, because it finds the optimal m-paramter for the weighting of the occurring components in order to judge the similarity of the species or samples. A high content of the fatty acid derivative protoanemonin was typical for the anther scent in Ranunculus and Pulsatilla species, relatively high amounts of the fatty acid derivative octanal and benzenoids were found in Aquilegia species, and in A. sylvestris. Sesquiterpenes were the dominant compounds in C. palustris and T. europaeus. These differences on the genus level may also be important for oligolectic (pollen specific) bees as cues for their host finding and recognition. Oligolectic bees collect pollen for their larvae only from a limited number of related taxa (Michener, 2000
). For example, Chelostoma rapunculi (Lepeletier) uses Campanula ssp., Chelostoma florisomne uses Ranunculus spp., and Megachile lapponica Thomson uses Epilobium ssp. as food resource (Westrich, 1989
). It is known that oligolectic bees use floral and pollen fragrances to locate their specific host plant (Dobson, 1987
; Bergstöm et al., 1995; Dobson et al., 1999
). Naïve females of Chelostoma florisomne recognize their host plant Ranunculus spp. on the basis of pollen volatiles alone (Dobson and Bergström, 2000
).
The most variable compound in our study was protoanemonin. It was found in all species studied, but dominated only the scent of the two Pulsatilla and the three Ranunculus species. Protoanemonin is widespread in Ranunculaceae (Ruijgrok, 1966
; Bonora et al., 1987a
, 1988
), and it is poisonous for humans and livestock (Bonora et al., 1987a
). In addition, this compound shows insecticidal activity against Drosophila melanogaster Meigen and Tribolium castaneum Herbst (Bhattacharya et al., 1993
), and a broad spectrum of antimicrobial activity (Martin et al., 1990
; Didry et al., 1993
). Bonora et al. (1987b)
found an elicitor-induced accumulation of protoanemonin in Caltha palustris, indicating that the formation of protoanemonin may be a defense-related process. Bonora et al. (1988)
and Bergström et al. (1995)
studied the organ-specific distribution of protoanemonin in Ranunculus ficaria L. and R. acris, respectively, and both found the highest amounts in reproductive organs. Protoanemonin may have its evolutionary origin in flower defense against destructive feeding of animals, especially phytophagous insects, as was suggested in general for flower volatiles (Pellmyr and Thien, 1986
). Such substances also are used by insect pollinators to identify their flower sources (Pellmyr and Thien, 1986
).
Anemone
The dominant compound in A. sylvestris was 2-phenyl ethanol. Anemone sylvestris is the only species in this investigation that has no nectaries and therefore solely depends on pollen as a food resource for flower visitors. Therefore, nectar-foraging moths are not expected to visit Anemone flowers and the occurrence of 2-phenyl ethanol cannot be explained by interaction with moths as in A. chrysantha. Unfortunately, to our knowledge, no data on flower visitors of A. sylvestris is available and further investigations are needed. However, the findings of Roy and Raguso (1997)
may offer a hypothesis for the role of high relative amounts of 2-phenyl ethanol in a pure pollen-flower such as A. sylvestris. They found that halictid bees were equally attracted to pseudoflowers and to a blend containing phenyl acetaldehyde, 2-phenyl ethanol, benzaldehyde, and methyl benzoate in the same relative concentration as in pseudoflowers. Interestingly, such pseudoflowers, which are formed by the crucifer hosts of the fungus Puccinia monoica Arth., are mimicking co-occurring yellow-flowered angiosperms such as Ranunculus inamoenus. From these facts, it can be hypothesized that 2-phenyl ethanol may be of importance for the interaction with pollen-collecting bees.
Besides, we found relatively low amounts of protoanemonin in A. sylvestris. It would be interesting to know if the low content of protoanemonin in anthers and pollen is typical in other Ranunculaceae species that lack nectaries, e.g., Clematis and Thalictrum. A low content of protoanemonin in these species would support the hypothesis that this compound has evolved only as a repellent against pollen-feeding in open flowers that offer nectar as food resource.
Aquilegia
In Aquilegia species nectar accessibility is restricted by the length of the spur into which the nectar is secreted. As stated by Miller and Willard (1983)
North American columbines may be divided into three groups on the basis of their floral color, morphology, and mode of pollination: (1) species in the A. vulgaris group (e.g., A. vulgaris, A. glandulosa) generally have blue or purple flowers with short, hooked spurs and are presumably pollinated by bumble bees, (2) members of the A. canadensis group (e.g., A. canadensis) have nodding, red flowers with short, straight spurs and are pollinated primarily by hummingbirds, (3) members of the A. caerula group (e.g., A. chrysantha) have longer, slightly curved spurs and are adapted for pollination by hawk moths. Despite the differences in pollination biology the scent of all studied Aquilegia species was dominated by the same compound: octanal. This fatty acid derivative, a compound found in the floral scent of different plant taxa (Knudsen et al., 1993
), was characteristic for the Aquilegia genus and responsible for a high interspecific and intergeneric variation.
Nevertheless, the number of scent compounds detected in Aquilegia species may be related to their pollination biology. In A. canadensis, the red coloration combined with production of dilute nectar (Macior, 1978
) suggests adaptation to hummingbird pollination (Grant and Grant, 1968
; Grant and Temeles, 1992
). However, A. canadensis has a well-developed capacity for autonomous selfing resulting from incomplete protogyny and close proximity of stigmas and anthers during dehiscence (Eckert and Schaefer, 1998
). The lower number of volatile compounds found in A. canadensis, compared to the other Aquilegia species, is consistent with either hummingbird pollination or selfing, neither of which should require odor emission. It is known that hummingbirds lack a well-developed sense of smell and that most hummingbird adapted flowers are scentless or reduced in scent (Faegri and van der Pijl, 1979
; Raguso et al., 2003
). In contrast to these findings, we detected high relative amounts of phenyl acetaldehyde, a compound shown to be strongly attractive for night- and day-active Lepidoptera species (Heath et al., 1992
; Honda et al., 1998
; Meagher, 2001
, 2002
). We find many cases where plants are visited by both, birds and Lepidoptera (e.g., Murawski and Gilbert, 1986
; Passos and Sazima, 1995
; Aigner and Scott, 2002
; Wolff et al., 2003
). Both, hummingbirds and many Lepidoptera need large nectar volumes, which are often deeply hidden in flower tubes or spurs. The occurrence of relative high amounts of phenyl acetaldehyde might indicate mixed pollinator guilds during the evolution of A. canadensis, or, as a possible preadaptation, it gives us at least a hint how hawk moth pollination may have evolved in this genus.
The anther odor of A. chrysantha is characterized by a relatively high amount of 2-phenyl ethanol, in comparison to the other investigated Aquilegia species. This benzenoid alcohol was found to be attractive or at least EAD-positive to noctuid moths (Haynes et al., 1991
; Plepys et al., 2002
), and may contribute to pollinator attraction in the sphingophilous A. chrysantha, where the hawk moths Eumorpha achemon (Drury) and Sphinx chersis (Hübner) have been recorded as the most important pollinators in southern Arizona (Miller, 1985
). Interestingly, Miller (1985)
observed that Dialictus species (Hymenoptera: Halictidae) are regular flower visitors of this species. Considering the investigations of Roy and Raguso (1997)
, who found a blend containing phenyl acetaldehyde, 2-phenyl ethanol, benzaldehyde, and methyl benzoate being attractive to halictid bees, it may be concluded that 2-phenyl ethanol plays a role in attracting these bees. However, Miller (1985)
observed that these bees are pollen thieves rather than pollinators because of their failure to consistently contact the stigmas of the flowers.
Caltha
Observed flower visitors at Caltha are Coleoptera, Diptera, Hymenoptera species and the moth Micropterix calthella L. (Micropterigidae) (Nebel, 1990
; Br
descu, 1994
). Dominant terpenoids in our study were linalool oxide (pyranoid) and -muurolene in C. palustris. Especially linalool oxide (pyranoid) is a typical and widespread floral scent compound (see Knudsen et al., 1993
), but also -muurolene was found in the floral scent of different plant taxa, e.g., Annonaceae (Jürgens et al., 2000
), Nyctaginaceae (Levin et al., 2001
), and Rubus idaeus (Robertson et al., 1995
). However, the relative amounts of -muurolene in these studies were very low in comparison to C. palustris. While linalool oxide (pyranoid) may be attractive for the observed moth, as it is electrophysiologically active in Lepidoptera (Raguso et al., 1996
; Andersson, 2003
), nothing is known about the biological significance of -muurolene.
Pulsatilla
The genus Pulsatilla is divided into two groups: a phylogenetically older group having white or yellow flowers without nectaries, which is pollinated by bumble bees and a more derived group with blue or violet flowers and nectaries, which is pollinated by small bees (Kratochwil, 1988
). The studied Pulsatilla vulgaris and probably also P. rubra belong to the latter. Especially P. vulgaris seems to be adapted to the pollination by small bees of the genera Lasioglossum and Andrena (Kratochwil, 1988
). We found high amounts of protoanemonin in both Pulsatilla species, but only in P. rubra pentadecane was another major compound. Both, protoanemonin and pentadecane, may originally act as defenses against phytophagous insects. Pentadecane can be found as floral volatile in different plant taxa (see Knudsen et al., 1993
), and is often used as a defense compound against predators of different insect taxa (Zinner, 1986
; Krall et al., 1999
; Eisner et al., 2000a
, b
; Williams et al., 2001
). It is not known whether pentadecane, found in flower volatiles, plays an additional role for the attraction of pollinators.
It would be interesting to know if we find in the genus Pulsatilla a general trend with species without nectaries having lower protoanemonin or pentadecane content, and with species that evolved nectaries having higher contents of these substances. In species with nectaries it is more reasonable to defend the pollen resource against pollen-collecting flower visitors, than in species that depend on pollen-collecting pollinators.
Ranunculus
Diptera, Hymenoptera, Coleoptera, and the micropterigid moth Micropterix calthella are the most abundant and characteristic flower visitors and probably pollinators of R. acris and other Ranunculus species (Harper, 1957
; Steinbach and Gottsberger, 1994
). In a special case, alpine R. acris, Muscidae and Anthomyiidae flies are by far the most important pollinators, but nevertheless a few visits by a nymphalid butterfly were recorded (Totland, 1994
). Besides generalistic flower visitors, some specialized pollinators exist. Chelostoma florisomne for example collects pollen for their brood solely at Ranunculus species (Westrich, 1989
). Dobson and Bergström (2000)
found out that foraging-naïve females of this bee species could recognize Ranunculus spp. more effectively when offered pollen odors than floral odors, and they found out a key component of the pollen odors without specifying this compound. It would not be surprising if protoanemonin were this key compound, because of its extremely dominant character in Ranunculus. However, in the same study foraging-experienced bees relied more on floral odors, which operate at longer distances.
Trollius
Trollius europaeus is pollinated by several species of Chiastocheta flies (Diptera: Anthomyiidae) that are both seed predators and pollinators. The flies mate in the globe-shaped flowers, consume pollen and nectar, and complete larval development by feeding on the seeds (Pellmyr, 1989
; Jaeger and Després, 1998
; Jaeger et al., 2001
). In T. europaeus (E,E)--farnesene, a typical and widespread floral scent compound (see Knudsen et al., 1993
), dominated the anther odor. This compound is attractive, or at least EAD-positive for insects of different orders, e.g., Lepidoptera (Hern and Dorn, 1999
; Bäckmann et al., 2001
). A second main volatile compound, cis-jasmone, is known to be induced on damage to repel aphids, and it acts additionally as an indirect plant defense by attracting aphid predators and parasitoids (Birkett et al., 2000
). The high proportion of cis-jasmone in Trollius anthers may be a cue signal for its specialized predating pollinators, which use this defense induced volatile to find their food source as well as their feeding mates in the flowers.
Summary and perspectives
The data shows that anther volatiles in Ranunculaceae are of chemotaxonomical value, but further detailed comparative studies of pollen scent chemistry and pollination biology are needed to gain a better understanding of the importance of these odors for the interaction with flower visitors. Especially Ranunculus acris and Trollius europaeus are known for their specific interactions with flower visitors that depend on Ranunculaceae pollen as food source. Experimental attempts to clarify the function of particular pollen odors, and to compare floral and pollen odors in these species are desirable. However, the difficult sampling of pollen odors via dynamic headspace methods, due to the low rate of volatile release, has hampered the progress in this field of research. Here we have shown that the ChromatoProbe technique allows the collection of odors from pollen amounts of single anthers. The ChromatoProbe is a very cost-effective alternative to other thermal desorption devices (see Amirav and Dagan, 1997
).
Our results might reflect stages in the evolution of chemical attractants for pollinators from herbivore deterrents as stated by Pellmyr and Thien (1986)
. The most variable compound in our study was protoanemonin, which may have its evolutionary origin in flower defense against phytophagous insects. In Anemone sylvestris, which depends on pollen-collecting pollinators, the protoanemonin content in anthers and pollen is low. In bee-visited Pulsatilla species the content is high, hence the evolution of nectaries in these particular species makes it reasonable to deter bees from pollen-collection, and in Ranunculus, specialized predating pollinators possibly use protoanemonin as key compound for host-finding.
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2 stefan.doetterl{at}uni-bayreuth.de
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LITERATURE CITED |
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Aigner P. A. P. E. Scott 2002 Use and pollination of a hawkmoth plant, Nicotiana attenuata, by migrant hummingbirds. Southwestern Naturalist 47: 1-11
Amirav A. S. Dagan 1997 A direct sample introduction device for mass spectrometry studies and GC-MS analysis. European Journal of Mass Spectrometry 3: 105-111
Andersson S. 2003 Antennal responses to floral scents in the butterflies Inachis io, Aglais urticae (Nymphylidae), and Gonopteryx rhamni (Pieridae). Chemoecology 13: 13-20[CrossRef][ISI]
Andersson S. L. A. Nilsson I. Groth G. Bergström 2002 Floral scents in butterfly-pollinated plants: possible convergence in chemical composition. Botanical Journal of the Linnean Society 140: 129-153[CrossRef]
Azuma H. M. Toyota Y. Asakawa 2001 Intraspecific variation of floral scent chemistry in Magnolia kobus DC. (Magnoliaceae). Journal of Plant Research 114: 411-422[CrossRef][ISI]
Bäckmann A. C. M. Bengtsson A. K. Borg-Karlsson I. Liblikas P. Witzgall 2001 Volatiles from apple (Malus domestica) eliciting antennal responses in female codling moth Cydia pomonella (L.) (Lepidoptera: Tortricidae): effect of plant injury and sampling technique. Zeitschrift für Naturforschung C 56 262-268
Barkman T. J. 2001 Character coding of secondary chemical variation for use in cladistic analyses. Biochemical Systematics and Ecology 29: 1-20[CrossRef][ISI][Medline]
Bartlet E. M. M. Blight A. J. Hick I. H. Williams 1993 The responses of the cabbage seed weevil (Ceutorhynchus assimilis) to the odour of oilseed rape (Brassica napus) and to some volatile isothiocyanates. Entomologia Experimentalis et Applicata 68: 295-302[CrossRef][ISI]
Bartlet E. M. M. Blight P. Lane I. H. Williams 1997 The responses of the cabbage seed weevil Ceutorhynchus assimilis to volatile compounds from oilseed rape in a linear track olfactometer. Entomologia Experimentalis et Applicata 85: 257-262[CrossRef][ISI]
Bergström G. H. E. M. Dobson I. Groth 1995 Spatial fragrance patterns within the flowers of Ranunculus acris (Ranunculaceae). Plant Systematics and Evolution 195: 221-242[CrossRef][ISI]
Bhattacharya P. R. S. C. Nath D. N. Bordoloi 1993 Insecticidal activity of Ranunculus sceleratus (L.) against Drosophila melanogaster and Tribolium castaneum. Journal of Experimental Biology 31: 85-86
Birkett M. A. C. A. M. Campbell K. Chamberlain E. Guerrieri A. J. Hick J. L. Martin M. Matthes J. A. Napier J. Pettersson J. A. Pickett G. M. Poppy E. M. Pow B. J. Pye L. E. Smart G. H. Wadhams L. J. Wadhams C. M. Woodcock 2000 New roles for cis-jasmone as an insect semiochemical and in plant defense. Proceedings of the National Academy of Sciences of the United States of America 97: 9329-9334
Boesch D. F. 1977 Application of numerical classification in ecological investigations of water pollution. Report prepared for US EPA Ecological Research Series (EPA-600/3–77–033). Available as PB269 604 from: National Technical Information Service. U.S. Dept. of Commerce, Springfield, Virginia, USA
Bonora A. B. Botta E. Menziani-Andreoli A. Bruni 1988 Organ-specific distribution and accumulation of protoanemonin in Ranunculus ficaria L. Biochemie und Physiologie der Pflanzen 183: 443-447[ISI]
Bonora A. G. Dall'Olio A. Donini A. Bruni 1987a An HPLC screening of some Italian Ranunculaceae for the lactone protoanemonin. Phytochemistry 26: 2277-2279[CrossRef][ISI]
Bonora A. B. Tosi A. Donini B. Botta A. Bruni 1987b Elicitor-induced accumulation of protoanemonin in Caltha palustris L. Journal of Plant Physiology 131: 489-494[ISI]
Borg I. J. Lingoes 1987 Multidimensional similarity structure analysis. Springer, New York, New York, USA
Brdescu V. 1994 Flower flies (Diptera: Syrphidae) on Caltha palustris L. Travaux du Museum d'Histoire Naturelle "Grigore Antipa" 34: 13-15
Buchmann S. L. 1983 Buzz pollination in angiosperms. In C. E. Jones and R. J. Little [eds.], Handbook of experimental pollination biology, 73– 113. Academic Press, New York, New York, USA
Clarke K. R. 1993 Non-parametric multivariate analyses of changes in community structure. Australian Journal of Ecology 18: 117-143
Cook S. M. E. Bartlet D. A. Murray I. H. Williams 2002 The role of pollen odour in the attraction of pollen beetles to oilseed rape flowers. Entomologia Experimentalis et Applicata 104: 43-50[CrossRef][ISI]
Dahl A. E. A.-B. Wassgren B. Bergström 1990 Floral scents in Hypecoum sect. Hypecoum (Papaveraceae): chemical composition and relevance to taxonomy and mating system. Biochemical Systematics and Ecology 18: 157-168
Davies N. W. 1990 Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. Journal of Chromatography 503: 1-24[CrossRef]
Didry N. L. Dubreuiland M. Pinkas 1993 Microbiological properties of protoanemonin isolated from Ranunculus bulbosus. Phytotherapy Research 7: 21-24[CrossRef][ISI]
Dobson H. E. M. 1987 Role of flower and pollen aromas in host-plant recognition by solitary bees. Oecologia 72: 618-623[CrossRef][ISI]
Dobson H. E. M. 1991 Pollen and flower fragrances in pollination. In C. Van Heemert and A. De Ruijter [eds.], Proceedings of the sixth international symposium on pollination, Tilburg 1990. Acta Horticulturae 288: 313-320
Dobson H. E. M. 1994 Floral volatiles in insect biology. In E. A. Bernays [ed.], Insect-plant interactions, vol. 5, 47–81. CRC Press, Boca Raton, Florida, USA
Dobson H. E. M. G. Bergström 2000 The ecology of pollen odors. Plant Systematics and Evolution 222: 63-87[CrossRef][ISI]
Dobson H. E. M. J. Bergström G. Bergström I. Groth 1987 Pollen and flower volatiles in two Rosa species. Phytochemistry 26: 3171-3173[CrossRef][ISI]
Dobson H. E. M. G. Bergström I. Groth 1990 Differences in fragrance chemistry between flower parts of Rosa rugosa. Israel Journal of Botany 39: 143-156[ISI]
Dobson H. E. M. E. M. Danielson I. D. van Wesep 1999 Pollen odor chemicals as modulators of bumble bee foraging on Rosa rugosa Thunb. (Rosaceae). Plant Species Biology 14: 153-166
Dobson H. E. M. I. Groth G. Bergström 1996 Pollen advertisement: chemical contrasts between whole-flower and pollen odors. American Journal of Botany 83: 877-885[CrossRef][ISI]
Doull K. M. 1966 The relative attractiveness to pollen-collecting honeybees of some different pollens. Journal of Apicultural Research 5: 9-14
Eckert C. G. A. Schaefer 1998 Does self-pollination provide reproductive assurance in wild columbine, Aquilegia canadensis (Ranunculaceae)?. American Journal of Botany 85: 919-924[Abstract]
Eisner T. D. J. Aneshansley M. Eisner A. B. Attygalle D. W. Alsop J. Meinwald 2000a Spray mechanism of the most primitive bombardier beetle (Metrius contractus). Journal of Experimental Biology 203: 1265-1275[Abstract]
Eisner T. C. Rossini M. Eisner 2000b Chemical defense of an earwig (Doru taeniatum). Chemoecology 10: 81-87[CrossRef][ISI]
Endress P. K. 2001 Origins of flower morphology. Journal of Experimental Zoology Part B—Molecular and Developmental Evolution 291: 105-115[CrossRef]
Faegri K. L. van der Pijl 1979 The principles of pollination ecology, 3rd ed. Pergamon Press, Oxford, UK
Flamini G. P. L. Cioni I. Morelli 2002 Differences in the fragrances of pollen and different floral parts of male and female flowers of Laurus nobilis. Journal of Agricultural and Food Chemistry 50: 4647-4652[CrossRef][ISI][Medline]
Gerlach G. R. Schill 1989 Fragrance analysis, an aid to taxonomic relationships of the genus Coryanthes (Orchidaceae). Plant Systematics and Evolution 168: 159-165[CrossRef][ISI]
Golding Y. C. M. S. Sullivan J. P. Sutherland 1999 Visits to manipulated flowers by Episyrphus balteatus (Diptera: Syrphidae): partitioning the signals of petals and anthers. Journal of Insect Behavior 12: 39-45
Grant K. A. V. Grant 1968 Hummingbirds and their flowers. Columbia University Press, New York, New York, USA
Grant V. E. J. Temeles 1992 Foraging ability of rufous hummingbirds on hummingbird flowers and hawkmoth flowers. Proceedings of the National Academy of SciencesUSA 89: 9400-9404[CrossRef]
Grassle J. F. W. Smith 1976 A similarity measure sensitive to the contribution of rare species and its use in investigation of variation in marine benthic communities. Oecologia 25: 13-25[CrossRef][ISI]
Groth I. G. Bergström O. Pellmyr 1987 Floral fragrances in Cimicifuga: chemical polymorphism and incipient speciation in Cimicifuga simplex. Biochemical Systematics and Ecology 15: 441-444[CrossRef]
Harborne J. B. 1993 Introduction to ecological biochemistry, 4th ed. Academic Press, London, UK
Harper J. L. 1957 Biological flora of the British Isles. Journal of Ecology 45: 289-342[CrossRef]
Haynes K. F. J. Z. Zhao A. Latif 1991 Identification of floral compounds from Abelia grandiflora that stimulate upwing flight in cabbage looper moths. Journal of Chemical Ecology 17: 637-646[CrossRef][ISI]
Heath R. R. P. J. Landolt B. Dueben B. Lenczewski 1992 Identification of floral compounds of night-blooming jessamine attractive to cabbage looper moths. Environmental Entomology 21: 854-859[ISI]
Hern A. S. Dorn 1999 Sexual dimorphism in the olfactory orientation of adult Cydia pomonella in response to alpha-farnesene. Entomologia Experimentalis et Applicata 92: 63-72[CrossRef][ISI]
Heywood V. H. 1993 Flowering plants of the world. Oxford University Press, New York, New York, USA
Hills H. G. N. H. Williams C. H. Dodson 1972 Floral fragrances and isolating mechanisms in the genus Catasetum (Orchidaceae). Biotropica 4: 61-76
Hodges S. A. 1997a Floral nectar spurs and diversification. International Journal of Plant Sciences 158: 81-88[CrossRef]
Hodges S. A. 1997b A rapid adaptive radiation via a key innovation in Aquilegia. In T. Givinish and K. Sytsma [eds.], Molecular evolution and adaptive radiations, 391–405. Cambridge University Press, Cambridge, UK
Honda K. H. Omura N. Hayashi 1998 Identification of floral volatiles from Ligustrum japonicum that stimulate flower-visiting by Cabbage butterfly, Pieris rapae. Journal of Chemical Ecology 24: 2167-2180[CrossRef][ISI]
Hoot S. B. 1995 Phylogeny of the Ranunculaceae based on preliminary atpB, rbcL and 18S nuclear ribosomal DNA sequence data. Plant Systematics and Evolution (Supplement) 9: 241-251
Hoot S. B. S. Magallon P. R. Crane 1999 Phylogeny of basal eudicots based on three molecular data sets: atpB, rbcL, and 18S nuclear ribosomal DNA sequences. Annals of the Missouri Botanical Garden 86: 1-32
Jaeger N. L. Després 1998 Obligate mutualism between Trollius europaeus and its seed-parasite pollinators Chiastocheta flies in the Alps. Comptes Rendus de l'Académie des Sciences Serie III 321: 789-796[CrossRef]
Jaeger N. F. Pompanon L. Després 2001 Variation in predation cost with Chiastocheta egg number on Trollius europaeus: how many seeds to pay for pollination?. Ecological Entomology 26: 56-62[CrossRef][ISI]
Jürgens A. A. C. Webber G. Gottsberger 2000 Floral scent compounds of Amazonian Annonaceae species pollinated by small beetles and thrips. Phytochemistry 55: 551-558[CrossRef][ISI][Medline]
Knudsen J. T. 1994 Floral scent variation in Pyrola rotundifolia complex in Scandinavia and Western Greenland. Nordic Journal of Botany 14: 277-282[ISI]
Knudsen J. T. S. A. Mori 1996 Floral scents and pollination in Neotropical Lecythidaceae. Biotropica 28: 42-60[CrossRef][ISI]
Knudsen J. T. L. Tollsten 1991 Floral scent and intrafloral scent differentiation in Monoses and Pyrola (Pyrolaceae). Plant Systematics and Evolution 177: 81-91[CrossRef][ISI]
Knudsen J. T. L. Tollsten 1993 Trends in floral scent chemistry in pollination syndromes: floral scent composition in moth-pollinated taxa. Botanical Journal of the Linnean Society 113: 263-284[CrossRef]
Knudsen J. T. L. Tollsten 1995 Floral scent in bat-pollinated plants. A case of convergent evolution. Botanical Journal of the Linnean Society 119: 45-57
Knudsen J. T. L. Tollsten G. Bergström 1993 Floral scents—a checklist of volatile compounds isolated by head-space techniques. Phytochemistry 33: 253-280[CrossRef][ISI]
Knuth P. 1898–1905 Handbuch der Blütenbiologie, I Bd. 1898 (Einleitung und Literatur). II Bd. 1898 (Die bisher in Europa und im arktischen Gebiet gemachten blütenbiologischen Beobachtungen. 1. Ranunculaceae bis Compositae, 2. Lobeliaceae bis Gnetaceae, 1899); III Bd. (Die bisher in außereuropäischen Gebieten gemachten blütenbiologischen Beobachtungen. 1. Cycadaceae bis Cornaceae, 1904, 2. Clethraceae bis Compositae. Nachträge, Rückblick, 1905). Engelmann Verlag, Leipzig, Germany
Krall B. S. R. J. Bartelt C. J. Lewis D. W. Whitman 1999 Chemical defense in the stink bug Cosmopepla bimaculata. Journal of Chemical Ecology 25: 2477-2494[CrossRef][ISI]
Kratochwil A. 1988 Funktionsmorphologische Veränderungen im Blütenbau innerhalb der Gattung Pulsatilla (Ranunculaceae)—Anpassung an neuen Bestäuberkreis. Verhandlungen der Deutschen Zoologischen Gesellschaft 81: 187
Kugler H. 1970 Einführung in die Blütenökologie. G. Fischer, Stuttgart, Germany
Levin R. A. A. R. Raguso L. A. McDade 2001 Fragrance chemistry and pollinator affinities in Nyctaginaceae. Phytochemistry 58: 429-440[CrossRef][ISI][Medline]
Lundqvist A. 1992 The self-incombatibility system in Caltha palustris (Ranunculaceae). Hereditas 117: 145-151[CrossRef][ISI]
Macior L. W. 1966 Foraging behaviour of Bombus (Hymenoptera: Apidae) in relation to Aquilegia pollination. American Journal of Botany 53: 302-309[CrossRef][ISI]
Macior L. W. 1978 The pollination of vernal angiosperms. Oikos 30: 452-460[CrossRef][ISI]
Martin M. L. L. san Roman A. Dominguez 1990 In vitro activity of protoanemonin, an antifungal agent. Planta Medica 56: 66-69[CrossRef][ISI][Medline]
Meagher R. L. 2001 Collection of soybean looper and other noctuids in phenylacetaldehyde-baited field traps. Florid
