Using the behavioural bioassay in situ we found that temporary ant-repellency resulting from VOC emission during dehiscence is common in young acacia flowers, but varies greatly in intensity between species. The flower volatiles tested also elicited different response levels in different resident ant species, with the rank-order being Crematogaster sjostedti most responsive, C. mimosae and then C. nigriceps intermediate, and Tetraponera penzigi least responsive (Fig. 2); these rank-orders were the same for all six acacia species providing adequate data for testing (Ho = rankings independent: W = 0·543, P < 0·05), and they match, in order of decreasing aggression, the dominance rank order for the ants from these acacias (Stanton, Palmer & Young 2002). Responses of non-resident ants (Camponotus) were usually undetectable above control levels. Matching of aggression and repellence hierarchies supports the hypothesis that floral repellents have been selected to keep the most aggressive ants away from flowers that require pollinating insect visitors.
Figure 2. Behavioural responses of different ants to flower volatiles from the eight acacia species collectively, showing both the hierarchy of response levels between ant species, and the greater responses during dehiscence. Responses of ants are means, corrected by subtraction of responses to clean air puffs (see Methods). The first four comparisons show different species of genus Crematogaster, the last one shows Tetraponera penzigi; and they derived from two different domatia-bearing host trees, Vachellia seyal fistula and V. drepanolobium. Hatched bars show mean responses during the dehiscence windows, and open bars are responses outside the windows; error bars are ± 1 SEM. Significant differences between these two periods (‘in’ vs ‘out’ dehiscence window) from GLM analyses are indicated: **P < 0.01, *P < 0.05.
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The ant-acacia Vachellia seyal fistula had the most ant-repellent flowers, eliciting responses significantly (n = 223, χ2 = 3·99, d.f. 1, P < 0·05) above the average level from all resident ant species, and with especially high responses when assayed within the dehiscence window. More generally, the responses for every instance of resident ant species tested on its own myrmecophyte acacia were always stronger when tested within the specific dehiscence windows, compared to using pre- or post-dehiscent flowers (see Fig. 2); and for 17 out of 26 other ant/acacia combinations tested the dehiscing flowers gave significantly stronger responses than the non-dehiscing flowers (with no significantly weaker responses). More specific time-based data from V. seyal fistula are shown in Fig. 3, including its volatile scent profile in successive 2-h samples.
Figure 3. Numbers (a) and durations (b) of ant visits to flowers of the myrmecophytic Vachellia seyal fistula, showing the few and very short visits to young flowers in the dehiscence window (09.00–11.00); and (c) the volatile emissions profile for young flowers of this species in 2-h sampling slots, with the peak of E,E-α-farnesene corresponding temporally with ant deterrence from flowers.
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Given that acacia flowers were more ant-repellent when pollen was dehiscing, the volatile signal is likely to derive from the anthers. This is confirmed by ant behaviour patterns; patrolling ants that walked onto the inflorescence ‘surface’ provided by the massed anther heads made long visits (mean 126 ± 27 s, n = 74) to old flowers, but only short visits to young flowers (mean 17 ± 9 s, n = 146, for all recording times). Specifically in the dehiscence window, the few ants seen on the surface of young flowers were visibly agitated and rapidly departed (mean visit 1·8 ± 1·1 s, n = 19). However, occasional ants (<10% of patrollers) instead foraged deep within a young inflorescence at the basal corollas, made longer visits (mean 245 ± 72 s, n = 34), and were unaffected by dehiscence. These patterns are consistent with repellent volatiles being emitted outwards from the peripheral cup-shaped anthers, rather than from the basal corollas. Two further lines of evidence locate the repellent signals specifically to the compound pollen grains (polyads):
(1) Timing of ant-repellence differed across species, but always matched the diurnal course of polyad availability for each of eight acacia species tested. For example, on V. seyal fistula, ants were more strongly repelled by inflorescences with a higher pollen standing crop so there was a strong positive correlation between percentage pollen availability and the magnitude of the ant response (R2 = 0·368, n = 302, P < 0·01). The only other possible volatile source in anthers is the anther gland ‘lids’ (see Methods); but for all eight species the correlations between % lids present and ant responses were weak and in nearly all cases non-significant.
(2) Ant-repellence persisted when polyads were artificially retained on inflorescences by bagging from dawn to exclude visitors. Vachellia etbaica inflorescences with 78% polyads retained, but with 87% anther glands shed (their stalks withered, and shrunken glands fallen from the muslin bag), elicited significantly higher ant responses (0·88 ± 0·12; see Methods) compared to the normally-visited inflorescences (10% polyads retained, 95% anther glands shed: ant response 0·18 ± 0·04).
We conclude that volatiles derive from polyads, so that sequential loss of pollen to visitors structures the time course of repellence, beginning when the ‘lids’ lift and expose the polyads and potential pollinators start arriving (Stone, Willmer & Nee 1996) and diminishing as the polyads are progressively removed by visitors. A pollen-based odour signal automatically and precisely provides the appropriate time-course to ant-deterrence. This specific floral VOC emission provides a transient, highly-focussed protection for the sparse and valuable young inflorescences, but allows ants to return and protect older post-pollination flowers as seed-set commences.
These results generalize our specific findings from 1997, and have been augmented by analysis of the VOCs concerned. GC-MS confirmed that old acacia inflorescences had much lower volatile emission levels than young ones (Table 1). The mean total volatile outputs detected per young inflorescence varied from a maximum of 1070 ng in V. etbaica to just 4 ng in V. drepanolobium. The mean ranks of repellence and of volatile output showed no match, so mere quantity of VOC emissions did not influence ant responses. However, the acacias showed qualitatively very different scent profiles, as did conspecific young and old inflorescences (notably V. seyal fistula where old inflorescences retained very little of the complex VOC profile of young ones). Several VOCs occurred in more than one acacia species, especially linalool and its derivatives, 2-ethylhexanoic acid, and pinenes (all being common floral volatiles: Knudsen et al. 2006). However, the only conspicuous temporal VOC effect occurred in V. seyal fistula, with a strong peak of E,E-α-farnesene dominating the 0900 and 1100 h samples (see Fig. 3c), and thus coincident with the dehiscence window and with maximum bioassay repellence. This was also the only VOC to show a significant negative correlation with recorded ant responses through time, as expected for an effective repellent (ant numbers, R2 = 0·49, P < 0·1 NS; ant visit duration, R2 = 0·59, P < 0·05; all other correlations NS).
Table 1. Volatiles present (ng per inflorescence) in different acacia species, as detected by GC-MS. (a) Samples gathered as five sequential: aliquots from young infloresences, then averaged; (b) Single samples gathered over 8 h, for young and old inflorescences. (Values are from at least 10 inflorescences, taken from 2 to 4 different trees)
|Vachellia seyal seyal||Vachellia seyal fistula||Senegalia mellifera||Vachellia etbaica||Vachellia brevispica||Vachellia drepanolobium||Vachellia seyal seyal||Vachellia seyal fistula|
| || || || || || ||Young||Old||Young||Old|
| β-pinene||–|| ||9||–||–||–||–||–||944||189|
| myrcene||–||–||27|| ||–||–||–||–||–||–|
| β-phellandrene||–||–||7|| ||4||–||–||–||–||–|
| Ocimene Z+E/ ocimenol|| || ||95|| ||163|| ||–||–||–||–|
| Linalool oxide pyranoid Z+E||21||23||23||22||65||–||–||–||272||–|
| Linalool oxide furanoid Z+E||2||–||15||82||339||–||183||31||62||–|
| Irregular terpenes|
| 4,8 dimethyl 1,3,7 nonatriene Z+E||–||–||–||–||92||–||–||–||–||–|
| Fatty acid derivatives|
| 2-ethylhexanoic acid||26||2||12||4||2||4||–||–||–||–|
| Cinnamic aldehyde/ alcohol Z+E||–||2||–||10||–||–||–||–||288||–|
| 4-methoxy benzoate/ aldehyde||–||24||–||21||–||–||–||–||292||–|
| N-containing compounds|
|Total ng per inflorescence||67||135||388||1070||918||4||304||31||3906||352|
E,E-α-farnesene is already known as a signalling molecule. In plants it is associated with anthers in several genera (Jürgens & Dotterl 2004), and is potentially inducible via the jasmonate pathway in response to herbivory (Rodriguez-Saona et al. 2001). In insects it is a known component of alarm pheromones in several taxa including aphids and some myrmecine and formicine ants (e.g. D’Ettore et al. 2000). Related volatiles from fruit extracts can repel Crematogaster opuntiae (Russell et al. 1994); and synthetic farnesol is repellent to the ant Linepithema humile (Shorey et al. 1996). Conversely, E,E-α-farnesene is attractive to bees, including Apis mellifera (Blight et al. 1997), and E,E-farnesol is a component of the foraging recruitment pheromone used by Bombus terrestris (Mena Granero et al. 2005). Thus our observations add a further nuance to the ways that plant VOCs can act as filters, manipulating insect behaviour by chemical mimicry and serving as dual function floral traits (Herrera et al. 2002).
Floral repellence is now also known in some Central American acacias (V. collinsii,Ghazoul 2001; V. hindsii, V. macracantha, Acaciella angustissima,Raine, Willmer & Stone 2002; and V. constricta, Nicklen & Wagner 2006). The last authors found that ants avoided protracted interactions with the youngest dehiscing flowers (though apparently only being repelled on contact), and concluded that repellence resided in pollen or anther glands. Intriguingly, E,E-α-farnesene has also been identified in V. collinsii from Costa Rica (NE Raine & D Edwards, unpublished data).
Thus at least 14 acacia species show some degree of floral ant-repellence, comprising six myrmecophytes and eight non-myrmecophytes. Patterns are beginning to emerge, since ant-repellence is generally higher in the myrmecophytes within related co-flowering communities (e.g. high in V. seyal fistula and V. hindsii, but lower in other species in Kenya and Mexico respectively). However the common East African V. drepanolobium, though heavily ant-defended, has low overall floral repellence and low volatile output per inflorescence (Table 1). This anomaly may relate to flowering regime; most species studied have sporadic sparse flowering, but V. drepanolobium shows intense mass-flowering, with old and young inflorescences in crowded contact and presumably indistinguishable by scent. High repellence from young inflorescences might then preclude ant-guard protection of older (seed-setting) flowers; in fact patrolling frequency of ant-guards was almost zero on heavily-flowering branches. Further investigation of the interactions of flowering regime, inflorescence density and repellence should clarify these effects.
We note also that volatile repellence is not the only tactic employed by acacias; some ant-guarded species use temporal and spatial patterning of their rewards to manipulate ant distributions and keep ants away from young inflorescences (Raine, Willmer & Stone 2002; Gaume, Zacharias & Borges 2005). Thus it is evident that ant-repellent ‘filters’ are not the whole story, and interact with other key aspects of the adaptive plant phenotype.
Formica aquilonia was more sensitive than L. niger, showing clear alarm/aggressive responses to about half of all floral species tested (Table 2), and with differences between overall responses to flowers for the three key behaviours (‘mandibles open’, ‘head-up’ and ‘charge’; see Methods). Again, responses were always greatest when flowers were at peak dehiscence, and were elicited to pollen alone when this was tested (Table 3), even though much smaller volumes of tissue were then used.
Table 2. Volatile reaction scores of Formica aquilonia (means, n = 6–22 for different species) to various floral volatiles, and the morphological score for physical barriers to ants in the same flowers (see Materials and Methods)
|Clade||Order||Genus and species||Volatile reaction score||Morphological score|
|Magnoliids||Magnoliales||Magnolia x soulangeana*||66||0|
|Monocots||Alismatales||Aponogeton x crispus||0||3|
|Symphytum x uplandicum||21||1|
|Forsythia x intermedia||35||0|
Table 3. Mean volatile reaction scores of Formica aquilonia (n ≥ 7) to volatiles from whole flowers and to separated anthers, either dehiscent or post-dehiscent, from five temperate plant species
| ||Volatile reaction score|
|Whole flower||Dehiscing anthers||Old anthers||Flower/ inflorescence with anthers removed|
For these plants, Fig. 4 indicates some trade-off between morphological barriers and the strength of volatile repellence in the flowers. No species tested showed high levels of both kinds of defence (i.e. none occur in the ‘upper right’ portion of the plot). This supports the original speculation on trade-offs by Guerrant & Fiedler (1981).
Figure 4. Trade-off effects between morphological defences and volatile floral repellence in 67 plants tested. While many species had low scores in both respects, those with high morphological defence scores never had strong VOC repellence, and those with strong repellence never had substantial morphological defence. (There were significant decreases in repellence between groups 0 and 2 (W = 35·0, P = 0·015); and between 2 and 1 (W = 40·5, P = 0·018); though not between 0 and 1 (W = 425·5, P = 0·825); with no tests using group 3 due to small sample size).
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As with acacias, it is likely that other variables influence both physical/decoy defences and VOCs in relation to ants, such as timing of anthesis and of nectar presentation (if any), and accounting for these might in practice reveal a multivariate trade-off (Agrawal & Fishbein 2006).
Floral volatile signals clearly have multiple functions as do all floral traits (Irwin, Adler & Brody 2004), including attracting pollinators, deterring casual visitors or thieves, and sometimes the specific deterrence of ants. Thus floral scents may function as allomones to deter enemies as well as being synomones to attract pollinating mutualists. But whilst it may be desirable to keep ants away from flowers, as with any mutualism there are associated costs for all the mechanisms described here, and in some cases there may be additional inherent risks of exploitation and cheating, so that the balance of costs and benefits will inevitably vary with circumstance and may change across evolutionary time. By keeping ants away, ‘windows of opportunity’ are provided for florivores, especially flower-feeding beetles. In an American ant-acacia, caterpillars of an unidentified moth exploit ant-repellency by constructing a protective case of acacia filaments so they can eat the foliage without being attacked by resident ants (Raine & Stone, pers. obs., Fig. 5a). Repelling the normally guarding ants may also alter the mutualism’s costs and benefits by allowing in enemies (predators and parasitoids) of herbivores, the enemies themselves often being recruited by plant leaf VOCs, so that the plant experiences multiple and conflicting selective pressures. Any ant-repellent filter is also likely to select for ants that can circumvent it, and ‘parasitic’ non-defensive ants commonly do exploit the normal ant-plant mutualisms (e.g. Gaume & McKey 1999; Raine et al. 2004; Clement et al. 2008). More specifically, Junker, Chung & Blüthgen (2007) found some ants resistant to the repellent effects of certain flowers; for example Dolichoderus thoracicus ants regularly foraged in Ipomea cairica flowers despite these eliciting strong repellence in other ant species; D. thoracicus thereby gain access to an otherwise under-utilized resource. Further studies of ant behaviours in natural encounters with living flowers whose repellence has been assayed would clearly be valuable and are underway.
Until recently, little was known about floral or pollen volatile effects except as attractants. Existing compilations of floral and/or pollen volatiles (Dobson & Bergström 2000; Knudsen et al. 2006) do contain some compounds that are known to affect some ant behaviours or to act as components of ant pheromones or defensive secretions. But our studies show that floral volatiles with a generalized role as filters against ants are far from characteristic of plants in general; they may be associated principally with species that recruit ants for defensive purposes or for seed-dispersal services, and/or with species lacking architectural defences for their flowers. Pollen-based compounds with defensive functions have been recorded in a few wind-pollinated plants (Jürgens & Dotterl 2004), but compounds deterrent to animal pollen-vectors were largely unreported until our work on acacias, and it is intriguing that one of the most effective compounds appears to be a pheromonal ‘mimic’. Since ants substantially pre-date much of the explosive radiation of flowering plants, they may have played a major role in selecting for dual-function VOCs that still attract pollinators, whether by influencing chemistry, dosage, or both. It has often been noted that ants’ pheromones can be perceived by other insects and so contribute to the ants’ protection of plants against herbivores (Offenberg et al. 2004); it now seems likely that floral release of pheromonal mimics can have similar effects, giving additional benefit to plants. Thus flower VOCs can be targeted such that particular animals are manipulated in time and space as either friend or enemy to a plant. Flowers are therefore emerging as hotspots for research into convergent mutualism management, with a diversity of subtle and interacting strategies that can be employed when simple morphological features are not enough.