1. Sexual or food deception
Sexual or food deception? Deconstructing the floral mimicry of Cephalanthera rubra (Orchidaceae)
First, Id like to thanks the opportunity to give this talk here and to present my work entitled: Sexual or food deception? Deconstructing the floral mimicry of Cephalanthera rubra (Orchidaceae)
Paulo Milet-Pinheiro
Manfred Ayasse
Stefan Dötterl

2. Pollination in Orchids
Orchidaceae has a vast array of pollination strategies and pollinators1
Attraction of pollinators:
Reward
Deception
1/3 of the estimated 25,000 species are thought to be pollinated by deceit1,2
In Europe, 80% of the allogamous species are deceptive3
Orchidaceae is a species-rich family that has a vast array of pollination mechanisms and pollinators.
In order to attract pollinators, orchids frequently offer reward, such as nectar, pollen, oil and even perfume, but may attract them by deception.
Pollination by deception in orchids is quite representative and 1/3 of the estimated 25,000 species are thought to be pollinated by deceit.
In Europe, 80% of the allogamous species are deceptive
(1) Van der Pijl & Dodson 1969, (2) Ackerman 1986, (3) Paulus 2005

3. Sexual and food deception in orchids
Sexual deception – insect males seeking for mates
Olfactory cues: central role in attracting males1,2
Visual cues: increase attraction of males3 or not4
Food deception – food-foraging insects
Visual cues: color as the main attractant5,6
Olfactory cues: no role5,6
Sexual deception involves the attraction of male by mimiking signals of con-specific females, and is very common in orchids of the genus Ophrys, for example.
In these systems, floral scents are known to play a central role in attracting the males, while visual cues may increase attraction or not…
Food deception involves the attraction of insects foraging for food (nectar, pollen, oil).
In these systems, colour is assumed to be the most important cue attracting pollinators, whereas olfactory cues are assumed to play no role.
(1) Schiestl 2005, (2) Ayasse 2006, (3) Spaethe et al. 2007, (4) Vereecken and Schiestl 2009, (5) Ackerman 1986, (6) Jersarkova et al. 2012

4. Cephalanthera rubra
Distribution: Europe, North Africa, and southwest Asia
Flowers of this allogamous species do not produce nectar
Pollinators: males of Chelostoma rapunculi1
Flight activity of males is strongly associated with bellflowers (Campanula), where they rest and seek for nectar and mates
Ch. rapunculi females are oligoleges on Campanula
Ce. Rubra occurs in Europe, North Africa and Southwest Asia.
This allogamous species does not produce nectar and depends on Ch. Rapunculi males as pollinators.
Nectar feeding, resting and mate-seeking flights of males is strongly associated with bellflowers (Campanula)
h. rapunculi females are oligoleges on Campanula
(1) Nilsson 1983

5. Deceptive pollination in Cephalanthera rubra
Ce. rubra flowers mimic color of Campanula and thereby attract males of Ch. rapunculi1
Rendezvous attraction: Females not observed
Experiments testing attractiveness of floral cues of Ce. rubra against those of Campanula were not performed
Possible role of floral scents was neglected
Are females really not attracted to Ce. rubra flowers or are they only less frequent than males?
In the eighties , Nilsson published its interesting paper about the deceptive pollination mechanism in Ce. Rubra. He suggested that flowers of Ce. Rubra would mimic the color of Campanula flowers to attract males of Ch. Rapunculi.
Since females were not observed on flowers, he suggested that flowers of Ce. Rubra would attract males of Ch. Rapunculi by means of rendezvous attraction, i.e. flowers of Ce. Rubra would mimic flowers of Campanula where mates frequently occur.
However, he did not perform behavioral experiments testing attractiveness of floral cues of Ce. rubra against those of Campanula and completely neglected the possible role of floral scents.
Furthermore, he could not explain well why females were not attracted, leaving some hesitation in his interpretation…I mean, are females really not attracted to….
(1) Nilsson 1983

6. Aim and hypotheses
To re-evaluate the kind of floral mimicry involved in this deceptive system:
Only males visit flowers:
Hypothesis 1: sexual deception
Hypothesis 2: rendezvous attraction
Females do visit flowers:
Hypothesis 3: food deception
In the present study, I aimed to re-evaluate the kind of floral mimicry involved in this deceptive pollination system. I considered two main scenarios,
1. in the first, only male visit flowers. If this is really true, then sexual deception or rendezvous attraction would be possible alternatives
2. Alternatively, if females do visit flowers, we could consider the hypothesis of food deception

7. Testing sexual deception
Bioassays with 1) Ch. rapunculi virgin females extracts 2) orchid flower extracts and 3) pentane (control) in a flight cage
Extracts applied on bee dummies
Male reactions recorded for 3 minutes - Approach: flight of the males towards the dummy - Pouncing: antennal inspection - Mounting: male mounts on the dummy - Copulation: copulation attempt
The first step of the work was to test sexual deception. For this purpose, we performed bioassays with pentane extracts of virgin females and flowers and with pentane as a control

8. Responses of males in bioassays
Control
Ce. rubra flower extract
Virgin Ch. rapunculi cuticle extract
Flowers of Ce. rubra do not mimic sex pheromones of Ch. rapunculi females *
Response of males
We observe that approach, pouncing and mounting responses did not differ significantly according to extracts, but copulation attempts did, i.e. males tried to copulate much more frequently with dummies impregnated with virgin female extracts.
Hypothesis 1: Sexual deception
approach pouncing mounting copulation
Mann-Whitney U-test, Benjamini-Hochberg correction, *: P<0.05

9. Testing rendezvous attraction and food deception
Two-choice bioassays: Olfactory cues
Ca. trachelium
Ce. rubra
30 min
Flower-naive bees
The next step, was to test the hypothesis of rendezvous attraction and food deception….
For this purposes, I performed two-choice bioassays testing the attractivenness of olfactory, with these black cylinder with holes....
Pump
Pump
1,5 m

10. Testing rendezvous attraction and food deception
Two-choice bioassays: Visual cues
Ca. trachelium
Ce. rubra
30 min
Flower-naive bees
visual, with these transparent closed cylinders....
1,5 m

11. Testing rendezvous attraction and food deception
Two-choice bioassays: Visual + olfactory cues
Ca. trachelium
Ce. rubra
30 min
Flower-naive bees
and a combination of both cues of of Ca. Trachelium vs. Those of Ce. Rubra to Ch. Rapunculi bees.
The bioassays were performed for 30 min and all responding bees were collected and stored in a ice box until the end of the experiments.
Pump
Pump
1,5 m

12. Responses of bees to floral cues of Ca. trachelium vs Ce. rubra
And in the nature?
The results showed that floral cues of Ca. trachelium and Ce. Rubra are equaly attractive to both male and female flower-inexperienced bees, indicating a flower mimikry by this orchid.
After finishing the bioassays, I placed a Ce. Rubra in the flight cage and both males and females promptly visited flowers.
At least in the flight cage, visits by females were very frequent, but I still had to confirm that they also visit flowers in the nature.
Binominal test, P > 0.05
00009_xvid.avi
EAD

13. Field observations
Three days of observation - eight females carrying pollinia
Fortunately, I did not need more than three days to observe eight females carrying pollinia of Ce. rubra, and one of these females even allowed me to take a picture.
Ok, we proved that floral signals of magnet and model are equally attractive to Ch. Rapunculi and that females do visit Ce. Rubra in the field.
Knowing this, the next step was to investigate more deeply the visual and olfactory cues of these plants.
EAD

14. Color spectra measurementsUV
Violet - blue
Green - yellow
Orange - red
We measured the color of 13 Campanula species and Ce. Rubra using a spectrophotometer. The similarity in colour reflectance between Ce. Rubra and Campanula species is very evident…

15. Flower colour of Ce. rubra and Campanula species
Bee colour hexagon
The color locus of Ce. rubra flowers is within the Campanula color space
Colour locus of Ce. rubra flowers resemble that of more Campanula spp. than any of the Campanula spp.
Based on hexagon distances, the colour of flowers of Ce. rubra resemble that a greater number of Campanula species than any Campanula species
EAD

16. Flower scents of Ce. rubra and Campanula species
Headspace samples of 11 Campanula species and Ce. rubra
Species-specific scent pattern (ANOSIM; Global R=0.99, P<0.001)
MDS with all compounds (ca. 150)
For evaluating similarity among floral scents, I collected headspace samples from 11 Campanula species and Ce. Rubra. An Analysis of similarities indicated that pattern of floral scent is species-specific as we can see in the figure.
We can also observe that floral scent of Ce. Rubra is not that similar, but is also not more dissimilar than some species, such as C. medium or C. punctata.
After that, I asked myself, how would this looks like if considering only compounds perceived by Ch. rapunculi.

17. GC/EAD analyses with headspace sample of Ca. trachelium1
a: (E)-β-ocimene + phenylacetaldehyde + 1,6-dioxaspiro[4.5]decane
b: (E)-Conophthorin
c: 2-nonanone + guaiacol + terpinolene + (E)-linalool oxide (furanoid)
d: linalool + nonanal (artifact as also present in ambient controls)
e: 2-phenylethanol
f: (E)-2-methyl-1,7-dioxaspiro[5.5]undecane +(E)-7-ethyl-1,6-dioxaspiro[4.5]decane
g: decanal (artifact as also present in ambient controls) + methyl salicylate
h: 2-phenylethyl acetate
i: α-copaene
j: geranyl acetone
I used the information provided by Milet-Pinheiro et al and runned again the analyses…
Milet-Pinheiro et al Journal of Chemical Ecology

18. Flower scents of Ce. rubra and Campanula species
Species-specific scent pattern (ANOSIM; Gl. R=0.94, P<0.001)
MDS only with EAD-active compounds (16)1
Ce. rubra mimics Campanula compounds that are perceived by Ch. rapunculi bees
Which compounds specifically?
And could observe that the analysis of similarities still indicate a species-specific scent pattern for all species, the samples of Ce. Rubra were plotted much closer to several Campanula species, indicating a mimesis of active floral scents by Ce. Rubra.
This result was quite promising, but it becomes even more interesting when having a deeper look at the EAD-active compounds
Milet-Pinheiro et al Journal of Chemical Ecology

19. GC/EAD analyses with headspace sample of Ca. trachelium1
a: (E)-β-ocimene + phenylacetaldehyde + 1,6-dioxaspiro[4.5]decane
b: (E)-Conophthorin
c: 2-nonanone + guaiacol + terpinolene + (E)-linalool oxide (furanoid)
d: linalool + nonanal (artifact as also present in ambient controls)
e: 2-phenylethanol
f: (E)-2-methyl-1,7-dioxaspiro[5.5]undecane + (E)-7-ethyl-1,6-dioxaspiro[4.5]decane
g: decanal (artifact as also present in ambient controls) + methyl salicylate
h: 2-phenylethyl acetate
i: α-copaene
j: geranyl acetone
In the headspace sample of Ca. trachelium we found 16 active compounds. Among them, very usual compounds in high amounts, such as……
But also…
Milet-Pinheiro et al Journal of Chemical Ecology

20. GC/EAD analyses with headspace sample of Ca. trachelium1
a: (E)-β-ocimene + phenylacetaldehyde + 1,6-dioxaspiro[4.5]decane
b: (E)-Conophthorin
c: 2-nonanone + guaiacol + terpinolene + (E)-linalool oxide (furanoid)
d: linalool + nonanal (artifact as also present in ambient controls)
e: 2-phenylethanol
f: (E)-2-methyl-1,7-dioxaspiro[5.5]undecane + (E)-7-ethyl-1,6-dioxaspiro[4.5]decane
g: decanal (artifact as also present in ambient controls) + methyl salicylate
h: 2-phenylethyl acetate
i: α-copaene
j: geranyl acetone
In the headspace sample of Ca. trachelium we found 16 active compounds. Among them, very usual compounds in high amounts, such as……
But also…
Milet-Pinheiro et al Journal of Chemical Ecology

21. GC/EAD analyses with headspace sample of Ca. trachelium1
a: (E)-β-ocimene + phenylacetaldehyde + 1,6-dioxaspiro[4.5]decane
b: (E)-Conophthorin
c: 2-nonanone + guaiacol + terpinolene + (E)-linalool oxide (furanoid)
d: linalool + nonanal (artifact as also present in ambient controls)
e: 2-phenylethanol
f: (E)-2-methyl-1,7-dioxaspiro[5.5]undecane + (E)-7-ethyl-1,6-dioxaspiro[4.5]decane
g: decanal (artifact as also present in ambient controls) + methyl salicylate
h: 2-phenylethyl acetate
i: α-copaene
j: geranyl acetone
But also very unusual floral scent compounds, such as these spiroacetals….
Milet-Pinheiro et al Journal of Chemical Ecology

22. GC/EAD analyses with headspace sample of Ca. trachelium1
a: (E)-β-ocimene + phenylacetaldehyde + 1,6-dioxaspiro[4.5]decane
b: (E)-Conophthorin
c: 2-nonanone + guaiacol + terpinolene + (E)-linalool oxide (furanoid)
d: linalool + nonanal (artifact as also present in ambient controls)
e: 2-phenylethanol
f: E-2-methyl-1,7-dioxaspiro[5.5]undecane +E-7-ethyl-1,6-dioxaspiro[4.5]decane
g: decanal (artifact as also present in ambient controls) + methyl salicylate
h: 2-phenylethyl acetate
i: α-copaene
j: geranyl acetone
In headspace samples of Ce. Rubra flowers, we recorded four compounds which are also found in Campanula flowers. All these compounds are common in floral scents of Campanula spp, but I would like to give more attention to two of them….
Milet-Pinheiro et al Journal of Chemical Ecology

23. GC/EAD analyses with headspace sample of Ca. trachelium1
Major compound of Ce. rubra, which is produced by 10 Campanula spp.
a: (E)-β-ocimene + phenylacetaldehyde + 1,6-dioxaspiro[4.5]decane
b: (E)-Conophthorin
c: 2-nonanone + guaiacol + terpinolene + (E)-linalool oxide (furanoid)
d: linalool + nonanal (artifact as also present in ambient controls)
e: 2-phenylethanol
f: E-2-methyl-1,7-dioxaspiro[5.5]undecane +E-7-ethyl-1,6-dioxaspiro[4.5]decane
g: decanal (artifact as also present in ambient controls) + methyl salicylate
h: 2-phenylethyl acetate
i: α-copaene
j: geranyl acetone
First 2-phenyl ethanol, the major compound of Ce. Rubra, which is produced by 10 of the 11 Campanula species investigated…
Milet-Pinheiro et al Journal of Chemical Ecology

24. GC/EAD analyses with headspace sample of Ca. trachelium1
The spiroacetal more commonly produced by Campanula (9 spp.).
Only mixture of spiroacetals (including E-conophthorin) attracts host-naive males and females of Ch. rapunculi1
a: (E)-β-ocimene + phenylacetaldehyde + 1,6-dioxaspiro[4.5]decane
b: (E)-Conophthorin
c: 2-nonanone + guaiacol + terpinolene + (E)-linalool oxide (furanoid)
d: linalool + nonanal (artifact as also present in ambient controls)
e: 2-phenylethanol
f: E-2-methyl-1,7-dioxaspiro[5.5]undecane +E-7-ethyl-1,6-dioxaspiro[4.5]decane
g: decanal (artifact as also present in ambient controls) + methyl salicylate
h: 2-phenylethyl acetate
i: α-copaene
j: geranyl acetone
And second, and more important, E-conophthorin. This is the spiroacetal more commonly produced by Campanula (9 from the 11 spp. investigated).
This is particularly relevant, because only a mixture of spiroacetals (including conphthorin) attracts host-naive males and females of Ch. Rapunculi.
Milet-Pinheiro et al Journal of Chemical Ecology

25. Take-home messages Ce. rubra is not pollinated by sexual deception
Orchid flowers mimic visual and olfactory cues of Campanula flowers to attract both males and females of Ch. rapunculi
Rendezvous attraction and food deception
Role of floral scent in food-deceptive flowers may have been frequently underestimated.

26. Thanks to… Rebecca Scholz (Ulm) Team of Botanical Garden in Ulm
Student helpers
German Science Foundation (DFG)

27. GC/EAD analyses with orchid and virgin female extracts
Flowers of Ce. rubra do not mimic sex pheromones of Ch. rapunculi females
Well, after these accumulating evidences, I was convinced that Cephalanthera rubra flowers do not mimic sex pheromones of Chelostoma rapunculi females and could definitely reject the hypothesis of sexual deception.
The next step was to test the hypothesis of rendezovous attraction and food deception….
Hypothesis 1: Sexual deception
EAD

28. Responses of bees to floral cues of Ca. trachelium vs Ce. rubra
Flower-experienced bees
Interestingly, the experienced bees discriminate floral cues of Ca. trachelium from Ce. Rubra by means of floral scents
Binominal test, *: P<0.01
EAD