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Introduction
Sexual deception of male bees is one of the
most remarkable mechanisms of pollination
and is exclusive to the Orchidaceae (Dafni
1984, Ackermann 1986, Nilson 1992). It has
been documented in Australia (Peakall 1990),
South America (van der Pijl and Dodson 1966,
Singer 2002), South Africa (Steiner et al. 1994)
and Europe (Kullenberg 1961, Kullenberg and
Bergstrom 1976, Paulus and Gack 1990a).
Ophrys orchids grow mainly around the Mediterranean (Europe, Northern Africa, Middle
East), but are also common in central and
northern Europe (Delforge 2001). They are
pollinated mostly by bees (Andrenidae, Anthophoridae, Colletidae, Megachilidae, and
Apidae) as well as by predatory and parasitic
wasps (Sphecidae and Scoliidae) and occasionally by beetles (Scarabaeidae, Kullenberg 1961,
1973; Borg-Karlson 1990; Paulus and Gack
1990a).
Ophrys owers imitate the pollinators
females in shape, colour and, most importantly, in scent; males of the pollinating bees
are lured to the owers by olfactory signals
106
107
Table 1. Investigated Ophrys species with their corresponding pollinators, and sample collection
information
Species
Pollinator
Sample Site
Date
O.
O.
O.
O.
O.
O.
A.
A.
A.
A.
A.
A.
Majorca
Crete
Majorca
Tunisia
Israel
Crete
Feb. 1998
Feb. 2001
Mar. 1998
Feb. 1996
Mar. 2001
Feb. 2001
9
15
13
11
13
13
fusca
sitiaca
bilunulata
africana
israelitica
herae
nigroaenea
nigroaenea
avipes
avipes
avipes
nigroaenea
108
Results
Biologically active compounds. We found 46
peaks, comprising 52 chemical compounds, to
be active in the simultaneous recordings of
FID and EAD signals. Some of the samples
were contaminated with silicones. These contaminations coeluted with some of the GCEAD active peaks (X1, X2, X3, X5, X9, X13,
(Z)-9-nonacosene) that were, therefore, excluded from further analyses and not listed in
Table 2. In O. sitiaca we registered 25 peaks
consisting of 28 active compounds (Table 2,
Fig. 1a), in O. herae 31 peaks comprising 37
compounds (Table 2, Fig. 1b). The highest
number of active compounds was found in
O. israelitica, with 39 compounds in 36 peaks
(Table 2, Fig. 1c). Although most of the
electrophysiologically active compounds were
present in all investigated Ophrys species, they
did not always trigger EAD responses. GCEAD runs with reference compounds showed
that this was due to the low concentration of
these compounds in the bouquets of certain
investigated species.
The biologically active compounds identied were mainly hydrocarbons, namely heneicosane to heptacosane (C21C27), alkenes
C25 to C31 showing the double bond at
positions 7, 9, 11 to 14, and alkadienes C27,
C29, and C31 (unknown double-bond positions). Among the non-hydrocarbons, aldehydes (chain lengths 9 to 26) were active, as
were also nonanoic acid and one wax-type
ester, 2-nonyl hexadecanoate (unknown enan-
Hexacosane
Heptacosane
Nonacosadieneb
Nonacosadieneb
(Z))14/(Z))13-/(Z)12/(Z)-11-Nonacosenea
Hentriacontadieneb
15.29 6.8 13.71 4.6
13
14
15
16
8.67 11.5* 6.15 5.7* 4.64 5.4* 2.16 1.2 0.68 0.2 AB
20.92 6.2* 20.17 5.6* 12.22 3.6* 15.04 8.1 17.47 4.9 FI;SI;HI
7.62 6.8*
18.56 5.6* 21.88 5.6* 19.66 6.6* 20.94 6.3 23.32 4.3 FA;FH;FB
0.00 0.0
0.51 0.3 4.46 2.9* 10.47 6.2 16.22 5.7 FA;AS;AH;AI;SH;SI;SB;
HI;HB
12.45 2.4 13.53 2.9* 16.10 6.1* 8.45 1.9 6.78 4.1 6.60 4.4 FA;FI;FB;AS;AH;SI;SB;
HI;HB
12.83 5.0 16.99 7.4* 14.48 4.4* 22.78 5.2* 16.79 5.9 14.37 3.1 FI;AI;HI
4.84 6.1 0.00 0.0
9.01 7.1* 8.70 9.9 13.98 11.5 14.86 5.6 FA;AS;SH;SB
Alkenes/ alkadienes
8
(Z))12/(Z))118.50 8.2
Pentacosenea
9
Heptacosadieneb
4.90 4.8
10 (Z))13/(Z))12/(Z)-11- 23.18 5.6
Heptacosenea
11 (Z))9-Heptacosene
13.71 3.5
12 (Z))7-Heptacosene
4.30 3.8
Tetracosane
Pentacosane
meanSE
4
5
meanSE
0.67 0.4 0.91 0.4* 1.54 0.3* 3.13 1.9* 3.77 2.5 1.59 0.7 FA;FH;FI;FB;SH;SI;SB
0.96 0.3 0.89 0.5
2.42 0.6* 1.37 0.4* 0.97 0.6 0.56 0.3 FH;AH;AI;SH;HI;HB
*
45.41 6.6 22.39 8.4 37.14 7.4* 27.62 7.5* 50.28 4.8 37.47 7.8 FS;FI;AS;AB;SH;SB;HI;
HB;IB
5.45 1.8 4.23 1.2* 5.82 0.9* 3.68 0.7* 3.44 0.5 3.23 0.5 AH;SH;HI;HB
19.75 3.1 36.49 4.7* 31.57 3.7* 27.60 3.8* 22.18 4.3 26.07 3.4 FA;FS;FH;FI;AS;AH;SI;
SB;HB;IB
1.38 0.6 2.13 0.5* 3.31 0.8* 3.77 0.8* 1.53 0.4 2.59 0.5 FA;FH;FI;AI;AB;SH;SI;
SB;HB;IB
26.38 6.2 32.95 8.4* 18.21 3.9* 32.82 6.6* 17.83 3.7 28.48 5.7 FB;AH;AB;SH;SB;HI;IB
meanSE
Ophrys
africana
Heneicosane
Docosane
Tricosane
meanSE
Ophrys
bilunulata
1
2
3
meanSE
Ophrys
israelitica
meanSE
Ophrys
herae
Alkanes
Ophrys
sitiaca
Ophrys
fusca
No. Compound
Table 2. Mean relative amounts (%) of the electrophysiologically active compounds in the labella extracts of the investigated Ophrys species.
Percentage was calculated separately for alkanes, alkenes/ alkadiens and non-hydrocarbons
X4
Eicosanal
X6
Heneicosanal
X9
X10
Docosanal
2-Nonyl
hexadecanoatec
X12
Tetracosanal
X14
X16
X17
Hexacosanal
26
27
28
29
30
31
32
33
2.1
3.1
2.6
1.3
0.8
1.7
5.6
4.81
3.71
3.66
1.24
2.16
6.43
9.64
3.7
3.7*
1.5*
0.5
0.8*
2.7*
3.6*
5.98
2.40
1.81
2.50
3.26
8.70
1.22
4.1
2.3*
0.6*
1.2*
0.9*
2.7*
0.5*
meanSE
Ophrys
herae
5.54
1.15
1.83
1.14
1.29
5.67
4.19
2.7
1.0*
0.8*
0.5*
0.5*
2.5*
2.6*
meanSE
Ophrys
israelitica
2.10
3.61
0.99
1.01
1.41
4.93
2.42
4.1
4.0
0.7
0.7
0.8
2.8
2.3
meanSE
Ophrys
bilunulata
4.05
1.62
0.40
1.11
1.34
4.41
0.64
1.9
0.7
0.1
0.3
0.4
1.2
0.1
meanSE
Ophrys
africana
0.31
6.56
0.39
9.10
0.33
7.09
0.43
4.97
1.64
2.41
3.61
2.88
0.3
2.9
0.4
3.8
0.4
4.2
0.3
2.6
1.3
4.6
2.0
2.6
0.69
7.18
0.20
8.83
0.43
2.84
0.25
3.48
0.48
0.00
8.74
1.23
0.7
5.2*
0.3
5.0
0.3*
2.2
0.3*
1.4
0.4
0.0
4.3
1.5*
0.48
2.30
0.09
11.22
1.79
1.60
0.01
2.18
1.83
0.95
6.59
4.47
0.22
9.41
0.42
15.47
0.83
7.41
0.5
0.64 0.6*
*
0.7
5.57 1.2*
0.3
1.54 1.0*
*
11.9 12.69 7.4
1.4*
0.50 0.2*
0.6
4.04 1.4*
0.08
1.04
1.63
0.03
12.87
0.64
0.16
2.21
0.93
0.20
10.82
1.46
0.2*
0.9*
0.9*
0.2*
4.3*
1.1*
0.0*
1.4
1.5*
0.3
2.3
4.2*
0.2
3.2
1.3
8.6
0.9
4.1
0.2
0.6
0.7
0.1
3.3
0.3
0.06
4.76
0.17
7.80
0.66
8.94
0.10
0.57
0.75
0.23
16.59
0.63
0.1
1.0
0.2
3.3
0.3
3.4
0.1
0.1
0.5
0.1
4.7
0.4
11.79 14.4 14.67 13.9* 11.16 11.8* 14.18 13.9* 14.53 14.2 5.89 3.0
5.84 2.3
9.58 4.0* 18.70 4.5* 15.63 5.5* 14.38 6.3 25.23 3.4
2.57
6.09
4.31
2.84
2.33
5.80
9.97
meanSE
Ophrys
sitiaca
AS
FH;AH;AB;SH;HI;HB;IB
FI;AI;SI;HI
FH
AS;AH;AI;SB;HI;HB
FA;FI
FA;AS;AH;AI;SH;SI;SB
FB;AH;SH;HI;HB
FI;AS;AH;SH;SI;HI;HB
AH;HB
FA;FH;FB;AS;AH;AI;SH;
SI;SB;HI
FA;FH;FI;FB;AS;AH;AI;
AB;SH;SI
FA;FB;AS;AH;AI;IB
AB;SB
AS;AH;AB;SH;HI;HB
FA;FS;FI;FB;AS;AH;HB
34
35
36
37
38
39
Nonanoic acid
Undecanal
Dodecanal
Tridecanal
Hexadecanal
Octadecanal
Nonadecanal
meanSE
Alkanes
19
20
21
22
23
24
25
Ophrys
fusca
No. Compound
Table 2. (Continued)
110
J. Stokl et al.: Pollinator attraction in sexually deceptive orchids
tiomeric composition, Table 2). Fourteen electrophysiologically active compounds could not
yet be identied. In all investigated Ophrys
species alkanes and alkenes released the strongest reactions in male antenna (Fig. 1ac).
The relative proportions of active alkanes,
alkenes and non-hydrocarbons of all investigated species are shown in Table 2. Alkanes
were dominated by the uneven numbered
111
tricosane, pentacosane and heptacosane. Alkenes present in the highest proportions in the
samples were (Z)-heptacosenes (double bond
positions 13, 12, 11, 9) and nonacosadiene.
Most of the non-active compounds belong
to the same chemical classes as the active
compounds and dierences were mainly in the
chain lengths and the double-bond positions
(Ibarra 2002). Further non-active compounds
3
7
10+11
17
25 26 27
19
20
21
23
24
6
8 33 35
9
28
14
13
FID
38
EAD
5
10+11 13+14
7
15
27
17
18 19 20
21
23
22
24
6
33 9
30
1 26
25 2
35
28 31 8
37
FID
38
EAD
18
19 20
21
23
22
24
1 26
25
27
2
10+11
16
32
29 4
9
33 6
30
FID
28 31 8 34 35 38 39
EAD
12
15
18
21
24
27 time [min]
112
113
114
115
116
117
extend neutral genotypic and phenotypic variation in oral scent is correlated in orchid
populations.
Conclusion and future investigations. Our
investigations have shown that allopatric and
sympatric Ophrys species with the same pollinator independent of their phylogenetic
relationship use the same volatiles in very
similar proportions for pollinator attraction.
Therefore, the odour bouquet of an Ophrys
species is under pollinator mediated stabilising
selection and quantitative dierences in scent
between species with the same pollinator are
probably driven by selection rather then
genetic drift. Negative frequency dependent
selection in response to odour learning in
deceptive systems may favour variability of the
pollinator attracting signal within orchid populations (Moya and Ackermann 1993, Ayasse
et al. 2000, Gigord et al. 2001) and Ayasse
et al. (2000) found considerable odour variation within O. sphegodes populations. This
variation may cause hybridisation and speciation, if the odour might by chance resemble the
sex pheromone of another species. Hybrids
that attract a new pollinator that is not
attracted by the parental species would successfully reproduce, being reproductively isolated from other sympatrically occurring
plants, and the new odour would be established by stabilising selection. To answer the
question of speciation by hybridisation, analyses of oral scent and plant material from the
same individuals are needed to answer the
issue of hybrid occurrence. In conclusion,
future investigations with molecular techniques and chemical analyses in combination
with behavioural experiments and electrophysiology will help to clarify the potential hybrid
origin of Ophrys populations and may demonstrate processes of sympatric speciation.
We want to thank Hans Nemeschkal for
statistical advice and Eva Zellinger and Gunda
Dutzler for technical assistance. This study was
nancially supported by the FWF Austria (P12275BIO), the DFG (Ay 12/11) and the Fonds der
Chemischen Industrie (Germany).
118
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Addresses of the authors: Johannes Stokl*, Hannes
Paulus, Manfred Ayasse* (e-mail: Manfred.Ayasse@
biologie.uni-ulm.de), Institute of Zoology, Department of Evolutionary Biology, University of Vienna,