Negative frequency-dependent selection maintains
a dramatic flower color polymorphism in the
rewardless orchid Dactylorhiza
sambucina (L.) Soò
Luc D. B. Gigord*, Mark R. Macnair, and Ann Smithson
Hatherly Laboratories, Department of Biological Sciences, University of Exeter, Prince of Wales Road, Exeter EX4 4PS, United Kingdom
Communicated by John L. Harper, University of Wales, Aberstwyth, United Kingdom, March 30, 2001 (received for review January 10, 2001)
The orchid Dactylorhiza sambucina shows a stable and dramatic
flower-color polymorphism, with both yellow- and purple-flow-
ered individuals present in natural populations throughout the
range of the species in Europe. The evolutionary significance of
flower-color polymorphisms found in many rewardless orchid
species has been discussed at length, but the mechanisms respon-
sible for their maintenance remain unclear. Laboratory experi-
ments have suggested that behavioral responses by pollinators to
lack of reward availability might result in a reproductive advantage
for rare-color morphs. Consequently, we performed an experiment
varying the relative frequency of the two color morphs of D.
sambucina to test whether rare morph advantage acted in the
natural habitat of the species. We show here clear evidence from
this manipulative experiment that rare-color morphs have repro-
ductive advantage through male and female components. This
is the first demonstration, to our knowledge, that negative
frequency-dependent selection through pollinator preference
for rare morphs can cause the maintenance of a flower-color
polymorphism.
T he evolutionary significance of floral polymorphisms has
excited biologists since Darwin (1). The advertisement pro-
vided by the corollas of angiosperms is vital in enabling polli-
nators to locate the source of food reward (2, 3). Although flower
color is diverse throughout the flowering plants, variation within
species is uncommon. Most reported cases of flower-color
polymorphisms cover a small color range, or the colors con-
cerned are not distinguished by pollinators and may simply
represent phenotypic plasticity or balance between mutation and
selection (4, 5). Species that show a true, stable, genetically based
polymorphism are comparatively rare (4).
Orchid species are unusual in that high levels of corolla-color
variability have been recorded within many species (6–9). These
variable species also are known not to produce a reward for their
pollinators, either through nectar or pollen (10). Does the
absence of reward play a role in the maintenance of these
corolla-color polymorphisms? It has been suggested that both
the rewardless characteristic of the plant and the naivete of
pollinators may contribute to their maintenance (7, 11, 12).
Rewardless orchids are usually pollinated by newly emerged
naive insects, typically bumblebees in Europe (6). When en-
countering a novel polymorphic rewardless food source, behav-
ioral experiments showed that bumblebees tend to sample
different color morphs in alternation, because visiting an empty
flower increases the probability of switching to a different color
morph (12). This behavior results in rare morphs being propor-
tionately over-visited (12). If the number of visits to a given
morph is correlated positively with male and female reproduc-
tive success in rewardless orchid populations, the relative fitness
of a morph is predicted to decrease as its relative frequency
increases. Such negative frequency-dependent selection could
maintain stable corolla-color polymorphisms in these species. To
www.pnas.org兾cgi兾doi兾10.1073兾pnas.111162598
maintain a polymorphism, the relative advantage for each morph
should reverse at some particular frequency. This frequency,
where each morph has an equal fitness, is the predicted equi-
librium morph frequency. This frequency will be reached re-
gardless of initial gene frequencies or the system of genetic
control of the color polymorphism, assuming that selection acts
only on the phenotype (13, 14).
The Elderflower orchid Dactylorhiza sambucina is a reward-
less species that has a striking purple-yellow flower-color poly-
EVOLUTION
morphism (Fig. 1). This polymorphism is present throughout the
European range of the species (15) and pollinators are able to
discriminate between the two distinct morphs (ref. 6; Fig. 2). To
test the negative frequency-dependent selection hypothesis, we
performed a manipulative experiment that used synthetic arrays
to vary the relative frequency of the two color morphs while
maintaining constant density.
Materials and Methods
Biological Model. D. sambucina is a widespread European orchid
with a boreal-alpine distribution (15). The mean frequency of
yellow-flowered D. sambucina across natural populations in
Europe is 53% ⫾ 2.6 (n ⫽ 174). We did not find any differences
between the two morphs for other traits such as plant height or
the number of leaves and flower size, and the two morphs are
identical for floral scent (6).
Experimental Design. We constructed 10 synthetic arrays of D.
sambucina, varying the morph frequency within the arrays to test
the influence of morph frequency on relative reproductive
success. The experiment was performed in France during May
and June 2000 on the central part of the Causse Noir (N 44° 10⬘,
E 03° 20⬘, Altitude: 1,000 ⫾ 11 m) in the Massif des Cévennes
where D. sambucina does not occur naturally, although popu-
lations are recorded frequently elsewhere in the Massif and at
the edge of the Causse (minimum 5 km from the experimental
areas). We chose such isolation to ensure naivete of the polli-
nators to D. sambucina. The predominant pollinators of D.
sambucina are bumblebees, principally Bombus lapidarius (6)
and Bombus terrestris (L.G. and A.S., personal observation),
which were abundant in the experimental areas. We removed
600 individual plants from a population ⬇12 km from the Causse
and potted them. Experimental arrays then were constructed in
10 locations on the Causse. Each location was chosen for habitat
similarity both to each other and to that of natural populations.
A minimum distance of 2 km separated the locations to ensure
isolation of the pollinator fauna. Arrays consisted of 50 plants
dispersed on a square grid. Grid size was 9.5 by 9.5 m, with grid
*To whom reprint requests should be addressed. E-mail: L.D.B.Gigord@exeter.ac.uk.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
PNAS 兩 May 22, 2001 兩 vol. 98 兩 no. 11 兩 6253– 6255

Fig. 1. Two individuals of D. sambucina are shown.
spacing of 0.5 m, giving 400 potential positions for plants. Plants
were allocated randomly to arrays after we controlled for flower
number, reallocating plants among arrays where necessary.
Plants then were allocated randomly to the 400 potential posi-
tions within arrays. Two replicates each of 0.1, 0.3, 0.5, 0.7, and
0.9 yellow morphs were used, with treatments randomly allo-
cated to arrays. We commenced experiments on May 6th, 2000,
the peak flowering time for D. sambucina in surrounding natural
plant populations. After 5 days, the number of pollinia removed
and the number of stigmas with pollen depositions were quan-
tified (all pollinator visitation had occurred by this point). Six
weeks later, fruit set was assessed by counting the total number
of fruits produced per plant.
Reproductive Traits. We calculated male reproductive success per
plant by dividing the total number of pollinia removed by those
available and the total number of pollen deposition recorded and
fruit set by the number of available flowers. These values then
were averaged separately for the two morphs within each array.
We found no significant differences in flower number between
morphs across arrays (F10,499 ⫽ 0.393, P ⬎ 0.05), nor between
morphs for fruit length (F10,723 ⫽ 1.519, P ⬎ 0.05), fruit width
(F10,723 ⫽ 0.630, P ⬎ 0.05), or dried weight of seeds per fruit
(F10,723 ⫽ 0.352, P ⬎ 0.05). The average reproductive success of
the arrays was very similar to those recorded in natural popu-
lations, which are limited by access to pollinators. The relative
reproductive success of the yellow morph for each array
was calculated as RRSy ⫽ 2.RSy兾(RSy ⫹ RSp). RRSy is the
6254 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.111162598
Fig. 2. The reflectance spectrum of the two flower colors is given as a
function of the wavelength (light gray for the yellow morph and black for the
purple morph). Two spectra are given for each color representing the range of
the spectrum found over 10 individuals. Spectra were recorded by using a
portable reflectance spectrophotometer (High Sensitivity Spectrophotometer
S2000, New Electro-Optical Concepts, Beaufays, Belgium) that gave a reflec-
tance spectrum across all of the wavelengths (including UV) to which insect
pollinators are sensitive (16).
relative reproductive success of the yellow morph, RSy is
the average reproductive success of the yellow morph, and RSp
is the average reproductive success of the purple morph. We then
tested the relationship between relative reproductive success of
the yellow morph and its frequency among arrays by correlation
and regression. After experiments were completed, all plants
were returned to their precise positions in the source population.
Results and Discussion
We found that relative male and female reproductive success of
the morphs, as measured by relative pollinia removal, relative
pollinia deposition, and fruit set, were all correlated significantly
negatively with morph frequency across experimental arrays
(Fig. 3). For all estimates of relative reproductive success, the
identity of the morph that has the relative advantage reverses as
the frequency of yellow plants increases. At both extreme
frequencies, the rare morph has a significantly higher value for
all indices of reproductive success considered, compared with
the common morph. To our knowledge, these results are the first
to clearly show that negative frequency-dependent selection,
mediated through pollinator foraging preference, has the po-
tential to maintain a color polymorphism in natural plant
populations. Assuming that pollinator-induced selection acts
alone on this polymorphism, we can use the relationship we
found between reproductive success and morph frequency to
predict the equilibrium morph frequency expected in nature.
The predicted morph frequencies (Fig. 3) were 69% for yellow
morphs, based on pollinia removal, and 72% and 61% for
pollinia deposition and fruit set, respectively (Fig. 3). In the
region of France where these array experiments were performed,
the mean morph frequency is 69% ⫾ 3% yellow morphs (n ⫽ 20
populations), which is entirely consistent with these predictions.
In populations elsewhere in South Europe, we have found similar
morph frequencies but higher levels of variation in morph
frequency among populations at high altitude and at the north-
ern edge of the species’ range in Scandinavia. Genetic drift may
result in such deviations from predicted equilibrium morph
frequencies. In natural populations, the relative reproductive
Gigord et al.

Fig. 3. Relative male (a) and female (b) reproductive success of the yellow
morph as a function of the relative frequency of the yellow morph in each
array. Male reproductive success for a morph was quantified as the average
proportion of pollinia removed from plants within each array (n ⫽ 5 to 45
plants) (F). Female reproductive success was measured as the average
proportion of stigmas receiving pollen (}) and setting fruits (䊐) for plants
within each array. Relative reproductive success was calculated as de-
scribed in Materials and Methods. Each data point represents the mean of
two arrays per frequency, with bars showing one SEM. Statistics were
calculated by using all values. r is the Spearman correlation coefficient
(Pearson correlation coefficients were identical). *, P ⬍ 0.05; **, P ⬍ 0.01.
The slopes given are the lines fitted in regression analysis: (a) y ⫽ ⫺0.66x ⫹
1.452; (b1, solid line for pollinia deposition) y ⫽ ⫺0.63x ⫹ 1.454, and (b2,
broken line for fruit set) y ⫽ ⫺0.59x ⫹ 1.354. The horizontal line corre-
sponds to equal reproductive success between the two morphs. The inter-
section between regression lines and the horizontal line gives the value
of predicted morph frequencies at equilibrium (represented by vertical
dotted lines).
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Kingdom (Murray, London).
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Gigord et al.
success of a given morph of D. sambucina also was found to
correlate negatively with morph frequency (17).
It can be seen that predicted morph frequencies are biased
constantly to yellow. This bias toward yellow morphs in terms of
the relative reproductive success and equilibrium morph fre-
quency is not unexpected. Pollinator-behavior experiments fre-
quently record innate pollinator bias to particular colors (12, 18),
and the bias could reflect foraging behavior that alternates
between morphs but favors one over the other. Alternatively, it
has been suggested that the predominant colors of coflowering
plants in the plant community surrounding rewardless orchid
populations may influence the color preferences of pollinators
(6). This behavior in turn could modify the predicted equilibrium
morph frequency, although not the prediction that negative
frequency dependence can maintain a polymorphism.
Negative frequency-dependent selection is often cited as a cen-
tral mechanism in maintaining phenotypic variability in natural
populations (19), but convincing demonstrations of negative fre-
quency-dependent selection acting in natural animal or plant
populations are, however, scarce (20–22). Frequency-dependent
selection caused by behavioral preferences is best known from
predator–prey systems, where predator behavior leads to prefer-
ence for common prey morphs (23–25). This preference would be
EVOLUTION
expected to lead to rare morph advantage through greater mortality
of common morphs in prey populations. In contrast, such prefer-
ences for common morphs among pollinators foraging on reward-
ing plants would tend to lead to an advantage for the common
morph. Such positive frequency-dependent selection is predicted to
lead to monomorphism (26–29). This study strongly suggests a
reversal in pollinator behavior for rewardless plants, giving negative
frequency-dependent selection that can lead to stable polymor-
phism. This study is a demonstration that pollinator behavior could
cause negative frequency-dependent selection. The role of such a
selection pattern deserves further attention, particularly when
considering its role in maintaining the exceptionally high levels of
variability recorded for flower color and other traits such as scent
and floral form among rewardless orchid species (7).
We thank the Parc National des Cévennes for permission to work in the
periphery of the park; C. Lee, C. Jelensperger, and R. Gala for their help
in the field; Mr. Rouzier and Mr. and Ms. Arjailles for allowing access
to the source population; and Mr. and Ms. Baraille, Mr. and Ms. Tritz,
the Passet family, and all the inhabitants of Meyrueis and the Causse
Noir for their participation in locating arrays. We also thank C. Lee for
the photograph of D. sambucina. L.G. was supported by National
Environment Research Council (London) Grant GR3兾12106 (to A.S.
and M.R.M.). A.S. was supported by National Environment Research
Council (London) Research Fellowship GT5兾98兾12兾TS.
15. Tutin, T. G., Heywood, V. H., Burges N. A., Moore, D. M., Valentine, D. H.,
Walters, S. M. & Webb, D. A., eds. (1980) Flora Europaea (Cambridge Univ.
Press, Cambridge, U.K.).
16. Peitsch, D., Fietz, A., Hertel, H., de Souza, J., Ventura, D. F. & Menzel, R.
(1992) J. Comp. Physiol. A 170, 23–40.
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Evolution, eds. Chittka, L. & Thomson, J.-D. (Cambridge Univ. Press, Cam-
bridge, U.K.), pp. 237–257.
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19. Ayala, F. J. & Campbell, D. (1974) Annu. Rev. Ecol. Syst. 5, 115–138.
20. Allen, J. A. (1988) Philos. Trans. R. Soc. London B 319, 485–503.
21. Sinervo, B. & Lively, C. M. (1996) Nature (London) 380, 240–243.
22. Eckert, C. G., Manicacci, D. & Barrett, S. C. H. (1996) Heredity 77, 581–588.
23. Allen, J. A. & Clarke, B. (1968) Nature (London) 220, 501–502.
24. Allen, J. A. (1972) Nature (London) 237, 348–349.
25. Gordon, I. J. (1987) Biol. J. Linn. Soc. 31, 1–23.
26. Levin, D. A. (1972) Am. Nat. 106, 453–460.
27. Smithson, A. & Macnair, M. R. (1996) J. Evol. Biol. 9, 571–588.
28. Thompson, V. (1984) Heredity 53, 677–686.
29. Cresswell, J. E. & Galen, C. (1991) Am. Nat. 138, 1342–1353.
PNAS 兩 May 22, 2001 兩 vol. 98 兩 no. 11 兩 6255
Corrections

EVOLUTION. For the article ‘‘Negative frequency-dependent se-

lection maintains a dramatic flower color polymorphism in the
rewardless orchid Dactylorhiza sambucina (L.) Soò,’’ by Luc
D. B. Gigord, Mark R. Macnair, and Ann Smithson, which
appeared in issue 11, May 22, 2001, of Proc. Natl. Acad. Sci. USA
(98, 6253– 6255; first published May 15, 2001; 10.1073兾
pnas.111162598), the authors note that the labels for the symbols
in the graph of Fig. 3b were switched. The open square should
have been labeled ‘‘fruit set r ⫽ ⫺0.85,’’ and the solid diamond
should have been labeled ‘‘pollinia deposition r ⫽ ⫺0.76.’’ The
corrected figure and its legend appear below.

CORRECTIONS
Fig. 3. Relative male (a) and female (b) reproductive success of the yellow
morph as a function of the relative frequency of the yellow morph in each
array. Male reproductive success for a morph was quantified as the average
proportion of pollinia removed from plants within each array (n ⫽ 5 to 45
plants) (F). Female reproductive success was measured as the average pro-
portion of stigmas receiving pollen (䉬) and setting fruits (䊐) for plants within
each array. Relative reproductive success was calculated as described in Ma-
terials and Methods. Each data point represents the mean of two arrays per
frequency, with bars showing one SEM. Statistics were calculated by using all
values. r is the Spearman correlation coefficient (Pearson correlation coeffi-
cients were identical). *, P ⬍ 0.05; **, P ⬍ 0.01. The slopes given are the lines
fitted in regression analysis: (a) y ⫽ ⫺0.66x ⫹ 1.452; (b1, solid line for pollinia
deposition) y ⫽ ⫺ 0.63x ⫹ 1.454, and (b2, broken line for fruit set) y ⫽ ⫺0.59x
⫹ 1.354. The horizontal line corresponds to equal reproductive success be-
tween the two morphs. The intersection between regression lines and the
horizontal line gives the value of predicted morph frequencies at equilibrium
(represented by vertical dotted lines).
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www.pnas.org PNAS 兩 May 18, 2004 兩 vol. 101 兩 no. 20 兩 7839 –7840
MEDICAL SCIENCES. For the article ‘‘Defective CD95兾APO-1兾Fas
signal complex formation in the human autoimmune lymphopro-
liferative syndrome, type Ia,’’ by David A. Martin, Lixin Zheng,
Richard M. Siegel, Baohua Huang, Galen H. Fisher, Jin Wang,
Christine E. Jackson, Jennifer M. Puck, Janet Dale, Stephen E.
Straus, Marcus E. Peter, Peter H. Krammer, Stephen Fesik, and
Michael J. Lenardo, which appeared in issue 8, April 13, 1999, of
Proc. Natl. Acad. Sci. USA (96, 4552–4557), the authors note the
following. For patient 31 in Table 1, the nucleotide change incor-
rectly appeared as ‘‘G943C’’ instead of ‘‘G934C,’’ and the amino
acid change appeared as ‘‘Arg-234-Pro’’ instead of ‘‘Gly-231-Ala.’’
The corrected table appears below.

Table 1. Clinical and molecular characteristics of nine ALPS patients with mutations in the CD95 death domain
CD4⫺/CD8⫺ Anti-CD3␧ Anti-CD95
Patient Nucleotide Amino acid ␣␤ T cells, % apoptosis,* % apoptosis,* % NMR
ID no. change change (⬍1.0%) (50–70%) (70–90%) structure Disease manifestations†

3 A915C Thr-225-Pro 15 14 35 Disrupted LY, SM, AIHA, NP

5 C1011T Gln-257-STOP 18 33 25 Disrupted LY, SM, AIHA, ITP, NP
6 C966A Ala-241-Asp 13 8 21 Intact LY, HSM, AIHA
17 T1123G Ile-294-Ser 11 9 18 Disrupted LY, HSM, AIHA, NP
26 A975T Asp-244-Val ND 16 1 Intact LY, HSM, AIHA
29 G943A Arg-234-Gln 2.2 15 18 Intact LY, SM, AIHA
30 ⌬T1074 Leu-278-STOP 7.4 ND 5 ND LY, HSM, AIHA, ITP, GN, NP
31 G934C Gly-231-Ala 2.3 ND 5 Disrupted LY, SM, AIHA
33 C1020T Gln-260-STOP 5.7 22 17 Disrupted LY, SM, AIHA

*Assays performed on PHA-stimulated peripheral blood lymphocytes as described in Materials and Methods. The percent apoptosis induced in T cells from normal
subjects under these assay conditions is shown in parentheses. All values reported are the results of the propidium iodide incorporation assay with the exception
of the anti-CD95 assay on patient 30, which were done by the DNA content assay as described in ref. 23. ND, not determined.
†LY, lymphadenopathy; SM, splenomegaly; HSM, hepatosplenomegaly; AIHA, autoimmune hemolytic anemia; ITP, immune thrombocytopenic purpura; GN,

glomerulonephritis; and NP, autoimmune neutropenia; direct antiglobulin—note that Coomb’s test was positive in all patients.

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7840 兩 www.pnas.org