Flowers of Cypripedium fargesii (Orchidaceae) fool
ﬂat-footed ﬂies (Platypezidae) by faking
fungus-infected foliage
Zong-Xin Rena,b, De-Zhu Lia, Peter Bernhardtc, and Hong Wanga,1aKey Laboratory of Biodiversity and Biogeography and Plant Germplasm and Genomics Center, Germplasm Bank of Wild Species, Kunming Institute ofBotany, Chinese Academy of Sciences, Kunming, Yunnan 650204, China; bGraduate University of the Chinese Academy of Sciences, Beijing 100049, China; andDepartment of Biology, Saint Louis University, St Louis, MO 63103

c

Edited by May R. Berenbaum, University of Illinois, Urbana, IL, and approved March 25, 2011 (received for review March 1, 2011)

Charles Darwin was fascinated by the orchid–pollinator interactions, but he did not realize that many orchid species are pollinated by deceit. Cypripedium, a model lineage of nonrewarding
orchid ﬂowers, is pollinated primarily by bees. Here we present
both an example of ﬂoral mimesis of fungus-infected foliage inorchids and an example of ﬂat-footed ﬂies (Agathomyia sp.; Platypezidae) as pollen vectors for angiosperms. Cypripedium fargesii
is a nectarless, terrestrial, endangered orchid from southwestern
China that requires cross-pollination to produce the maximum
number of viable embryos. All insects caught entering or leaving
the labellum sac were Agathomyia sp. carrying conidia of Cladosporium sp. on their mouthparts and legs, suggesting mycophagy.
Blackish hairy spots on the upper surface of foliage may imitate
black mold spots, serving as short-term visual lures. Some odor
molecules also associated with Cladosporium cultures were isolated in the ﬂoral scent. Mimesis of fungus-infected foliage probably represents an overlooked but important option inangiosperm diversiﬁcation, because there are three to ﬁve more
Cypripedium spp. in southwestern China with the same mode of
ﬂoral presentation and black-spotted hairy leaves.
deceptive pollination| mimicryC

harles Darwin described and identiﬁed most of the functional ﬂoral morphology and biomechanics in orchid pollination without ever recognizing that many of the ﬂowers that he
examined lacked edible rewards (e.g., nectar, granular pollen)(1). In fact, ﬂoral evolution in the Orchidaceae appears to be
dominated by modes of pollination by deceit (2), in which visualand/or olfactory cues mimic food sources, bodies of receptive
females, and oviposition sites. Variation in ﬂoral mimesis
appears to drive speciation in many lineages within theOrchidaceae (3–5).
The genus Cypripedium is regarded as a model lineage of fooddeceptive orchids restricted to temperate regions of theNorthern Hemisphere (6, 7). No Cypripedium ﬂower studied todate has been found to secrete nectar or to offer accessible,
granular pollen. However, observers continue to record insects
entering their inﬂated labellum sacs (6), with the exception of
three autonomously self-pollinating species (8, 9). The ﬂower
often produces a discernible and pleasant aroma, whereas thelabellum is often a vivid and contrasting color from that of thebroad, solitary staminode and the other ﬁve perianth segments.
Insects that pollinate these ﬂowers do not escape from the sac
until they pass under the receptive stigma and then out throughone of two rear apertures. This escape pathway forces the insect
to contact one of two dehiscent anthers ﬁxing either sticky,
amorphous lumps of pollen or whole pollinia to the dorsum of
the insect’s thorax or head. With a few important exceptions (10,11), bees of various sizes dominate the pollination of Cypripedium spp. (6, 7, 12, 13).
The biodiversity hotspot of the mountains of southwestern
China is the center of diversity for this genus (8). It is here where
7478–7480 | PNAS | May 3, 2011 | vol. 108 | no. 18

we ﬁnd most of the small, single-ﬂowered, geoﬂorous, dark dullcolored, unpleasantly scented species bearing labellum
1sculptures that resemble the surfaces of fungal sporocarps (14,15). Vogel suggested that these species are pollinated by small
ﬂies, particularly fungus gnats (Sciaridae and Mycetophilidae)(13–16), but he was unable to prove it during his lifetime. Pollination by fungus gnats has been shown in some genera ofmonandrous orchids (4, 17), although some of these ﬂowers
mimic the bodies of female gnats, not mushrooms (2).
Here we focus on Cypripedium fargesii, a critically endangered
species endemic to southwestern China (18), to elucidate its
mechanism of ﬂoral mimesis and to interpret the function of
black spots on its leaves. In this species, each short ﬂoweringstem bears two leaves with rows of black spots on the uppersurfaces (Fig. 1 A and B). The stem terminates in a small, solitary, dark-red to dull-yellow ﬂower that produces a faint but
unpleasant odor reminiscent of rotting leaves. We examined
ﬂower and leaf traits and conducted hand-pollination experiments to identify the breeding system. We tested embryo viability
using a modiﬁed tetrazolium method (19). Pollinators were observed, captured by clogging ﬂoral apertures, and identiﬁed (20).
Floral scent was collected outdoors by dynamic headspace
adsorption methods and analyzed by GC-MS.
Results
The hand-pollination experiments showed that bagged controls
never set fruit, but C. fargesii was self-compatible (Fig. 2), withfruit set rates for hand-mediated self- and cross-pollination of
80.0% and 77.3%, respectively, with no signiﬁcant difference
between treatments (P > 0.05, χ2 test). The seed viability tests
showed that cross-pollinated ﬂowers produced signiﬁcantly
higher proportions of viable embryos compared with self-pollinated ﬂowers (t = 21.231; df = 6; P < 0.001). Fruit set in ﬂowers
exposed to insects was low over four seasons (7.3%, 2.5%, 5.9%,and 3.3%, respectively; Fig. 2). This suggests that this populationof C. fargesii is pollinator-limited, like most Cypripedium spp. (6).
As predicted by such low rates of insect-mediated fruit set, wesaw few insects entering or escaping from these ﬂowers despite
70 daytime and 15 nighttime h of observation at the ﬁeld site. We
collected and euthanized only three female and twomale specimens in the orchids’ labella or in the process of escaping from the interior of a labellum over a 4-y period. Allspecimens were ﬂat-footed ﬂies (Agathomyia sp., Platypezidae)(Fig. 1E). Specimen identiﬁcation was based on morphology andconﬁrmed by DNA barcoding. We observed that these ﬂies entered and escaped from the labellum sac following the same

Author contributions: D.-Z.L., P.B., and H.W. designed research; Z.-X.R. and H.W. performed research; and Z.-X.R., D.-Z.L., P.B., and H.W. wrote the paper.
The authors declare no conﬂict of interest.
This article is a PNAS Direct Submission.
1

To whom correspondence should be addressed. E-mail: wanghong@mail.kib.ac.cn.

www.pnas.org/cgi/doi/10.1073/pnas.1103384108