Content uploaded by Kenji Suetsugu
Author content
All content in this area was uploaded by Kenji Suetsugu on Apr 08, 2015
Content may be subject to copyright.
REGULAR PAPER
Autonomous self-pollination and insect visitors in partially
and fully mycoheterotrophic species of Cymbidium (Orchidaceae)
Kenji Suetsugu
Received: 25 February 2014 / Accepted: 14 August 2014 / Published online: 7 October 2014
ÓThe Botanical Society of Japan and Springer Japan 2014
Abstract Few studies have examined the reproductive
ecology of mycoheterotrophic plants, but the existing lit-
erature hypothesizes that they adopt a self-pollinating
strategy. Although growing evidence indicates that some
rewarding mycoheterotrophic plants depend (at least par-
tially) on an insect-mediated pollination system, it remains
unclear whether some mycoheterotrophic plants can attract
pollinators without nectar or other rewards. Moreover, in a
broader evolutionary/ecological context, the question of
whether the evolution of mycoheterotrophy induces a shift
in pollination pattern is still unknown. Here I present a
comparative investigation into the breeding system of two
fully mycoheterotrophic orchids, Cymbidium macrorhizon
and C. aberrans, and their closest extant relative, the
mixotrophic C. lancifolium. Pollination experiments were
conducted to determine the breeding system of these plants.
In addition, flower visitors that might contribute to polli-
nation were recorded. Flowers at different maturity stages
were examined to investigate mechanisms enabling or
limiting self-fertilization. While nectarless flowers of
C. lancifolium and C. macrorhizon can successfully attract
potential pollinator honeybees, all three Cymbidium pos-
sess an effective self-pollination system in which the ros-
tellum that physically separates the stigma and pollinia is
absent. Because mixotrophic and mycoheterotrophic
Cymbidium occupy low-light niches, pollinator foraging
would be negatively influenced by low-light intensity. In
partial and fully mycoheterotrophic Cymbidium, autogamy
would likely be favoured as a reproductive assurance to
compensate for pollinator limitation due to their lack of
nectar and pollinators’ hostile habitat preferences.
Keywords Autogamy Mixotrophy Mycoheterotrophy
Orchidaceae Pollination biology Self-pollination
Introduction
A significant number of terrestrial plant species have
abandoned photosynthesis and evolved total dependence on
fungal-derived energy sources (Leake 1994). These plants,
which are ecologically distinct from those that parasitize
other plants (e.g. Suetsugu et al. 2008,2012), depend upon
their mycorrhizal associations and are therefore referred to
as mycoheterotrophs (Leake 1994). Mycoheterotrophy has
evolved in over 400 angiosperm species belonging to many
different plant families, including the Ericaceae, Polygal-
aceae, Gentianaceae, Burmanniaceae, Thismiaceae, Corsi-
aceae, Orchidaceae, Petrosaviaceae, Iridaceae and
Triuridaceae (Leake 1994). While mycoheterotrophs have
long attracted the curiosity of botanists and mycologists,
and much is known about their mycorrhizal associations
and convergent life-history traits (reviewed by Bidartondo
2005), there have only been a few, mostly anecdotal reports
regarding their pollination biology (Hentrich et al. 2010
and reference therein: Klooster and Culley 2009).
Several reviews have suggested that the limitations of
such highly specialized life histories could be the strongest
determinant influencing the reproductive biology of my-
coheterotrophs (Bidartondo 2005; Waterman and Bidart-
ondo 2008). In particular, it has been reasoned that
pollinator deception would be unlikely, as no successful
plant lineage would be able to cheat both its mycorrhizal
fungi (by failing to provide photosynthates) and insect
K. Suetsugu (&)
Graduate School of Human and Environmental Studies, Kyoto
University, Yoshida-Nihonmatsu-cho, Sakyo, Kyoto 606-8501,
Japan
e-mail: kenji.suetsugu@gmail.com
123
J Plant Res (2015) 128:115–125
DOI 10.1007/s10265-014-0669-4
pollinators (by failing to provide nectar or other rewards),
since such a bifurcated life-history would ultimately lead to
evolutionary instability (Bidartondo 2005; Waterman and
Bidartondo 2008). It has therefore been suggested that, in
contrast to autotrophic plants, mycoheterotrophic species
have a disproportionate dependence on non-specific poll-
inators and self-pollination (Bidartondo 2005; Klooster and
Culley 2009; Leake 1994; Waterman and Bidartondo 2008).
These concepts highlight the potential reproductive strate-
gies of plants possessing multiple life-history constraints
and have been suggested many times with regard to my-
coheterotrophic plants as a whole, as well as specifically to
mycoheterotrophic orchids (Bidartondo 2005; Klooster and
Culley 2009; Leake 1994; Waterman and Bidartondo 2008).
Indeed, most detailed reports investigating mycohet-
erotrophic species have found them to be exclusively aut-
ogamous (e.g. Hentrich et al. 2010 and reference therein,
Suetsugu 2013a; Zhou et al. 2012), reinforcing the view
that most mycoheterotrophic plants employ a self-polli-
nating strategy. However, it has also been noted that sev-
eral of the autogamous species have flower visitors that
could mediate outcrossing (Takahashi et al. 1993), and it
has been suggested that this combination of outcrossing
and self-pollination could be utilized as reproductive
assurance with self-pollination only occurring when poll-
inators have been unsuccessful in transferring pollen
(Hentrich et al. 2010; Lehnebach et al. 2005; Zhang and
Saunders 2000). There have also been several reports of
xenogamous mycoheterotrophic plants that depend com-
pletely on insect pollination (Burns-Balogh et al. 1987;
Hentrich et al. 2010; Klooster and Culley 2009). However,
most species that utilize some degree of insect-mediated
pollination provide nectar and other rewards, with exam-
ples being found in several genera of mycoheterotrophic
plants, including Monotropa and Monotropsis (Klooster
and Culley 2009, Wallace 1977), Burmannia (Zhang and
Saunders 2000), Petrosavia (Takahashi et al. 1993), Voy-
ria, (Hentrich et al. 2010), and Gastrodia (Jones 1985,
Kato et al. 2006). Therefore, it remains unclear whether
some mycoheterotrophic plants can be pollinated by floral
visitors without producing any rewards. Moreover, in a
broader evolutionary/ecological context, the question of
whether the evolution of mycoheterotrophy induces a shift
in pollination pattern is still unknown, since there have
been no studies that have simultaneously evaluated both of
these life history traits (Klooster and Culley 2009).
The genus Cymbidium (Orchidaceae), which includes
both mixotrophic (partially mycoheterotrophic) and fully
mycoheterotrophic species, is an ideal model to study the
evolution of characteristics associated with mycohetero-
trophy. The genus comprises approximately 50 species
(Yukawa and Stern 2002; Yukawa et al. 2002) distributed
from East and Southeast Asia to Australia, exhibiting
terrestrial, lithophytic and epiphytic growth habits (Mo-
tomura et al. 2008). Two species, C. aberrans and C.
macrorhizon, completely lack foliage, and a comprehen-
sive phylogenetic analysis of the genus based on nuclear
and plastid DNA has revealed that (1) the two leafless
species form a sister pair, (2) the leafy species C. lan-
cifolium is sister to them, and (3) C. lancifolium and the
two leafless species form a clade with two other species: C.
goeringii and C. kanran (Yukawa and Stern 2002; Yukawa
et al. 2002).
Through isotopic abundance analysis of their stable
nitrogen and carbon isotopes, Motomura et al. (2010)
demonstrated that the two leafless species can be consid-
ered as fully mycoheterotrophic, although they retain low
level of chlorophyll pigments and photosynthetic ability on
their stems and ovaries (Suetsugu unpublished data). Given
that the outgroups of these five Cymbidium species are
autotrophic epiphytes (Motomura et al. 2008; Yukawa and
Stern 2002), it is likely that mycoheterotrophy in Cym-
bidium evolved from autotrophy via a mixotrophic transi-
tional stage (Motomura et al. 2010).
To date, there have been few studies of the pollination
biology of the mycoheterotrophic and mixotrophic Cym-
bidium species. However, it has been noted that male bees
of Anthophora plumipes villosula and the worker bees of
Apis cerana japonica can act as pollinators for the mixo-
trophic species C. goeringii and the putative mixotrophic C.
kanran, respectively (Tsuji and Kato 2010). In addition, it
has also been reported that a Chinese population of the
mixotrophic species C. lancifolium is exclusively pollinated
by the workers of A. cerana cerana (Cheng et al. 2007).
However, there have been no detailed investigations into
the leafless species C. macrorhizon and C. aberrans. In
addition, C. lancifolium is a species complex that contains
several indistinct, cryptic taxa, and the Japanese C. lan-
cifolium populations are morphologically, genetically and
phenologically distinct entities from Chinese populations
(Yukawa 2000). Although Japanese C. lancifolium popu-
lations often grow sympatrically with leafless C. macrorh-
izon and C. aberrans, there have been no investigations into
the breeding system of the Japanese populations.
Despite the current lack of data, the sympatric populations
of these Cymbidium species provide an ideal opportunity to
investigate the variation in insect visitors and breeding sys-
tems of the mixotrophic species C. lancifolium and the two
mycoheterotrophic species C. macrorhizon and C. aberrans.
The current study investigated several populations of these
species, including one population in which all three grew
sympatrically, to address the following three questions: (1)
Do these three Cymbidium species have the potential for
autonomous self-pollination? (2) If so, how is autonomous
self-pollination accomplished? And (3) do they successfully
attract insect visitors that can act as pollinators?
116 J Plant Res (2015) 128:115–125
123
Materials and methods
Study species and sites
C. macrorhizon and C. aberrans are rootless mycohetero-
trophic plants occurring in dense evergreen or deciduous
broadleaf forests and mixed pine forests (Fig. 1a, c). C.
macrorhizon produces 2–8 nectarless flowers on a lax
inflorescence that grows to ca. 10–30 cm tall. The sepals
and lateral petals are pale brownish-olive green with a
purplish-red longitudinal line (Liu et al. 2009). The label-
lum is whitish, marked with crimson or purplish blotches
(Liu et al. 2009). The scent of the flowers is faintly fragrant
and perceptible only by smelling them closely during high
temperatures in the daytime. C. aberrans bears 1–7 nect-
arless flowers on a lax inflorescence that grows to ca.
10–20 cm tall. The sepals and petals are white to pale
brownish-olive green (Liu et al. 2009). The labellum is also
Fig. 1 Flowers and pollinator of Cymbidium. aFlowering individual of C. macrorhizon.bApis cerana cerana visiting the C. macrorhizon
flower. cFlowering individual of C. aberrans. dFlowering individuals of C. lancifolium
J Plant Res (2015) 128:115–125 117
123
whitish to pale olive green without any colored blotches
(Liu et al. 2009). The scent of the flowers is sometimes
slightly fragrant and perceptible by smelling them closely,
but some of the flowers have no detectable scent as per-
ceived by humans, even during high temperatures in the
daytime. C. lancifolium are partial mycoheterotrophic
orchids and grow in densely shaded, evergreen, broad-
leaved forests and mixed pine forests (Fig. 1d). C. lan-
cifolium produces a lax inflorescence of 10–25 cm in length
with 2–9 nectarless flowers. The sepals and lateral petals are
pale green, and the midvein is sometimes purplish-brown
(Liu et al. 2009). The labellum is whitish to pale green, with
purplish-brown markings (Liu et al. 2009). The scent of the
flowers is faintly fragrant and perceptible only by smelling
them closely during high temperatures in the daytime.
The pollination biology of these three species was
investigated in central Japan from early to late July from
2008 to 2012 (Table 1), which covered the peak of the
flowering season. All populations were located in dense
forests dominated by broad-leaved trees such as Quercus
serrata,Castanopsis cuspidata, Carpinus tschonoskii and
Machilus thunbergii with sparse herbaceous understories,
and Cymbidium plants were typically observed growing in
close proximity to ectomycorrhizal trees such as Q. ser-
rata,C. cuspidata, C. tschonoskii and Pinus thunbergii.
Observation of insect visitors
Flower-visiting insects were studied in early to late July
from 2008 to 2012. I walked around the study populations
or sat near flowering individuals to observe the intrafloral
behavior of insect visitors. Pollinator observations covered
the peak activity hours of diurnal insects (08:00–17:00 h)
in every population. The floral visitors were carefully
observed to assess visiting duration and intrafloral polli-
nation behavior, and some were captured for identification
immediately after leaving a flower.
Pollination experiment
I selected 10 inflorescences with more than four buds each
for pollination experiments in the Ichikawa (IC) population
of C. macrorhizon and C. aberrans in 2009, and the Ash-
igara (AK) population of C. lancifolium in 2011. To test
whether these three Cymbidium species rely on pollinators
for fruit set, a breeding system experiment was set up with
four treatments per plant, and apparent cases of contact
between pollinia and stigmatic fluid were excluded from the
pollination experiments. (1) Autonomous self-pollination
treatment: flowers were bagged with a fine mesh net before
anthesis to exclude pollinators. This treatment was used to
test whether fruit set could occur by autonomous self-pol-
lination. (2) Agamospermy treatment: the entire pollinaria
were removed before anthesis using forceps, and the flowers
were then bagged. (3) Artificial self-pollinated treatment:
the pollinaria were removed and used to hand-pollinate the
same flower before being bagged. (4) Artificial cross-pol-
linated treatment: the same as treatment 3, but using the
pollinia from a different plant. To avoid sampling within a
clonal plant, all pollinia were taken from donor plants at
least 1 m away from the intended recipient plants during the
cross-pollination experiments. In addition, another 10–35
flowering individuals were randomly tagged and allowed to
develop and fruit under natural pollination conditions. The
experimental plants were monitored intermittently over the
following 4–6 weeks and then scored for fruit set once
capsules had formed. In order to minimize the impact of this
study on these endangered plant populations, only small
numbers of plants (i.e. 10 inflorescences for each species)
were used for controlled pollination experiments. To
determine pollen limitation, I compared the fruit set under
natural conditions with artificial self- and cross-pollinated
treatments by Fisher’s exact test.
I also determined the difference in the quality of
seeds resulting from self- and cross-pollinations. For
Table 1 Study populations of three Cymbidium species
Population Site Plant species Population size
a
Investigated year Observation time
IS Inukami, Shiga C. macrorhizon 12 2008 30 h
HO Higahsi-Osaka, Osaka C. macrorhizon 7 2009 30 h
IC Ichikawa, Chiba C. macrorhizon ca. 50 2009–2011 70 h
C. aberrans ca. 100
MT Mitaka, Tokyo C. macrorhizon [100 2009–2012 50 h
C. aberrans [100
AK Ashigara, Kanagawa C. macrorhizon ca. 10 2011–2012 44 h
C. aberrans ca. 10
C. lancifolium ca. 20
YK Yokosuka, Kanagawa C. lancifolium ca. 10 2011 30 h
a
Population size is given as the number of flowering plants
118 J Plant Res (2015) 128:115–125
123
C. macrorhizon and C. aberrans in December 2009, and
for C. lancifolium in December 2011, all of the mature but
non-dehisced fruits (eight to eleven per treatment) were
collected from flowers of artificial self- and cross-polli-
nated treatment and autonomous self-pollination treatment.
It should be noted that one to three fruits per species for
each treatment were excluded from the analysis, as the
mature fruits could not be detected. It is possible that these
fruits had aborted subsequent to the initial survey. After the
fruits were silica-dried, I measured the total mass of dry
seeds freed from each capsule to the nearest 0.0001 g. A
subsample of the seeds was then taken for assessment of
seed quality. I examined 500 seeds from each capsule by
stereoscopic microscope to calculate the ratio of seeds with
embryos. The effect of the pollination treatments on seed
weight and the proportion of seeds having a well-devel-
oped embryo were tested with ANOVA, followed by
Fisher’s multiple comparison. The inbreeding depression
index (d) was calculated in the following equation by
Charlesworth and Charlesworth (1987):
1. (Proportion of well-developed seeds after artificial
self-pollination/proportion of well-developed seeds
after artificial cross-pollination).
Self-pollination mechanism
A well-developed rostellum structure is considered the most
important physical barrier between the male and female
parts of the flower, preventing self-fertilization (e.g. Gale
2007; Peter and Johnson 2009; Suetsugu 2013a,2014a, but
also see Suetsugu 2013b,b; Zhou et al. 2012). In most self-
pollinating orchids, this structure either does not develop or
disintegrates during flowering (Catling 1990). Column
morphology was checked in terms of whether the archi-
tecture might promote or protect against spontaneous
autogamy. In every species, inflorescences at different
developmental stages (at ca. 1 day before, and 1 day,
3 days, and 1 week after flower opening) were randomly
chosen and checked for the presence of the rostellum and
contact between the stigma and pollinia. At least 20 flowers
from 5 individuals at each developmental stage were
observed in every species at all of the population sites.
Results
Observation of insect visitors
While I performed intensive observations of the flowers,
there were very few insect visitors to these three Cym-
bidium species (Table 2). The fly Japanagromyza tok-
unagai, whose larvae feed on orchid seeds, occasionally
visited the plants to lay eggs on the young ovaries and/or
stems in all of these orchids. However, they did not go
inside the inner portion of the flower or touch the column.
In C. aberrans, no insects were observed entering the
flower. Thus, there were no effective pollinators observed
in C. aberrans. The only insect observed actually entering
the flowers of C. lancifolium and C. macrorhizon was A.
cerana cerana (Fig. 1b; Table 2). After landing directly
upon the mid-lobe, the bee changed course before
crawling into the flower (Fig. 1b). Upon discovering that
there were no rewards to be found in the flower, the bee
exited, holding the mid-lobe tightly with its back legs.
However, there were no massulae introduced to the
flowers, and the pollinia remained intact within the anther
in all three visits to C. lancifolium and two visits to C.
macrorhizon at the AK population. In contrast, eight
individuals of A. cerana cerana visited the orchid flowers
in C. macrorhizon of the MT population, and two indi-
viduals with pollinia attached to their thorax were
observed during the retreating process. The total time
spent per flower was typically \10 s, and honeybees were
found to leave non-rewarding flowers immediately after
escaping from the labellum. Rare pollinia removal
occurrences in C. lancifolium and C. macrorhizon were
due to existing contact between the pollinia and stigmatic
fluid within the same flower in the early and middle
stages of flowering.
Breeding system
Relatively high levels of fruiting (60.0–76.9 %) in bag-
ged conditions were recorded in all of the species
(Table 3). In contrast, emasculated flowers did not show
fruit set, except for one case of C. aberrans. The fruit
sets seen in an emasculated flower of C. aberrans were
Table 2 The identity of insect taxa and total number of floral visi-
tations on flowers of three Cymbidium species
Plant species Population Insect
species
Times
visited
Pollinia
removal
C. macrorhizon MT A. cerana cerana 8 Yes
J. tokunagai 9No
IS J. tokunagai 6No
IC J. tokunagai 8No
HO None – –
AK A. cerana cerana 2No
C. lancifolium YK none – –
AK A. cerana cerana 3No
J. tokunagai 1No
C. aberrans MT J. tokunagai 11 No
IC J. tokunagai 5No
AK J. tokunagai 2No
J Plant Res (2015) 128:115–125 119
123
probably due to cleistogamous fertilization, rather than
apogamy. Although apparent cases of contact between
the stigma and pollinia before flower opening were
excluded from the emasculated pollination experiments,
contact between the pollinia and stigmatic fluid some-
times occurs at the bud stage. Fruit set in these three
species was not significantly different between treat-
ments, except for the agamospermy treatment (Table 3).
It should be noted that the small sample size may not be
enough to precisely determine the fruit set ratio on each
treatment. The open treatment resulted in the lowest fruit
formation of any other treatment in these three species.
This is partially explained by the infestation of J. tok-
unagai in the natural treatment cohort, while the plants
bagged before anthesis completely avoided infestation.
The proportion of seeds with well-developed embryos
also did not differ significantly between artificial self-
pollinated, artificial outcrossed and autonomous self-
pollination treatments (P[0.2 in all the treatments;
Table 4). The calculated inbreeding depression (d)
obtained by the proportion of well-developed seeds was
-0.012 in C. macrorhizon, 0.052 in C. aberrans and
0.078 in C. lancifolium.
Self-pollination mechanism
Investigation into the column morphology suggested that
there is no rostellum in these three Cymbidium species.
Consequently, there’s no physical barrier between the
stigma and the pollinia. Autonomous self-pollination
mainly occurs within one day after the flower opens, and
occasionally in bud stage, and there are no clear dif-
ferences in the column morphology (i.e. ability to be
autogamous) between populations in each species. The
stigma is separated from the pollinia during the bud
stage. After the flower opens, pollinia with the anther
cap begin to drop downward into the stigma. Due to the
lack of a rostellum, contact between the pollinia and
stigma can happen, allowing autonomous self-pollination
(Fig. 2a–f).
Discussion
Self-pollination was detected in all three of the Cymbidium
species examined. The mechanism of autonomous self-
pollination was facilitated by the absence of a rostellum,
which allowed contact between the stigma and pollinia.
This is the most common mode of autonomous self-polli-
nation in the Orchidaceae and has been found in approxi-
mately half of the self-pollinating orchid species studied to
date (Catling 1990).
Although it has previously been reported that self-pol-
lination tends to result in significantly lower levels of
embryo formation compared to cross-pollination, particu-
larly in outcrossing species (Tremblay et al. 2005), this
effect was not detected in any of the Cymbidium species. It
has been suggested that autogamous species are less prone
to inbreeding depression than outcrossing species because
any lethal or highly deleterious recessive alleles would be
Table 3 Effects of pollination treatments on fruit set of Cymbidium macrorhizon, C. aberrans and C. lancifolium
Species Open Selfed Crossed Bagged Emasculated
C. macrorhizon N/n 26/133 10/17 10/16 10/16 10/15
% 54.9
a
76.5
a
68.8
a
75.0
a
0
b
C. aberrans N/n 35/146 10/15 10/12 10/13 10/12
% 50.7
a
66.7
a
75.0
a
76.9
a
0
b
C. lancifolium N/n 10/41 10/15 10/16 10/15 10/20
% 56.1
a
73.3
a
75.0
a
60.0
a
10.0
b
N/n Examined ramets/flowers, %mean value of fruit set in percentage
Pollination treatments that showed significant differences (Fisher’s exact probability test, P\0.05) are indicated by superscript letters
Table 4 Effects of pollination
treatments on, seed mass and
proportion of seeds of
Cymbidium macrorhizon, C.
aberrans and C. lancifolium
Species Open Selfed Crossed
C. macrorhizon Seed mass (mg) 15.8 ±4.4 16.6 ±5.1 16.2 ±5.3
Seed with embryo (%) 78.8 ±15.10 78.1 ±16.3 77.2 ±16.7
C. aberrans Seed mass (mg) 15.7 ±3.9 14.8 ±4.8 15.1 ±4.5
Seed with embryo (%) 80.5 ±13.3 77.1 ±13.8 81.4 ±13.4
C. lancifolium Seed mass (mg) 17.3 ±4.0 18.3 ±4.9 18.1 ±4.7
Seed with embryo (%) 81.2 ±12.8 77.5 ±15.0 84.1 ±11.1
120 J Plant Res (2015) 128:115–125
123
purged upon self-pollination (Bellusci et al. 2009;
Charlesworth and Charlesworth 1987). The lack of any
apparent effect of inbreeding depression on seed fertility
observed in the current study may therefore indicate that all
three of the Cymbidium species routinely reproduce by
self-pollination.
Despite this, their fruit set was found to be lower than
that of many other obligate autogamous taxa, which pro-
duce almost 100 % fruit set (Table 2; Tremblay et al.
2005). While a relatively low level of fruit set has been also
recorded for other species of autonomous self-pollinating
orchids, in many cases this appeared to be the result of a
failure of the pollinia to reach the stigma (Liu et al. 2006;
Peter and Johnson 2009). However, this explanation would
not apply to the Cymbidium species, as their poorly
developed rostellum, which usually functions as a physical
barrier separating the stigma and pollinia, resulted in nearly
all stigmas being pollinated by the middle of flowering
stage. Furthermore, there was no significant difference
between the fruit set of artificially-pollinated flowers and
bagged flowers. Therefore, low fruit set could be caused by
another mechanism, such as resource limitation. It has been
considered that mycoheterotrophs are limited in their
growth and reproduction not only by phosphorus and
nitrogen, but also by the flow of carbon from their fungal
hosts (Shefferson et al. 2011). Relatively low levels of fruit
Fig. 2 Column morphology
and autogamy in C.
macrorhizon,C. aberrans and
C. lancifolium. a–cColumn
from a flower 24 h after flower
opening. d–fColumn from
7 days after flower opening;
arrows indicate pollinia a,d
C. macrorhizon. b,eC.
aberrans.c,fC. lancifolium
J Plant Res (2015) 128:115–125 121
123
set have also been observed in another mycoheterotrophic
orchid, Cyrtosia septentrionalis, which is autogamous
(Suetsugu 2013a). Thus, it is possible that their pollination
biology can be affected by resource limitations due to their
mycoheterotrophic nature.
The three Cymbidium species were also found to lack
food rewards, such as nectar or edible pollen. This suggests
that if pollination were to occur, it would likely be the
result of a deceptive pollination strategy. Despite the
potential for selfing in the Cymbidium species studied, it
was found that both C. macrorhizon and C. lancifolium
were able to successfully attract the worker bees of A.
cerana cerana. In contrast, no insects were observed
entering the flowers of C. aberrans, perhaps as a conse-
quence of its white flowers lacking purplish chestnut spots
on the labellum, characteristic of false nectar guides that C.
macrorhizon and C. lancifolium use to exploit the foraging
behavior of bees (Cheng et al. 2007). The entirely white
flowers of C. aberrans may therefore denote a greater
specialization to an autonomous self-pollination strategy,
although more intensive observations might still lead to the
identification of a potential pollinator. In addition, the
limited observation of the current study found no evidence
of pollinia removal in the Japanese populations of C. lan-
cifolium. However, honeybees can work as pollinators,
given that honeybees were reported as a pollinator in
Chinese C. lancifolium (Cheng et al. 2007) and were also
observed as floral visitors to the Japanese population of C.
lancifolium.
The present study suggests, contrary to previous asser-
tions (Bidartondo 2005), that this fully mycoheterotrophic
species can attract potential pollinators without nectar
rewards. While honeybees visiting C. macrorhizon and C.
lancifolium left the non-rewarding flowers immediately
without visiting any of the surrounding conspecific flowers,
honeybees were observed leaving the flowers of C. mac-
rorhizon carrying attached pollinia. This behavior is nor-
mal for the pollinators of food-deceptive flowers (e.g.
Suetsugu and Fukushima 2013,2014; Sugiura 2013), and it
is thought that fewer flower visits could actually benefit
deceptive plants by reducing the risk of geitonogamy
(Johnson et al. 2004; Jersa
´kova
´and Johnson 2006). How-
ever, the importance of insect-mediated pollination to the
three Cymbidium species in the current investigation is
probably relatively low, given the high propensity for self-
pollination at the early stage of flowering. Furthermore, it
was also noted that their stigmas often became covered
with pollinia still attached to their anther caps (Fig. 2d–f),
leaving no space for the adhesion of pollinia transported by
bees. Further investigation using water-resistant stains for
pollen-tracking would be helpful in precisely determining
the relative importance of insect-mediated pollination in
these species (Kropf and Renner 2008; e.g. Peakall 1989).
Autonomous self-pollination has often been proposed as
an evolutionary response to a lack of pollinators, providing
reproductive assurance when the frequency of pollination
is regularly very low (Baker 1955; Lloyd 1992; Wyatt
1988). Pollinator limitation is known to be a factor that
frequently affects deceptive orchids (Neiland and Wilcock
1998; Tremblay et al. 2005), and it has been shown that
deceptively-pollinated orchids achieve, in general, about
half the fruit set of rewarding ones (20.7 ±1.7 vs
37.1 ±3.2 %; Tremblay et al. 2005). However, the level
of fruit set found in the mixotrophic entomophilous species
C. goeringii is far lower than average, even amongst
nectarless orchids, with one Korean population exhibiting
only 0.4–0.6 % fruit set under natural conditions, even
though artificial self- and cross-pollination both resulted in
nearly 100 % fruit set (Chung and Chung 2003). Further-
more, despite intensive observation (132 h), it was not
possible to detect effective pollinators in two Japanese
populations of C. goeringii that also failed to fruit suc-
cessfully (Tsuji and Kato 2010), presumably as a result of
pollination failure. It is likely that a similar pollinator
limitation severely affects the reproductive success of the
mixotrophic Cymbidium species. Indeed, the present study
found that the frequency of insect visitation for the mixo-
trophic species C. lancifolium was extremely low.
Mixotrophic and mycoheterotrophic Cymbidium species
often grow on the forest floor of evergreen broadleaved
forests, but also in temperate, deciduous, broadleaved or
Pinus forests (Maekawa 1971). The light intensity of these
habitats, which are shaded by woodland or scrub (except
during winter in the case of deciduous forests) is dim to
dark, and it is thought that mixotrophy and mycohetero-
trophy could be an adaptation that has enabled these
Cymbidium species to survive in such low-light conditions
(Bidartondo et al. 2004). However, low-light environments
present problems for plant reproduction, as pollinator for-
aging can be negatively influenced by light intensity
(Herrera 1995a,1997) and the density of forest canopies
(Lee et al. 2001). Bees in particular tend to be restricted to
areas of high light intensity, and it is possible that the
colonization of shaded habitats results in reduced pollina-
tion by bees, which are scarce in the forest understory.
Temperature also influences pollinator behavior, and it has
been noted that the number of foraging bouts for many
ectothermic insects is positively correlated with tempera-
ture (Comba 1999; Herrera 1995a,b; Lee et al. 2001;
Totland 2001). As a result, temperature might also be an
important factor affecting the reproduction of mycohet-
erotrophic plants, since shaded environments have a lower
temperature (Herrera 1997; Lee et al. 2001; Newmark
2001).
Although there have only been a few reports regarding
the reproductive biology of mycoheterotrophic plants
122 J Plant Res (2015) 128:115–125
123
(Hentrich et al. 2010 and reference therein; Klooster and
Culley 2009), it appears that most of the species investi-
gated to date (especially nectarless species) have indeed
abandoned bee pollinators in favor of self-pollination.
Although there are few firm hypotheses regarding the
ecological constraints that favor a self-pollination strategy,
it is possible that the occupation of low-light niches could
be one of the causes. In addition, as mentioned before,
pollen limitation is generally known to be frequent among
deceptive orchids (Neiland and Wilcock 1998; Tremblay
et al. 2005). Thus, pollen limitation due to lack of nectar is
also probably a driving force in the autonomous self-pol-
lination mechanism in the three Cymbidium. It is intriguing
that almost all entomophilous mycoheterotrophic plants
provide nectar and other rewards (Hentrich et al. 2010;
Klooster and Culley 2009; Takahashi et al. 1993; e.g.
Wallace 1977; Zhang and Saunders 2000;) that can work as
a buffer to pollinator limitation.
The present study suggests that in partial and fully
mycoheterotrophic Cymbidium, autogamy has evolved as a
reproductive assurance. Autonomous self-pollination has
also been found in other mixotrophic genera, including
Cephalanthera and Epipactis (Bonatti et al. 2006; Burns-
Balogh et al. 1987; van der Pijl and Dodson 1966; Pedersen
and Ehlers 2000; Suetsugu 2013b; Tałałaj and Brzosko
2008). Taken together, these observations provide further
evidence that autogamous species do indeed have some
advantage for species growing in low-light conditions.
However, this is the first study to test for pollination
strategy shift in accordance with the evolution of myco-
heterotrophy, since pollination biology in the genus Epi-
pactis and Cephalanthera has never been discussed on the
robust phylogenetic framework.
Mycoheterotrophy has often been considered an adap-
tation to low-irradiance niches that possess few auto-
trophic competitors (Bidartondo et al. 2004). However,
having evolved, mycoheterotrophs tend to become
restricted to these habitats as a consequence of their
dependence on the mycorrhizal fungi that inhabit dense
forests. In fact, it has been suggested that unique micro-
climates, such as dark and moist forests, are necessary for
mycoheterotrophic growth and development (Leake 1994).
It is therefore reasonable to assume that the evolution of
additional traits (in this case, autogamy) is required during
adaptation to shaded environments. The observation in the
current study that two Japanese populations of C. lan-
cifolium have evolved autonomous self-pollination dem-
onstrates that the shift from entomophily to autogamy has
also occurred in the mixotrophic species C. lancifolium
that inhabits shaded habitats. In contrast, at least one
Chinese population of C. lancifolium is completely
dependent on pollinators (Cheng et al. 2007). This may
indicate that a shift in the reproductive strategy of
mixotrophic populations of C. lancifolium is an on-going
process of adaption that parallels the incomplete shift in
their mycorrhizal associations (i.e. mixotrophic Cymbid-
ium species harboring both ectomycorrhizal and sapro-
trophic fungi; Ogura-Tsujita et al. 2012). It is also
interesting to note that the mixotrophic species C. goer-
ingii and C. kanran, which are outgroups of the three
Cymbidium species investigated in the current study,
flower in winter to early spring coinciding with a period of
high-irradiance before canopy closure. It has been noted
that, plants that flower in the winter can experience a
shortage of insect pollinators, and that the winter flowering
phenology is often an attempt to attract bird pollinators in
cold weather, when insect activity is limited (Fang et al.
2012). However, since both C. goeringii and C. kanran are
pollinated by the usual bee pollinators (Tsuji and Kato
2010) it is more likely that their unique flowering phe-
nology is an adaptation to maximize light availability in
temperate, deciduous forest floor environments to increase
the efficiency of pollination.
In summary, the present study suggests that it is not only
the capacity for mycoheterotrophic, but also a shift to
autogamy that contribute to the successful invasion of low-
light niches. However, given that there are examples of
both xenogamous and autogamous species, it should be
noted that there seems to be no fixed link between the
breeding systems and trophic strategies of mycohetero-
trophic plants (Hentrich et al. 2010 and reference therein;
Klooster and Culley 2009). Given that complete myco-
heterotrophy has evolved independently in the Orchidaceae
on multiple occasions (Bidartondo 2005; Merckx and
Freudenstein 2010), it would be interesting to investigate
how and when a shift in pollination strategy has occurred in
other lineages containing both mixotrophic and mycohet-
erotrophic species.
Acknowledgments I thank Y. Kitada, T. Yamamoto, K. Onuki and
S. Mori for habitat information and/or field study assistance, and K.
Onuki, Drs. M. Kato and A. Kawakita for helpful discussions. This
study was partly supported by a Japan Society for the Promotion of
Science Research Fellowships for Young Scientists Grant.
References
Baker HG (1955) Self-compatibility and establishment after ‘‘long-
distance’’ dispersal. Evolution 9:347–348
Bellusci F, Pellegrino G, Musacchio A (2009) Different levels of
inbreeding depression between outcrossing and selfing Serapias
species. Biol Plant 53:175–178
Bidartondo MI (2005) The evolutionary ecology of myco-heterotro-
phy. New Phytol 167:335–352
Bidartondo MI, Burghardt B, Gebauer G, Bruns TD, Read DJ (2004)
Changing partners in the dark: isotopic and molecular evidence
of ectomycorrhizal liaisons between forest orchids and trees.
Proc R Soc B 271:1799–1806
J Plant Res (2015) 128:115–125 123
123
Bonatti PM, Sgarbi E, Del Prete C (2006) Gynostemium micromor-
phology and pollination in Epipactis microphylla (Orchidaceae).
J Plant Res 119:431–437
Burns-Balogh P, Szlachetko DL, Dafni A (1987) Evolution, pollina-
tion, and systematics of the tribe Neottieae (Orchidaceae). Plant
Syst Evol 156:91–115
Catling PM (1990) Auto-pollination in the Orchidaceae. Orchid Biol
Rev Perspect 5:121–158
Charlesworth D, Charlesworth B (1987) Inbreeding depression and its
evolutionary consequences. Ann Rev Ecol Evol Syst 18:237–268
Cheng J, Liu SY, He R, Wei XJ, Luo YB (2007) Food-deceptive
pollination in Cymbidium lancifolium (Orchidaceae) in Guangxi,
China. Biodiver Sci 15:608–617
Chung MY, Chung MG (2003) The breeding systems of Cremastra
appendiculata and Cymbidium goeringii: high levels of annual
fruit failure in two self-compatible orchids. Ann Bot Fenn
40:81–85
Comba L (1999) Patch use by bumblebees (Hymenoptera Apidae):
temperature, wind, flower density and traplining. Ethol Ecol
Evol 11:243–264
Fang Q, Chen YZ, Huang SQ (2012) Generalist passerine pollination
of a winter-flowering fruit tree in central China. Ann Bot
109:379–384
Gale S (2007) Autogamous seed set in a critically endangered orchid
in Japan: pollination studies for the conservation of Nervilia
nipponica. Plant Syst Evol 268:59–73
Hentrich H, Kaiser R, Gottsberger G (2010) The reproductive biology
of Voyria (Gentianaceae) species in French Guiana. Taxon
59:867–880
Herrera CM (1995a) Floral biology, microclimate, and pollination by
ectothermic bees in an early-blooming herb. Ecology
76:218–228
Herrera CM (1995b) Microclimate and individual variation in
pollinators: flowering plants are more than their flowers. Ecology
76:1516–1524
Herrera CM (1997) Thermal biology and foraging responses of insect
pollinators to the forest floor irradiance mosaic. Oikos 78:601–611
Jersa
´kova
´J, Johnson SD (2006) Lack of floral nectar reduces self-
pollination in a fly-pollinated orchid. Oecologia 147:60–68
Johnson SD, Peter CI, A
˚gren J (2004) The effects of nectar addition
on pollen removal and geitonogamy in the non-rewarding orchid
Anacamptis morio. Proc R Soc B 271:803–809
Jones DL (1985) The pollination of Gastrodia sesamoides R. Br. in
southern Victoria. Victorian Nat102:52–54
Kato M, Tsuji K, Kawakita A (2006) Pollinator and stem-and corm-
boring insects associated with mycoheterotrophic orchid Gas-
trodia elata. Ann Entomol Soc Am 99:851–858
Klooster MR, Culley TM (2009) Comparative analysis of the
reproductive ecology of Monotropa and Monotropsis: two
mycoheterotrophic genera in the Monotropoideae (Ericaceae).
Am J Bot 96:1337–1347
Kropf M, Renner SS (2008) Pollinator-mediated selfing in two
deceptive orchids and a review of pollinium tracking studies
addressing geitonogamy. Oecologia 155:497–508
Leake JR (1994) The biology of myco-heterotrophic (‘saprophytic’)
plants. New Phytol 127:171–216
Lee HL, Sodhi NS, Elmqvist T (2001) Bee diversity along a
disturbance gradient in tropical lowland forests of south-east
Asia. J Appl Ecol 38:180–192
Lehnebach CA, Robertson AW, Hedderley D (2005) Pollination
studies of four New Zealand terrestrial orchids and the impli-
cation for their conservation. NZ J Bot 43:467–477
Liu KW, Liu ZJ, Huang L, Li LQ, Chen LJ, Tang GD (2006)
Pollination: self-fertilization strategy in an orchid. Nature
441:945–946
Liu Z, Chen X, Chen S, Cribb PJ (2009) Cymbidium. In: Wu ZY,
Raven PH, Hong DY (eds) Flora of China, vol 25., Orchida-
ceaeScience Press and Missouri Botanical Garden Press, Beijing,
pp 260–281
Lloyd DG (1992) Self- and cross-fertilization in plants. II. The
selection of self-fertilization. Int J Plant Sci 153:370–380
Maekawa F (1971) The wild orchids of Japan in colour. Seibundo-
shinkousha, Tokyo
Merckx V, Freudenstein JV (2010) Evolution of mycoheterotrophy
in plants: a phylogenetic perspective. New Phytologist
185:605–609
Motomura H, Yukawa T, Ueno O, Kagawa A (2008) The occurrence
of crassulacean acid metabolism in Cymbidium (Orchidaceae)
and its ecological and evolutionary implications. J Plant Res
121:163–177
Motomura H, Selosse MA, Martos F, Kagawa A, Yukawa T (2010)
Mycoheterotrophy evolved from mixotrophic ancestors: evi-
dence in Cymbidium (Orchidaceae). Ann Bot 106:573–581
Neiland MRM, Wilcock CC (1998) Fruit set, nectar reward, and rarity
in the Orchidaceae. Am J Bot 85:1657–1671
Newmark WD (2001) Tanzanian forest edge microclimatic gradients:
dynamic patterns. Biotropica 33:2–11
Ogura-Tsujita Y, Yokoyama J, Miyoshi K, Yukawa T (2012) Shifts
in mycorrhizal fungi during the evolution of autotrophy to
mycoheterotrophy in Cymbidium (Orchidaceae). Am J Bot
99:1158–1176
Peakall R (1989) A new technique for monitoring pollen flow in
orchids. Oecologia 7:361–365
Pedersen HAE, Ehlers BK (2000) Local evolution of obligate
autogamy in Epipactis helleborine subsp. neerlandica (Orchid-
aceae). Plant Syst Evol 223:173–183
Peter CI, Johnson SD (2009) Autonomous self-pollination and
pseudo-fruit set in South African species of Eulophia (Orchid-
aceae). S Afr J Bot 75:791–797
Shefferson RP, McCormick MK, Whigham DF, O’Neill JP (2011)
Life history strategy in herbaceous perennials: inferring demo-
graphic patterns from the aboveground dynamics of a primarily
subterranean, mycoheterotrophic orchid. Oikos 120:1291–1300
Suetsugu K (2013a) Autogamous fruit set in a mycoheterotrophic
orchid Cyrtosia septentrionalis. Plant Syst Evol 299:481–486
Suetsugu K (2013b) Delayed autonomous self-pollination in two
Japanese varieties of Epipactis helleborine (Orchidaceae). Bot J
Linn Soc 173:733–743
Suetsugu K (2014a) Autonomous self-pollination in the nectarless
orchid Pogonia minor. Plant Spec Biol. doi:10.1111/1442-1984.
12037
Suetsugu K (2014b) Gastrodia flexistyloides (Orchidaceae), a new
mycoheterotrophic plant with complete cleistogamy from Japan.
Phytotaxa 175:270–274
Suetsugu K, Fukushima S (2013) Pollination biology of the endan-
gered orchid Cypripedium japonicum in a fragmented forest of
Japan. Plant Spec Biol. doi:10.1111/1442-1984.12016
Suetsugu K, Fukushima S (2014) Bee pollination of the endangered
orchid Calanthe discolor through a generalized food-deceptive
system. Plant Syst Evol 300:453–459
Suetsugu K, Kawakita A, Kato M (2008) Host range and selectivity of
the hemiparasitic plant Thesium chinense (Santalaceae). Ann Bot
102:49–55
Suetsugu K, Takeuchi Y, Futai K, Kato M (2012) Host selectivity,
haustorial anatomy and impact of the invasive parasite Parentu-
cellia viscosa on floodplain vegetative communities in Japan.
Bot J Linn Soc 170:69–78
Sugiura N (2013) Specialized pollination by carpenter bees in
Calanthe striata (Orchidaceae), with a review of carpenter bee
pollination in orchids. Bot J Linn Soc 171:730–743
124 J Plant Res (2015) 128:115–125
123
Takahashi H, Nishio E, Hayashi H (1993) Pollination biology of the
saprophytic species Petrosavia sakuraii (Makino) van Steenis in
Central Japan. J Plant Res 106:213–217
Tałałaj I, Brzosko E (2008) Selfing potential in Epipactis palustris,E.
helleborine and E. atrorubens (Orchidaceae). Plant Syst Evol
276:21–29
Totland Ø (2001) Environment-dependent pollen limitation and
selection on floral traits in an alpine species. Ecology
82:2233–2244
Tremblay RL, Ackerman JD, Zimmerman JK, Calvo RN (2005)
Variation in sexual reproduction in orchids and its evolutionary
consequences: a spasmodic journey to diversification. Bio J Linn
Soc 84:1–54
Tsuji K, Kato M (2010) Odor-guided bee pollinators of two
endangered winter/early spring blooming orchids, Cymbidium
kanran and Cymbidium goeringii, in Japan. Plant Spec Biol
25:249–253
van der Pijl L, Dodson CH (1966) Orchid flowers, their pollination
and evolution. University of Miami Press, Florida
Wallace GD (1977) Studies of the Monotropoideae (Ericaceae).
Floral nectaries: anatomy and function in pollination ecology.
Am J Bot 64:199–206
Waterman RJ, Bidartondo MI (2008) Deception above, deception
below: linking pollination and mycorrhizal biology of orchids.
J Exp Bot 59:1085–1096
Wyatt R (1988) Phylogenetic aspects of the evolution of self-
pollination. In: Gottlieb LD, Jain SK (eds) Plant evolutionary
biology. Chapman and Hall, London, pp 109–131
Yukawa T (2000) Identity of Cymbidium lancifolium complex.
J Plant Res 64:135
Yukawa T, Stern WL (2002) Comparative vegetative anatomy and
systematics of Cymbidium (Cymbidieae: Orchidaceae). Bot J
Linn Soc 138:383–419
Yukawa T, Miyoshi K, Yokohama J (2002) Molecular phylogeny and
character evolution of Cymbidium (Orchidaceae). Bull Natl Sci
Mus Tokyo 28:129–139
Zhang D, Saunders RMK (2000) Reproductive biology of a myco-
heterotrophic species, Burmannia wallichii (Burmanniaceae).
Bot J Linn Soc 132:359–367
Zhou X, Lin H, Fan XL, Gao JY (2012) Autonomous self-pollination
and insect visitation in a saprophytic orchid, Epipogium roseum
(D.Don) Lindl. Aust J Bot 60:154–159
J Plant Res (2015) 128:115–125 125
123
