Botanical Journal of the Linnean Society, 2009, 161, 78–88. With 4 figures
Confronting assumptions about spontaneous autogamy
in populations of Eulophia alta (Orchidaceae) in south
Florida: assessing the effect of pollination treatments
on seed formation, seed germination and seedling
development
boj_992
78..88
TIMOTHY R. JOHNSON1*, SCOTT L. STEWART2, PHILIP KAUTH1,
MICHAEL E. KANE1 and NANCY PHILMAN1
1
Plant Restoration, Conservation and Propagation Biotechnology Lab, Department of Environmental
Horticulture, University of Florida, Gainesville, FL 32611-0675, USA
2
Horticulture and Agriculture Programs, Kankakee Community College, 100 College Drive, Kankakee,
IL 60901, USA
Received 1 June 2009; accepted for publication 7 July 2009
The breeding system of the terrestrial orchid Eulophia alta was investigated in south Florida where it has
previously been reported as an auto-pollinated species. The effect of breeding system on seed viability and
germinability and seedling development was also investigated. Incidences of spontaneous autogamy in E. alta were
rare at the study site, resulting in only 7.1% of observed flowers forming capsules. In addition, hand pollination
resulted in significantly greater capsule formation when flowers were subjected to induced autogamy (46.4%),
artificial geitonogamy (64.3%) and xenogamy at both short (pollen source 10–100 m away; 42.9%) and long (pollen
source > 10 km away; 67.9%) distances. Pollen source had little effect on seed viability and germinability or
seedling growth rates. However, seed resulting from spontaneous autogamy developed more slowly than seed
originating from the other treatments. These data indicate that spontaneous autogamy is rare in E. alta and that
naturally forming capsules may be the result of unobserved pollination events. © 2009 The Linnean Society of
London, Botanical Journal of the Linnean Society, 2009, 161, 78–88.
ADDITIONAL KEYWORDS: asymbiotic germination – auto-pollination – breeding system – fitness – optimal
out-crossing distance – orchid – sexual reproduction.
INTRODUCTION
Pollination can be a limiting stage in the life cycles of
flowering plants, especially for those for which fitness
depends on pollination by insect vectors (Weekley &
Brothers, 2006). Recent evidence of pollinator decline
attributed to anthropogenic habitat loss (Biesmeijer
et al., 2006; Taki & Kevan, 2007) has drawn attention
from conservation and pollination biologists alike.
Understanding threats to the reproductive success of
*Corresponding author. E-mail: timjohn@ufl.edu
78
plants is vital to conservation planning and implementation. The importance of maintaining functional
breeding systems may be particularly relevant in
Orchidaceae as pollinators have played a critical role
in the diversification and speciation of the family
(Darwin, 1885; Dressler, 1981; Tremblay et al., 2005).
Orchids have also evolved many mechanisms to
enhance out-crossing and prevent or reduce incidents
of self-pollination, but genetically regulated selfincompatibility appears to be rare in the family, or at
least rarely reported (see Tremblay et al., 2005). This
may reflect the effectiveness with which orchid floral
structures, such as pollinia and rostellae, prevent
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88
BREEDING SYSTEM OF EULOPHIA ALTA
self-fertilization by effectively separating pollen from
the stigma on the same flower during flower maturation and immediately after pollen removal (Dressler,
1981). Other structural, phenological and evolutionary mechanisms that promote out-crossing without
the need for genetic incompatibility include delayed
caudicle taxis, shrinkage of pollinia after removal,
incomplete flowers, dichogamy and attraction of pollinators with large foraging ranges (Dressler, 1981;
Borba & Semir, 1999; Johnson & Edwards, 2000;
Tremblay et al., 2005; Tremblay, Pomales-Hernandez
& Mendez-Cintron, 2006). Evidence from nectar
addition studies with orchid species also indicates
that the evolution of deceptive pollination strategies,
strategies in which pollinators are attracted to
flowers with the promise of a reward without one
being supplied, may have evolved to enhance outcrossing, as nectar rewards increase pollinator visitation time and lead to increased self-pollination
(Johnson & Nilsson, 1999; Johnson, Peter & Agren,
2004; Cozzolino & Widmer, 2005; Jersáková &
Johnson, 2006; Jersáková et al., 2008).
Early studies of orchid pollination systems in
Florida and elsewhere have emphasized the identification of visitors but, in many cases, do not report
capsule formation data (see, for example, Dodson &
Frymire, 1961; Dodson, 1962). Undoubtedly, identification of visitors and pollinators is an important
aspect of integrated orchid conservation, as protecting
breeding systems is vital to the in situ persistence
of populations and species (Swarts & Dixon, 2009).
However, confirming successful breeding by quantifying capsule formation and identifying potential postpollination reproductive barriers, such as genetic
incompatibility, inbreeding depression and/or outbreeding depression, is equally as important. Given
many recent reports of successful asymbiotic and
symbiotic culture of rare, threatened and endangered
orchid taxa (see, for example, Stenberg & Kane, 1998;
Huynh et al., 2004; Zettler et al., 2005, 2007;
Znaniecka et al., 2005; Stewart & Kane, 2006, 2007;
Dutra, 2008; Dutra et al., 2008; Kauth et al., 2008;
Dutra, Kane & Richardson, 2009b), examining the
effects of breeding system studies on effective capsule
formation, seed viability and germinability and
seedling vigour could be particular illuminating in
investigations of orchid breeding systems. Although
self-fertilization has been shown to decrease the seed
production and seed weight of some orchid species
(Beardsell et al., 1986; Gonzalez-Diaz & Ackerman,
1988; Peakall, 1989; Robertson & Wyatt, 1990),
virtually nothing is known about how breeding
system affects germinability and early seedling development in orchids. Such integrated data would help
to clarify possible effects of different pollination
systems on reproductive success and stand establish-
79
ment, leading to more informed decisions about the
status of orchid populations, conservation concerns
and conservation management.
In contrast with the numerous examples of floral
adaptations in orchids that promote out-crossing,
some orchid species have evolved various methods of
ensuring pollination in the absence of pollinators. In
auto-pollination (= spontaneous autogamy) systems,
pollinia are deposited on the stigmatic surface
without a pollinator through a number of mechanisms, including development of powdery pollinia,
expansion of the pollinia, malformation of the rostellum and bending of the caudicle (Gonzalez-Diaz &
Ackerman, 1988; Catling & Lefkovitch, 1989; Catling,
1990; Gale, 2007; Micheneau et al., 2008). Autogamy
is thought to facilitate (or to be a consequence of)
range expansion into areas in which pollinators are
rare or absent (Catling, 1990). Autogamy may be
more widespread than previously thought; Fenster &
Martén-Rodriguez (2007) suggested that pollinator
specificity and auto-pollination may have co-evolved
as adaptations to the same selection pressures: pollinator limitation and the uncertainty of seed set. In
this scenario, auto-pollination could be a means of
reproductive assurance given the ‘risky’ strategy of
specialized pollinator attraction exhibited by many
orchid species.
Reports of auto-pollination are not uncommon in
Orchidaceae. Over 350 species have been reported to
be autogamous in some part of their range, and
estimates of the number of autogamous orchid species
of some regional floras, such as Ecuador and Puerto
Rico, are 20% or greater (Ackerman, 1985; Catling,
1990). Several species of orchids in peninsular Florida
are reportedly autogamous (Leur, 1971; Leur, 1972;
Goss, 1973; Dressler, 1981; McCartney, 1985; Catling,
1987, 1990; Calvo, 1990). However, most reports of
autogamy do not include investigations of capsule
formation under experimental conditions. Such
studies are needed to confirm that auto-pollination is
a successful mechanism of fruit production.
To quantify the rates of spontaneous autogamy and
post-pollination reproductive isolating mechanisms,
the pollination system of Eulophia alta (L.) Fawc. &
Rendle (subfamily Epidendroideae, tribe Cymbidieae;
Cameron et al., 1999) was studied. This terrestrial
species is distributed in Africa, the neotropics and
Florida, USA. It has been reported to be autopollinated in Mexico, South Africa, Zambia and
Florida (Goss, 1973; Williamson, 1984; Catling, 1990),
but its breeding system has not yet been fully studied.
Consequently, several questions remain. (1) How
common is spontaneous autogamy in this species in a
single population? (2) How efficient is spontaneous
autogamy as a mode of fertilization and capsule
formation? (3) Does out-breeding enhance capsule
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88
80
T. R. JOHNSON ET AL.
flatwoods (Brown, 2005). Although not listed as state
threatened, E. alta is state protected, listed by the
Federal Government as a wetlands indicator species
(USDA, 2009) and described as commercially exploitable (Brown, 2005). At the study location, the Florida
Panther National Wildlife Refuge (FPNWR), E. alta is
locally abundant, but patchy in distribution (Stewart
& Richardson, 2008). Concentrated populations are
found in only five of 54 burn units on FPNWR, totalling approximately 2000 plants (S.L. Stewart, pers.
observ.). Plants are found in the understorey of pine
flatwoods, especially along mowed access roads.
Eulophia alta is among the largest North American
terrestrial orchids. Indeterminate, unbranched
racemes can be up to 2 m in height and contain over
50 flowers (although some carry as few as five flowers;
Fig. 1A) that are 3.5–4.5 cm wide (Brown, 2005).
Flowers open in succession and often have red hues
(pink, red and maroon flowers are common). Green
flowering phenotypes can also be found. The pollen of
E. alta flowers is coalesced into two hard pollinia
joined by a single motile stipe. The base of the pollinarium has a sticky viscidium that readily attaches to
contacting objects. Flowers have no discernible scent.
DETERMINATION
Figure 1. Study of the breeding system of Eulophia alta.
(A) Flower. Scale bar, 1 cm. (B) Inflorescence with pollinator exclusion netting. (C) Infructescence. (D) Capsule.
Scale bar, 1.5 cm. (E) Seeds stained with tetrazolium
chloride. Scale bar, 250 mm. (F) Germinated seeds at
week 8. Scale bar, 250 mm.
formation frequency? (4) Are there indications of
genetic post-pollination reproductive barriers, such
as self-incompatibility, in this species? In order to
examine these questions, several experiments were
designed with the objective of describing the pollination mechanisms of E. alta in south Florida. In addition, pollination treatment effects on seed viability,
germination and early seedling development were
also examined.
MATERIALS AND METHODS
STUDY SPECIES
Eulophia alta (common name: wild coco; Fig. 1A) is a
terrestrial orchid found in Africa, the West Indies and
the American tropics and subtropics. The occurrence
of E. alta in the USA is restricted to south Florida,
where it is found in moist to saturated soils of pine
OF POLLINATION MECHANISM
Pollination experiments following the methods of
Wong & Sun (1999) were conducted at the FPNWR
(Collier County, FL, USA). In order to assess the
breeding system of E. alta, the effects of seven pollination treatments (Table 1) on capsule formation,
capsule length and capsule width were examined.
Pollinia were harvested by touching a toothpick to the
viscidium of flower pollinaria (to which the viscidium
readily adheres), removing the anther cap and placing
the harvested pollinia into a plastic bag for transport.
Harvested pollinia were used within 8 h of collection.
To test for asexual fruit set (agamospermy), flowers
were emasculated and covered (bagged) with pollinator exclusion netting (2 mm2 mesh fabric; Fig. 1B).
The ability of flowers to transfer pollinia to the stigma
without a pollination vector (spontaneous autogamy)
was tested by bagging flowers with their pollinia left
in place and without hand pollination. Within-flower
self-incompatibility was tested for by emasculating flowers and pollinating them with their own
pollinia (induced autogamy) before bagging. Selfincompatibility among flowers of the same inflorescence was tested for by pollinating flowers with
pollinia from another flower on the same inflorescence
(induced geitonogamy) before bagging. The effectiveness of outbreeding at great distances on capsule
formation (artificial xenogamy) was examined by
emasculating flowers and pollinating them with pollinia harvested from plants > 10 km from the study
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88
BREEDING SYSTEM OF EULOPHIA ALTA
81
Table 1. Pollination treatments tested in the study of the pollination mechanism of Eulophia alta
Treatment
number
Pollination treatment
Pollinators
excluded
Pollinia
removed
Pollen treatment
Assessment
1
Control (open pollination)
No
No
No pollination
2
Agamospermy
Yes
Yes
No pollination
3
Spontaneous autogamy
Yes
No
No pollination
4
5
Induced autogamy
Artificial geitonogamy
Yes
Yes
Yes
Yes
6
Artificial xenogamy
Yes
Yes
Same flower
Different flower on
same plant
Flower from a distant
population
Capsule set under natural
conditions
Frequency of non-sexual
capsule set
Need for pollinators for
reproduction
Self-compatibility of flowers
Self-compatibility of plants
7
Induced xenogamy
Yes
Yes
site before bagging. The effect of out-crossing at short
distances (induced xenogamy) was examined by pollinating emasculated flowers with pollinia collected
from plants at a distance of 10–100 m away from
manipulated plants.
Fourteen inflorescences from 14 individual plants
were selected with at least seven open, unpollinated
flowers. Each inflorescence received all seven previously described pollination treatments. Treatments
were randomly assigned to seven flowers on each
inflorescence. Senesced lower flowers were removed
as they were encountered and 0–5 flowers immediately above manipulated flowers were removed as
needed for the attachment of pollinator exclusion
netting. Upper flowers that opened later in the season
were not removed. For each treatment, n = 14 for each
of two study years, the first beginning on 9 October
2006 and then again on 11 November 2008. Potential
pollinators were excluded by securing 2 mm2 mesh
fabric around all flowers, except those in treatment 1
(Fig. 1B), the open pollination control. Capsules
(Fig. 1C, D) were examined 3, 6 and 12 weeks after
pollination treatments were initiated.
Inflorescences were used as block treatments. This
design was chosen because of the limited number of
flowering individuals and to avoid possible effects of
individual plant genotypes on the results. Logistic
regression was used to assess the effect of pollination
treatment on capsule formation using proc glimmix in
SAS v.9.1 (SAS Institute, 2003). Only capsules that
developed to at least week 11 were used in this
analysis. Least-squares means were used for mean
separation (a = 0.05). The effect of pollination treatment on capsule dimensions (length and diameter)
Distant plant in the
same population
Potential for out-breeding
at long distances (pollen
source > 10 km)
Potential for out-breeding
at short distances (pollen
source 10–100 m)
was analysed using general linear modelling and
Waller–Duncan mean separation (a = 0.05). All data
were analysed using SAS v.9.1 (SAS Institute).
SEED
COLLECTION, STORAGE AND GERMINATION
In order to examine the effect of pollination treatment
on seed viability, germinability and seedling development, capsules formed in 2006 as the result of the
pollination treatments were collected after 12 weeks
and dried over silica gel desiccant at room temperature until the capsules dehisced. At least one capsule
was collected from six of the pollination treatments.
Seed from each capsule was stored separately over
silica gel desiccant at room temperature for up to 2
months prior to experimentation. For each of the
treatments that produced more than one capsule,
5.0 mg samples from all capsules were collected and
pooled to create a proportional representative sample.
Approximately 5.0 mg of bulked seed from each treatment was surface sterilized for 45 s in a solution of
100% ethanol–6.0% NaOCl–sterile distilled deionized
water (1 : 1 : 20), and then rinsed three times in
sterile distilled water.
PhytoTechnology Orchid Seed Sowing Medium
(P723) was used for asymbiotic germination. This has
been shown previously to support germination and
early seedling development of E. alta (Johnson et al.,
2007). The medium was adjusted to pH 5.7 with 1.0 M
NaOH and autoclaved for 40 min at 121 °C and
117.1 kPa. Sterilized medium was dispensed as
30 mL aliquots into Petri plates (diameter, 9.0 cm).
Approximately 30 surface-sterilized seeds from each
pollination treatment were sown separately onto
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88
82
T. R. JOHNSON ET AL.
Table 2. Developmental stages used to score Eulophia
alta seed germination and seedling development
Stage
Description
0
1
2
3
4
Hyaline embryo, testa intact
Embryo swollen, rhizoids present (= germination)
Continued embryo enlargement, testa ruptured
Appearance of protomeristem
Emergence of first leaf
eight replicate Petri plates. The plates were then
sealed with a single layer of Nesco film (Karlan
Research Products Corp.), wrapped in two layers of
aluminium foil to exclude light and incubated at
22 ± 3 °C for 12 weeks. Seeds were exposed to short
periods of light (less than 30 min) during data collection. Germination and seedling development was
scored on a scale of 0–5 (Table 2) with a dissecting
microscope after 4, 8 and 12 weeks of culture. The
percentage seed and seedlings in each developmental
stage and the percentage germination were calculated. Germination and developmental data were
arcsine transformed and analysed using general
linear modelling and Waller–Duncan mean separation (a = 0.05).
SEED
VIABILITY TESTING
Tetrazolium chloride staining was used to assess seed
viability by pretreating three replicates of 100–200
seeds from each pollen treatment with 0.5% calcium
hypochlorite for 3 h. The seed was then rinsed three
times in sterile distilled deionized water and soaked
for 24 h in darkness at 22 ± 2 °C. The seed was then
soaked for 24 h in 1.0% tetrazolium chloride at
30 ± 2 °C in darkness. The percentage viability was
calculated for each replicate by dividing the number
of pink/red-stained embryos (viable; Fig. 1E) by the
total number of embryos counted. Three replications
were performed for each treatment and this experiment was repeated once (n = 6). Statistical analysis
was conducted as described for the germination data.
RESULTS
POLLINATION
Figure 2. Effect of pollination treatment on capsule formation 10 weeks after pollination treatments. Data from
two study years are combined. Error bars represent standard error of means for each treatment. Means with the
same letters are not significantly different at P > 0.05.
SYSTEM AND CAPSULE FORMATION
In 2008, a late frost destroyed developing capsules on
all but five plants 11 weeks after the initiation of
experimentation. All capsules resulting from agamospermy (1) and artificial xenogamy (4) were damaged,
as well as six of seven induced autogamy capsules,
seven of 10 artificial geitonogamy capsules and seven
of nine artificial xenogamy capsules. The presence of
capsules at the time of frost 11 weeks after experi-
mentation began was confirmed by observing frostdamaged capsules at week 12. Undamaged capsules
were used in the analysis of the effect of pollination
on capsule dimensions.
Statistical analysis indicated that pollination treatment had a significant effect on capsule formation
(F6,6 = 6.29, P = 0.02). Data on the effect of pollination
treatment on capsule formation from both observation
years were combined because significant differences
were not observed between years (F1,6 = 0.28,
P = 0.62). All pollen treatments resulted in capsule
formation in 2006, but capsules that formed from
spontaneous agamospermy aborted before 6 weeks
of development. Similarly, the spontaneous agamospermy treatment resulted in only a single capsule
being formed in 2008. Overall, agamospermy only
produced a single mature capsule (Fig. 2; 3.6% ±
3.6%; mean ± 3.6%). Open pollination resulted in
14.3% ± 6.7% capsule formation and was not significantly different from the observed capsule formation
from spontaneous autogamy (7.1% ± 5.0%) or agamospermy. A greater proportion of capsules formed from
induced xenogamy (42.9% ± 9.5%) than spontaneous
autogamy or agamospermy. Rates of capsule formation resulting from induced xenogamy, induced autogamy (46.4% ± 9.6%), geitonogamy (64.3% ± 9.2%)
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88
BREEDING SYSTEM OF EULOPHIA ALTA
83
Figure 3. Effect of pollination treatment on Eulophia alta capsule length (A) and diameter (B). Data from two study
years are combined. The data for agamospermy (*) at week 6 are for the length and width of a single capsule (a single
capsule formed in 2006, but aborted after week 3 and a single capsule that formed in 2008 was destroyed by frost).
and artificial xenogamy (67.9% ± 9.0%) were not
significantly different.
The study year did not have a significant effect on
capsule length or capsule diameter at week 3 (length:
F1,72 = 0.00; P = 0.99; diameter: F1,72 = 1.62; P = 0.21)
or week 12 (length: F1,72 = 1.13; P = 0.30; diameter:
F1,72 = 1.33; P = 0.26). Significant differences were
detected between study years during week 6 observations (length: F1,57 = 15.32; P = 0.00; diameter:
F1,57 = 26.31; P = 0.00). However, when the capsule
length and capsule diameter data for each study year
were analysed separately, the same result was
obtained for both study years; no significant differences were detected among treatments in 2006
(length: F6,30 = 0.60; P = 0.70; diameter: F6,30 = 0.46;
P = 0.80) or 2008 (length: F6,27 = 0.88; P = 0.52;
diameter: F6,27 = 0.66; P = 0.68) at week six. Because
of these results, capsule dimension data for both
study years were combined.
Pollination treatment did not have a significant
effect on capsule dimensions at weeks 3 (length:
F6,72 = 0.92; P = 0.48; diameter: F6,72 = 0.54; P = 0.77), 6
(length: F6,72 = 1.33; P = 0.26; diameter: F6,30 = 0.65;
P = 0.69) or 12 (length: F6,33 = 0.82; P = 0.55; diameter:
F6, 33 = 1.00; P = 0.43). Capsule lengths were not
significantly different among treatments at 6 and 12
weeks (Fig. 3A). At week 3, the capsule diameters of
the agamospermous treatment were not statistically
siginificantly different from those of capsules resulting from spontaneous autogamy treatment, but were
significantly lower than those of all other treatments.
The diameters of capsules resulting from different
pollination treatments were not significantly different
at any time examined (Fig. 3B).
EFFECT
OF POLLINATION TREATMENT ON SEED
VIABILITY AND EARLY SEEDLING DEVELOPMENT
In 2006, seed was generated from all pollination
treatments except agamospermy. Seed viability was
not significantly affected by pollination treatment
(F5,30 = 0.57; P = 0.57) and ranged from 88.2% ± 2.4%
to 92.7% ± 1.8%. Pollination treatment had a significant effect on germination after 12 weeks of culture
(F5,37 = 2.88; P = 0.03) and ranged from 94.7% ± 1.4%
to 99.3% ± 0.5% (Fig. 4). At week 4, most seeds were
observed at stage 1 (swollen embryo; 74.2% ± 3.4% to
86.8% ± 2.4%) and were considered to have germinated. Pollination treatment had a significant affect
on the percentage of seedlings at stage 2 at this time
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88
84
T. R. JOHNSON ET AL.
Figure 4. Effect of pollination treatment on Eulophia alta seed germination and seedling development after 4, 8 and 12
weeks culture. Seed and seedling stages are reported in Table 2. Capsules resulting from the 2006–2007 pollination
experiment were used. Error bars represent the upper limit of the standard error. Treatments within each stage with
different letters are significantly different (a = 0.05).
(F5,37 = 3.08; P = 0.02); significantly fewer seeds from
the spontaneous autogamy treatment had developed
to stage 2 (6.8% ± 2.5%) than all other treatments,
except for artificial geitonogamy (11.5% ± 2.5%). At
week 8 (Fig. 1F), pollination treatment had a significant effect on the percentage of seeds/seedlings at
stage 0 (F5,37 = 6.41; P = 0.00) and stage 3 (F5,37 = 7.85;
P = 0.00). Significantly more nongerminated seeds
(stage 0) were observed from spontaneous autogamy
(6.7% ± 1.7%) and artificial autogamy (6.7% ± 1.3%)
treatments. Fewer seeds from the spontaneous
autogamy treatment had developed to stage 3 (7.3% ±
2.5%) compared with other treatments. Statistically
significant differences were not found among treatments at stages 1 (F5,37 = 0.86; P = 0.51), 2 (F5,37 =
0.48; P = 0.79) or 4 (F5,37 = 0.86; P = 0.51) after 8
weeks of culture. After 12 weeks of culture, pollination treatment was found to have a significant effect
on the percentage of seedlings at stages 0 (F5,37 = 3.10;
P = 0.02), 2 (F5,37 = 7.10; P = 0.00), 3 (F5,37 = 5.96;
P = 0.00) and 4 (F5,37 = 38.20; P = 0.00), but not at
stage 1 (F5,37 = 0.37; P = 0.86). At this time, signifi-
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88
BREEDING SYSTEM OF EULOPHIA ALTA
cantly fewer seedlings from the spontaneous autogamy treatment were observed at stage 4 (7.2% ±
1.2%) compared with other treatments. Seed generated from the spontaneous autogamy treatment also
had significantly lower seedling development than
other treatments, as evidenced by a significantly
higher percentage of seedlings at stage 3 (56.2% ±
4.1%; F5,37 = 5.96; P = 0.00).
DISCUSSION
Eulophia alta infructescences with abundant capsules
are often observed at FPNWR (T.R. Johnson pers.
observ.; Fig. 1C, D). Goss (1973) reported that, soon
after flowers open, the caudicle of the pollinarium of
south Florida E. alta plants bends forwards bringing
the pollinia into contact with the stigma. However,
this mechanism was not observed during the course of
our study. Given previous reports of an autogamous
breeding mechanism of E. alta in Florida (Goss, 1973)
and the lack of documented E. alta pollinators in
North America, there has been little reason to investigate the pollination biology of this species. However,
the low success rate of induced autogamy in forming
capsules during this investigation indicates that fieldobserved capsules could be the result of unobserved
pollinator visitation.
Further evidence of the scarcity or lack of autopollination in E. alta at the study site comes from the
observation that glasshouse-grown plants do not
produce capsules unless pollinated by hand (T.R.
Johnson, pers. observ.). Discrepancies between this
report and previous reports of autogamy in E. alta
could be a result of differences in the frequency of
auto-pollination of E. alta throughout its range in
Florida, although this requires further study. Capsule
formation in other auto-pollinated orchid species is
typically much more efficient than observed for E.
alta (Tremblay et al., 2005). The fruit set of Nervilia
nipponica Makino under pollinator exclusion netting
was 93% and that of Jumellea stenophylla Schltr. was
66.7%–83.9% (Gale, 2007; Micheneau et al., 2008).
This is to be expected given the strong selection
pressure that pollinator absence would impose.
Seed that formed from the autogamous treatment
in 2006 exhibited a slower rate of germination and
seedling development compared with seed resulting
from other pollination treatments. This is an indication that rare spontaneous autogamy may produce
seed of lower fitness than other pollination treatments. It is possible that the difference in capsule
formation and seedling development between spontaneous autogamy and induced autogamy treatments is
a consequence of pollination with aged pollen (Rosell,
Galan Sauco & Herrero, 2006; Castro, Silveira &
Navarro, 2008). More pronounced effects of inbreed-
85
ing have been reported for other orchid genera. Seed
germination of Dactylorhiza sambucina (L.) Soó and
the subsequent survival of reintroduced plants produced from induced autogamy were lower than that of
seeds and seedlings produced from out-crossing pollination treatments (Juillet, Dunand-Martin & Gigord,
2007). Induced autogamy also decreased the seed
viability of Barlia robertiana (Loisel.) Greuter and
Anacamptis morio (L.) R.M.Bateman, Pridgeon &
M.W.Chase and reduced seed weight and the number
of seeds with embryos of Platanthera ciliaris Lindl.
(Robertson & Wyatt, 1990; Smithson, 2006).
Most pollination treatments did not have a significant effect on seed viability of E. alta. In all pollination
treatments, tetrazolium chloride staining underestimated the observed germination by as much as
10.8% (artificial geitonogamy: estimated, 88.2% ±
2.4%; observed, 99.0% ± 0.7%), but provided a good
relative estimate of germinability among treatments.
This difference in tetrazolium chloride-estimated viability and germinability highlights the need to perform
tests of both seed respiration and germinability.
One consequence of using a blocked design in the
current study was the inability to control for the
possible selective abortion of low-quality fruits. It is
possible that low capsule formation in spontaneous
autogamous treatments is reduced when higher
quality pollen is available for fertilization (Bookman,
1984). However, the observations that glasshousegrown plants do not form capsules without hand
pollination suggests that resource limitation does not
regulate spontaneous autogamy. In addition, selfpollination resulted in a high degree of capsule formation and vigorous seed on tested plants. These
data indicate that E. alta populations at FPNWR are
not autogamous, as has been reported previously for
the species in Florida (Goss, 1973; Catling, 1990).
Whether autogamy of E. alta is common throughout
its range or limited to certain populations also
remains to be studied.
Capsule formation resulting from induced autogamy in self-compatible orchids can be highly effective: 85% in Listera ovata (L.) R.Br. and 71.9–90% in
Platanthera ciliaris at two study sites (Ackerman &
Mesler, 1979; Robertson & Wyatt, 1990). Although
induced autogamy in E. alta resulted in fewer capsules than did artificial geitonogamy and induced
xenogamy, the means were not statistically different.
These data indicate that there are no significant
reproductive
barriers
to
within-flower
selffertilization for E. alta in Florida. However, the transfer of pollinia to stigma may still be vital to capsule
formation, given the significant difference in capsule
formation between treatments that did not involve
hand pollination (control, agamospermy and spontaneous autogamy) and those that did (induced
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88
86
T. R. JOHNSON ET AL.
autogamy, artificial geitonogamy, artificial xenogamy
and induced xenogamy).
Pollination by induced autogamy did not significantly reduce seed viability, germination or the development of seeds and seedlings relative to other pollen
supplementation treatments. Agamospermy appears
to be a rare event in this species as no capsules
formed from emasculated flowers in 2006 and a single
capsule formed in this treatment in 2008. Although
only visibly unpollinated flowers were used during
the experimental set-up, undetected pollination of
some flowers could have occurred. Agamospermy
appears to be rare in E. alta and is not expected to
contribute to viable seed production.
The hypothesis of insect-mediated pollination of E.
alta in Florida may be speculative, but there is evidence that plants could be pollinated by an unidentified vector. Comparison with other Eulophia spp.
suggests that E. alta could be pollinated by beetles (as
is Eulophia foliosa Bolus) or Xylocopa bees (as are E.
cristata Lindl. and E. rosea (Lindl.) A.D.Hawkes [= E.
horsfallii (Bateman) Summerh.]) (Kullenberg, 1961;
Lock & Profita, 1975; Peter & Johnson, 2006). Scarce
pollinators have been linked to poor capsule formation in Cyrtopodium punctatum (L.) Lindl., an orchid
found in the vicinity of E. alta at FPNWR. Cyrtopodium punctatum is hypothesized to be pollinated by
Xylocopa bees, populations of which may be declining
in south Florida as a result of agricultural pesticide
application (Dutra et al., 2009a). If Xylocopa bees are
responsible for the pollination of E. alta in Florida, as
they are for other Eulophia species in other parts of
the world, pollinator observations would be expected
to be exceedingly rare (Dutra et al., 2009a). As spontaneous autogamy (the previously reported mode of
pollination) was inefficient in producing capsules, a
new priority in efforts to manage and protect E. alta
populations in south Florida is to determine whether
a pollinator exists.
ACKNOWLEDGEMENTS
Partial project funding was provided by the US Fish
and Wildlife Service. The authors wish to thank
Danielle P. Johnson for field support and Larry Richardson (Florida Panther National Wildlife Refuge) for
logistical support during this project. The authors also
wish to thank Meghan Brennan (Institute of Food and
Agriculture Science Statistics Department, University
of Florida) for assistance with statistical analysis.
REFERENCES
Ackerman JD. 1985. Pollination of tropical and temperate
orchids. In: Tan KW, ed. Proceedings of the eleventh world
orchid conference. Miami, FL: International Press Co Ltd.,
98–101.
Ackerman JD, Mesler MR. 1979. Pollination biology of
Listera cordata (Orchidaceae). American Journal of Botany
66: 820–824.
Beardsell DV, Clements MA, Hutchinson JF, Williams
EG. 1986. Pollination of Diuris maculata R Br (Orchidaceae) by floral mimicry of the native legumes Daviesia spp
and Pultenaea scabra R Br. Australian Journal of Botany
34: 165–173.
Biesmeijer JC, Roberts SPM, Reemer M, Ohlemuller R,
Edwards M, Peeters T, Schaffers AP, Potts SG,
Kleukers R, Thomas CD, Settele J, Kunin WE. 2006.
Parallel declines in pollinators and insect-pollinated plants
in Britain and the Netherlands. Science 313: 351–354.
Bookman SS. 1984. Evidence for selective fruit production in
Asclepias. Evolution 38: 72–86.
Borba EL, Semir J. 1999. Temporal variation in pollinarium
size after its removal in species of Bulbophyllum: a different
mechanism preventing self-pollination in Orchidaceae.
Plant Systematics and Evolution 217: 197–204.
Brown PM. 2005. Wild orchids of Florida. Gainesville, FL:
University Press of Florida.
Calvo RN. 1990. Inflorescence size and fruit distribution
among individuals in three orchid species. American
Journal of Botany 77: 1378–1381.
Cameron KM, Chase MW, Whitten WM, Kores PJ,
Jarrell DC, Albert VA, Yukawa T, Hills HG, Goldman
DH. 1999. A phylogenetic analysis of the Orchidaceae: evidence from rbcL nucleotide sequences. American Journal of
Botany 86: 208–224.
Castro S, Silveira P, Navarro L. 2008. Effect of pollination
on floral longevity and costs of delaying fertilization in
the out-crossing Polygala vayredae Costa (Polygalaceae).
Annals of Botany 102: 1043–1048.
Catling PM. 1987. Notes on the breeding systems of Sacoila
lanceolata (Aublet) Garay (Orchidaceae). Annals of the
Missouri Botanical Garden 74: 58–68.
Catling PM. 1990. Auto-pollination in the Orchidaceae. In:
Arditti J, ed. Orchid biology: reviews and perspectives V.
Portland, OR: Timber Press, 121–158.
Catling PM, Lefkovitch LP. 1989. Associations of vascular
epiphytes in a Guatemalan cloud forest. Biotropica 21:
35–40.
Cozzolino S, Widmer A. 2005. Orchid diversity: an evolutionary consequence of deception? Trends in Ecology and
Evolution 20: 487–494.
Darwin C. 1885. The various contrivances by which orchids
are fertilized by insects. London: John Murray.
Dodson CH. 1962. The importance of pollination in the
evolution of the orchids of tropical America. American
Orchid Society Bulletin 31: 525–534, 641–649, 731–735.
Dodson CH, Frymire GP. 1961. Natural pollination
of orchids. Missouri Botanical Garden Bulletin 49: 133–
152.
Dressler RL. 1981. The orchids: natural history and classification. Cambridge: Harvard University Press.
Dutra D. 2008. Reproductive biology and asymbiotic seed
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88
BREEDING SYSTEM OF EULOPHIA ALTA
germination of Cyrtopodium punctatum, an endangered
Florida orchid. Masters Thesis. University of Florida.
Dutra D, Johnson TR, Kauth PJ, Stewart SL, Kane ME,
Richardson L. 2008. Asymbiotic seed germination, in vitro
seedling development, and greenhouse acclimatization of
the threatened terrestrial orchid Bletia purpurea. Plant
Cell, Tissue and Organ Culture 94: 11–21.
Dutra D, Kane ME, Reinhardt Adams C, Richardson LR.
2009a. Reproductive biology of Cyrtopodium punctatum in
situ: implications for conservation of an endangered Florida
orchid. Plant Species Biology 24: 92–103.
Dutra D, Kane ME, Richardson L. 2009b. Asymbiotic seed
germination and in vitro seedling development of Cyrtopodium punctatum: a propagation protocol for an endangered
Florida native orchid. Plant Cell, Tissue and Organ Culture
96: 235–243.
Fenster CB, Martén-Rodriguez S. 2007. Reproductive
assurance and the evolution of pollination specialization.
International Journal of Plant Sciences 168: 215–228.
Gale S. 2007. Autogamous seed set in a critically endangered
orchid in Japan: pollination studies for the conservation of
Nervilia nipponica. Plant Systematics and Evolution 268:
59–73.
Gonzalez-Diaz N, Ackerman JD. 1988. Pollination fruit set
and seed production in the orchid Oeceoclades maculata.
Lindleyana 3: 150–155.
Goss GJ. 1973. Pollination biology in the Orchidaceae: Polystachia flavescens, Epidendrum difforme, and Eulophia alta
from South Florida; Encyclia gracilis, Encyclia altissima,
and Encyclia rufa from Great Inagua, Bahamas. Masters
Thesis. Florida Atlantic University.
Huynh TT, McLean CB, Coates F, Lawrie AC. 2004. Effect
of developmental stage and peloton morphology on success
in isolation of mycorrhizal fungi in Caladenia formosa
(Orchidaceae). Australian Journal of Botany 52: 231–241.
Jersáková J, Johnson SD. 2006. Lack of floral nectar
reduces self-pollination in a fly-pollinated orchid. Oecologia
147: 60–68.
Jersáková J, Johnson SD, Kindlmann P, Pupin A-C.
2008. Effect of nectar supplementation on male and female
components of pollination success in the deceptive orchid
Dactylorhiza sambucina. Acta Oecologica 33: 300–306.
Johnson SD, Edwards TM. 2000. The structure and function of orchid pollinaria. Plant Systematics and Evolution
222: 243–269.
Johnson SD, Nilsson LA. 1999. Pollen carryover, geitonogamy, and the evolution of deceptive pollination systems
in orchids. Ecology 80: 2607–2619.
Johnson SD, Peter CI, Agren J. 2004. The effects of nectar
addition on pollen removal and geitonogamy in the nonrewarding orchid Anacamptis morio. Proceedings of the
Royal Society of London Series B – Biological Sciences 271:
803–809.
Johnson TR, Stewart SL, Dutra D, Kane ME, Richardson L. 2007. Asymbiotic and symbiotic seed germination of
Eulophia alta (Orchidaceae) – preliminary evidence for the
symbiotic culture advantage. Plant Cell, Tissue and Organ
Culture 90: 313–323.
87
Juillet N, Dunand-Martin S, Gigord LDB. 2007. Evidence
for inbreeding depression in the food-deceptive colourdimorphic orchid Dactylorhiza sambucina (L.) Soo. Plant
Biology 9: 147–151.
Kauth PJ, Kane ME, Vendrame WA, Reinhardt-Adams
C. 2008. Asymbiotic germination response to photoperiod
and nutritional media in six populations of Calopogon
tuberosus var. tuberosus (Orchidaceae): evidence for ecotypic
differentiation. Annals of Botany 102: 783–793.
Kullenberg B. 1961. Studies in Ophrys pollination. Zoologiska Bidrag 34: 1–330.
Leur CA. 1971. Abnormal development of the anther – a
report of two cases. Florida Orchidist 14: 26–29.
Leur CA. 1972. The native orchids of Florida. Ipswich: W.S.
Cowell Ltd.
Lock JM, Profita JC. 1975. Pollination of Eulophia cristata
(Sw.) Steud. (Orchidaceae) in southern Ghana. Acta
Botanica Neerlandica 24: 135–138.
McCartney C. 1985. The orchids of Everglades National
Park – 1. American Orchid Society Bulletin 54: 265–
276.
Micheneau C, Fournel J, Gauvin-Bialecki A, Pailler T.
2008. Auto-pollination in a long-spurred endemic orchid
(Jumellea stenophylla) on Reunion Island (Mascarene Archipelago, Indian Ocean). Plant Systematics and Evolution
272: 11–22.
Peakall R. 1989. Outcrossing in an ant pollinated clonal
orchid. Heredity 62: 161–167.
Peter CI, Johnson SD. 2006. Anther cap retention presents
self-pollination by elaterid beetles in the South African
orchid Eulophia foliosa. Annals of Botany 97: 347–355.
Robertson JL, Wyatt R. 1990. Reproductive biology of
the yellow-fringed orchid, Platanthera ciliaris. American
Journal of Botany 77: 388–398.
Rosell P, Galan Sauco V, Herrero M. 2006. Pollen germination as affected by pollen age in cherimoya. Scientia
Horticulturae 109: 97–100.
SAS Institute. 2003. SAS for Windows, version 9.1. Cary, NC:
SAS Institute.
Smithson A. 2006. Pollinator limitation and inbreeding
depression in orchid species with and without nectar
rewards. New Phytologist 169: 419–430.
Stenberg ML, Kane ME. 1998. In vitro seed germination
and greenhouse cultivation of Encyclia boothiana var. erythronioides, an endangered Florida orchid. Lindleyana 13:
101–112.
Stewart SL, Kane ME. 2006. Asymbiotic seed germination
and in vitro seedling development of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant
Cell, Tissue and Organ. Culture 86: 147–158.
Stewart SL, Kane ME. 2007. Symbiotic seed germination
and evidence for in vitro mycobiont specificity in Spiranthes
brevilabris (Orchidaceae) and its implications for specieslevel conservation. In Vitro Cellular and Developmental
Biology-Plant 43: 178–186.
Stewart SL, Richardson LW. 2008. Orchid flora of the
Florida Panther National Wildlife Refuge. North American
Native Orchid Journal 14: 70–104.
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88
88
T. R. JOHNSON ET AL.
Swarts ND, Dixon KW. 2009. Terrestrial orchid conservation
in the age of extinction. Annals of Botany (in press).
Taki H, Kevan PG. 2007. Does habitat loss affect the community of plants and insects equally in plant-pollinator
interactions? Preliminary findings. Biodiversity and Conservation 16: 3147–3161.
Tremblay RL, Ackerman JD, Zimmerman JK, Calvo RN.
2005. Variation in sexual reproduction in orchids and its
evolutionary consequences: a spasmodic journey to diversification. Biological Journal of the Linnean Society 84: 1–
54.
Tremblay RL, Pomales-Hernandez G, Mendez-Cintron
ML. 2006. Flower phenology and sexual maturation: partial
protandrous behavior in three species of orchids. Caribbean
Journal of Science 42: 75–80.
USDA (United States Department of Agriculture
Natural Resource Conservation Service). 2009. Plants
Database. United States Department of Agriculture. Available at http://plants.usda.gov/index.html [accessed 14 May
2009].
Weekley CW, Brothers A. 2006. Failure of reproductive
assurance in the chasmogamous flowers of Polygala lewtonii
(Polygalaceae), an endangered sandhill herb. American
Journal of Botany 93: 245–253.
Williamson G. 1984. Observations of a mechanism by which
self-pollination may occur in Eulophia (Orchidaceae).
Journal of South African Botany 50: 417–423.
Wong KC, Sun M. 1999. Reproductive biology and conservation genetics of Goodyera procera (Orchidaceae). American
Journal of Botany 86: 1406–1413.
Zettler LW, Piskin KA, Stewart SL, Hartsock JJ, Bowles
ML, Bell TJ. 2005. Protocorm mycobionts of the federally
threatened eastern prairie fringed orchid, Platanthera
leucophaea (Nutt.) Lindley, and a technique to prompt leaf
elongation in seedlings. Studies in Mycology 53: 163–171.
Zettler LW, Poulter SB, McDonald KI, Stewart SL. 2007.
Conservation-driven propagation of an epiphytic orchid
(Epidendrum nocturnum) with a mycorrhizal fungus. HortScience 42: 135–139.
Znaniecka J, Krolicka A, Sidwa-Gorycka M, Rybczynski
JJ, Szlachetko DL, Lojkowska E. 2005. Asymbiotic germination, seedling development and plantlet propagation
of Encyclia aff. oncidioides – an endangered orchid. Acta
Societatis Botanicorum Poloniae 74: 193–198.
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88