Plant Species Biology (2009) 24, 92–103
doi: 10.1111/j.1442-1984.2009.00242.x
Reproductive biology of Cyrtopodium punctatum
in situ: implications for conservation of an endangered
Florida orchid
DANIELA DUTRA,*1 MICHAEL E. KANE,* CARRIE REINHARDT ADAMS* and LARRY RICHARDSON†
*Department of Environmental Horticulture, University of Florida, PO Box 110675, Gainesville, Florida 32611, USA and
†Florida Panther National Wildlife Refuge, US Fish and Wildlife Service, 3860 Tollgate Boulevard, Suite 300, Naples,
Florida 34114, USA
Abstract
Cyrtopodium punctatum (Linnaeus) Lindley is an endangered epiphytic orchid restricted
in the USA to southern Florida. This species has been extensively collected from the wild
since the early 1900s, and today only a few plants remain in protected areas. As part of a
conservation plan, a reproductive biology study was conducted to better understand the
ecology of this species in Florida. Cyrtopodium punctatum relies on a deceit pollination
system using aromatic compounds to attract pollinators. Nine aromatic compounds were
identified as components of the fragrance of C. punctatum inflorescences, including two
compounds that are known to be Euglossine bee attractants. However, this group of bees
is not native to Florida. Of the four bee species observed to visit C. punctatum flowers in
the present study, carpenter bees (Xylocopa spp.) are likely to be the main pollinators.
Pollination experiments demonstrated that C. puntatum is self-compatible, but requires a
pollinator and thus does not exhibit spontaneous autogamy. In addition, the rates of fruit
set were significantly higher for flowers that were outcrossed (xenogamy) than for those
that were self-crossed. Thus, the species has evolved a degree of incompatibility.
Examples of natural pollination and fruit set were observed during the present study
(2007–2008), but the rates of reproduction were modest as a consequence of the low plant
numbers and possible changes in insect densities as a result of anthropogenic influences.
Keywords: breeding system, Cyrtopodium punctatum, Orchidaceae, pollination.
Received 11 March 2009; accepted 21 May 2009
Introduction
Several types of plant rarity exist and different natural
processes may affect species density and distribution
(Rabinowitz 1981; Fiedler & Ahouse 1992). Species may be
considered rare based on habitat specificity, local population size and/or the size of their geographical range.
However, plants that were once common in their range
may now be rare as a result of anthropogenic influences
(Partel et al. 2005). The factors influencing rarity need to
be considered for an effective conservation plan to be
designed for any target species. Pollination systems are
Correspondence: Daniela Dutra
Email: ddutra@hawaii.edu
1
Present address: Botany Department, University of Hawai’i at
Manoa, 3190 Maile Way, Room 101, Honolulu, HI 96822, USA.
often linked to plant rarity given the dependence on
insect species for cross pollination as this relationship is
required for seed production and fruit set to take place.
Decreased abundance or loss of pollinators often results in
increased rarity of the plant. The Orchidaceae is a very
diverse family, and pollination systems may range from
being very specific with a plant being pollinated by only
one insect, to very generalized with a plant being pollinated by many species (van der Pijl & Dodson 1966;
Dressler 1981; van der Cingel 1995). A comprehensive
investigation of the pollination system is a prerequisite to
developing a conservation plan for any rare species.
Cyrtopodium punctatum (L.) Lindl. is an epiphytic orchid
found in southern Florida, Cuba, Hispaniola, Puerto
Rico and the north-western Caribbean coast of South
America (Romero-González & Fernández-Concha 1999).
© 2009 The Authors
Journal compilation © 2009 The Society for the Study of Species Biology
REPRODUCTIVE BIOLOGY OF CYRTOPODIUM PUNCTATUM
In Florida, the species is listed as endangered (Coile &
Garland 2003); populations have been depleted because of
cypress logging and over-collecting during the past
century. Early accounts in the literature to C. punctatum as
abundant throughout south and south-west Florida, particularly in the cypress swamps in the Big Cypress region
(Ames 1904; Luer 1972). Photographs from the early 1900s
show wagons loaded with plants being extracted from the
wild. Consequently, only a few plants still remain in very
small populations within inaccessible protected areas,
such as Everglades National Park, Big Cypress National
Preserve and the Florida Panther National Wildlife Refuge
(FPNWR). Over the past 10 years, observations of plant
populations in the FPNWR have indicated very low rates
of capsule formation and seed production in the remaining plants (L. Richardson, pers. comm. 2005). Additional
information regarding the breeding strategies and pollination mechanisms of natural populations of C. punctatum
in Florida is critical to develop strategies for the recovery
of the species.
The study of pollination systems, including breeding
systems, is critical in rare plant conservation because it
may influence the genetic diversity of populations
(Hamrick & Godt 1996). Pollination aids the long-term
survival of most species because population persistence
depends on the recruitment of new plants from genetically diverse seeds (Dixon et al. 2003). Most of the pertinent literature on the pollination of C. punctatum refers to
observations conducted in only one area of the species’
range (e.g. Puerto Rico; Ackerman 1995). In different parts
of its geographical range, a species may possess different
breeding strategies, which is not uncommon for tropical
orchid species in Florida. For example, both Epidendrum
nocturnum and Secoila lanceolata produce seeds by agamospermy in Florida, but use animal cross-pollination in the
more southern part of their range (Catling 1987; Brown
2002).
Pollinator species may also vary from one location in the
geographical range to another. Pollination ecotypes have
been reported by Robertson and Wyatt (1990a) in Plantanthera ciliaris. In the present study, the pollination ecology
of two disjunct populations was compared and the
primary pollinators were found to differ between the
sites. Cyrtopodium punctatum is reported to be pollinated
by Euglossa bees (van der Pijl & Dodson 1966; Jeffrey et al.
1970; Luer 1972; Dressler 1993). However, as Florida lies
outside the range of euglossine bees, C. punctatum’s pollinator(s) in Florida may vary (Chase & Hills 1992). In
Puerto Rico, C. punctatum is reported to be pollinated by
Centris or Xylocopa bees (Ackerman 1995). Bees of the
genus Centris were reported by Dodson and Adams (in
Luer 1972) to visit flowers of C. punctatum in Florida.
However, these observations have not been fully
described or verified.
Plant Species Biology 24, 92–103
93
Fragrances produced by plants attract pollinators and
may function to select the type and number of visitors
(Hills et al. 1972). Floral fragrances have been associated
with deception pollination systems based on generalized
food deception (Little 1983) and can be an extra tool in the
determination of pollination systems. Different fragrance
compounds or groups of compounds may attract different
pollinators. A larger array of compounds may be beneficial in deceit pollination systems because a higher number
of visitors will be attracted to the plant.
In many orchid pollination strategy studies, capsule
formation is commonly observed as the final result of the
breeding system study without taking into consideration
seed viability (Wong & Sun 1999; Lehnebach & Robertson
2004). Other researchers have used seed number and
weight as a further measurement for breeding system
determination (Robertson & Wyatt 1990b; Sipes & Tepedino 1995). However, this type of measurement produces
uncertain results because of differences in capsule maturity. Sipes and Tepedino (1995) reported that fruit weight
and fruit size were not correlated with seed number, and
instead used seed number as another measurement
for breeding system determination. As capsules of C.
punctatum require approximately 11 months to mature,
repeated capsule measurement over the course of capsule
development may minimize this limitation.
Another tool used to evaluate breeding systems is tetrazolium seed viability testing (TZ) and it has been used
to assess the viability of European and North American
temperate orchid species (Van Waes & Debergh 1986;
Lauzer et al. 1994; Vujanovic et al. 2000; Bowles et al. 2002)
and tropical epiphytic species (Singh 1981). Assessing
seed viability and germinability is crucial for in vitro
propagation of orchid species and their conservation and
may be a helpful tool to more precisely determine effective pollination strategies for threatened and endangered
taxa.
As a prerequisite to developing a comprehensive conservation plan for C. punctatum, integrated field and laboratory studies were conducted with the objectives of: (i)
determining the breeding system of C. punctatum through
controlled pollination experiments and the effects on both
capsule formation and seed viability; (ii) determining the
pollinator(s) of C. punctatum in Florida; and (iii) determining the volatile potential attractant compounds present in
C. punctatum flowers.
Materials and methods
Study species
In the FPNWR (Collier County, FL, USA), C. punctatum
plants are found growing epiphytically on cypress (Taxodium distichum) and less often on cabbage palm (Sabal
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Journal compilation © 2009 The Society for the Study of Species Biology
94 D . D U T R A E T A L .
palmetto) or old stumps of massive cypress trees that were
logged in the 1940s. The vegetation classification for the
habitat is cypress forest moderately to densely distributed
cypress with an open canopy. The understory is composed of Fraxinus caroliniana, Cladium jamaicense and
Annona glabra, among others. In other areas of Florida
(e.g. Everglades National Park), C. punctatum can also be
found growing on Buttonwood trees (Conocarpus erecta) in
full sun. The inflorescences of C. punctatum are up to 1 m
long and can have 30–40 flowers.
Breeding system determination
One population with 15 plants located in the FPNWR
served as the source of plants for the determination of the
breeding system. Six plants located approximately 30 m
apart were used for the breeding system experiment
during two consecutive years (2006–2007 and 2007–2008).
Seven breeding system treatments (Table 1) were applied
to five flowers of each plant. Flowers were randomly
selected on the inflorescences. A total of 245 flowers were
used in this study. A plant located approximately 1.61 km
from the breeding system study population served as the
pollen donor. Pollen was applied to flowers within 1 h.
The breeding system determination methods were
adapted from Wong and Sun (1999). An extra treatment
(induced xenogamy) was added based on Stewart (2007).
The breeding system treatments are described in Table 1.
Pollen used in the artificial xenogamy treatment was
obtained from a plant originating from a different population in the FPNWR. Inflorescences were produced in
February and were bagged before the flowers opened
with 95 mm nylon mesh to prevent pollination events
prior to initiating the experiment (Fig. 1). Plants remained
bagged until signs of capsule formation were observed.
The percentage of flowers forming capsules was recorded
and capsule development was recorded bimonthly for
1 year by recording capsule length, width and abortion.
The breeding system study was conducted during 2006–
2007 and 2007–2008. Data were analyzed using general
linear procedures and Waller–Duncan mean separation
(a = 0.05) with SAS v. 9.1 (SAS Institute 2003).
Tetrazolium seed viability test
Seed capsules produced from the breeding system study
treatments (2006–2007) were collected on 23 February
2007 and dried over silica desiccant for 70 days at
23 ⫾ 2°C. The seeds harvested from each capsule collected were transferred to 20 mL scintillation vials. The
vials were then maintained in cold storage (-10 ⫾ 2°C).
Three 5 mg seed subsamples (75–100 seeds) from each
capsule of each treatment were dispensed into a 1.5 mL
microcentrifuge tube and homogenized with the aid of a
vortex shaker. The methods of tetrazolium viability
testing follow Lakon (1949). A pretreatment test conducted prior to experimentation indicated that a 15 min
treatment in a seed scarification solution of 5% CaOCl2
was ideal for C. punctatum seeds. Percentage seed viability
data were collected for each of the three subsamples per
capsule by placing seeds that were suspended in water
droplets into Petri dishes and the seeds were then scored
with the aid of a dissecting microscope. Approximately
100 seeds were counted per subsample. Embryos that contained any degree of red or pink coloration were scored as
viable and white embryos were scored as non-viable
(Fig. 2). Percentage viability was calculated for each replicate by dividing the number of stained embryos by the
total number of embryos. Data were analyzed using
general linear procedures and Waller–Duncan mean separation (a = 0.05) using SAS v. 9.1.
Asymbiotic seed germination test
Seeds from the capsules produced from each specific
breeding system treatment were sampled (5 mg subsamples of 75–100 seeds). Seeds from the same treatment
were then pooled and dispensed into 1.5 mL microcentrifuge tubes. The seeds were surface sterilized in a solution
containing 5 mL ethanol (100%), 5 mL 6.0% (v/v) sodium
Table 1 Pollination treatments used to determine orchid breeding systems
Breeding system test
Control
Agamospermy
Spontaneous autogamy
Induced autogamy
Artificial geitonogamy
Artificial xenogamy
Induced xenogamy
Flowers bagged
Treatment
Pollen source
No
Yes
Yes
Yes
Yes
Yes
Yes
None
Emasculate
None
Emasculate
Emasculate
Emasculate
Emasculate
Open pollination
No pollination
Same flower
Same flower
Different flower, same plant
Different population
Same population, different plant
Adapted from Wong and Sun (1999) and Stewart (2007).
© 2009 The Authors
Journal compilation © 2009 The Society for the Study of Species Biology
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95
Fig. 1 Breeding system experiment. (a) Flower, (b) artificial removal of pollinia, (c) capsule measurement, (d) Cyrtopodium. punctatum
plant with pollination bag in natural habitat at the Florida Panther National Wildlife Refuge (Collier County, FL, USA).
hypochlorite and 90 mL sterile distilled water for 3 min,
followed by three repetitive 30 s rinses in sterile distilled
water.
P723 Orchid Seed Sowing Medium was prepared from
concentrated stock solutions using the formulation developed by PhytoTechnology Laboratories, LLC (Shawnee
Mission, KS, USA) and adjusted to pH 5.8 with 0.1 mol/L
Plant Species Biology 24, 92–103
KOH prior to the addition of 0.8% TC agar (PhytoTechnology Laboratories, LLC) and autoclaving at
117.7 kPa for 40 min at 121°C. The autoclaved medium
(approximately 50 mL medium/plate) was dispersed into
square 100 mm ¥ 15 mm Petri plates (Falcon ‘Integrid’
Petri Dishes; Becton Dickinson Woburn, MA, USA). The
bottom of each dish was divided into 36, 13 mm ¥ 13 mm
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96 D . D U T R A E T A L .
Fig. 2 Seed viability and asymbiotic germination. (a) Germinating seeds (stage 2). (b) Protocorm at stage 4 of development showing two
leaves. (c) Stained and unstained seeds from tetrazolium. (d) Falcon ‘Integrid’ Petri dishes showing cells.
cells (Fig. 2). Only the 16 cells in the middle of the plate
were considered for inoculation because the cells at the
outer edges of the plate were more susceptible to drying.
Five of the 16 interior cells were selected randomly for
sowing using a computerized random number generator.
Surface-disinfected seeds were sown onto the surface of
the sterile germination medium using a sterile bacterial
inoculating loop. Plates were sealed with NescoFilm
(Karlan Research Products, Santa Rosa, CA, USA) and
incubated under 12/12 h light/dark (60 mmol/m2/s) photoperiod at 25 ⫾ 3°C. Approximately 82 seeds were sown
onto each plate (mean seeds/plate = 82, mean seeds per
cell = 16.4). Five replicate plates were used for each breeding system treatment. Seed germination and protocorm
development stage percentages were recorded weekly for
10 weeks. Seedling development was scored on a scale of
1–5 (Table 2; modified from Stewart et al. 2003). Germina-
Table 2 Seed developmental stages of Cyrtopodium punctatum
(modified from Stewart et al. 2003)
Stage
Description
1
2
3
4
5
Intact testa
Embryo enlarged, testa ruptured (= germination)
Appearance of protomeristem
Emergence of two first leaf primordia
Elongation of shoot and further development
tion percentages were calculated by dividing the number
of seeds in each germination and development stage by
the total number of viable seeds in the subsample. Data
were analyzed using general linear model procedures and
Waller–Duncan at a = 0.05 with SAS v. 9.1. Percentage
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Journal compilation © 2009 The Society for the Study of Species Biology
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97
germination counts were arcsine transformed to normalize variation.
Pollinator identification
Pollinator observations were conducted during the 2007
and 2008 flowering seasons on a population consisting of
15 plants. Observations took place on plants in the population that had the largest number of inflorescences
during both flowering seasons (12 and 14 inflorescences
in 2007 and 2008, respectively). Observations took place
from 07.00 to 18.00 hours for the first 2 days (13 and 14
March 2007). When peak visitation time was identified,
observations took place from 10:00 am to 4:00 pm for the
remaining days (15, 23–25 March 2007). In 2008, observations took place on 18–19 March (10.00–15.00 hours) and
26 March (11.00–15.00 hours). Flower visitors were photographed, collected and identified, and their behavior in
the flowers was documented. Flower visitors capable of
pollinating the flowers were determined either by direct
observation or by the detection of pollinaria on their
bodies. The insects collected were identified at the Florida
State Collection of Arthropods (Gainesville, FL, USA).
Fragrance analysis
Two inflorescences with 30 open flowers each from two C.
punctatum plants were collected from the FPNWR during
the 2007 flowering season. The inflorescences were collected while still attached to the pseudobulbs to preserve
the flowers and taken to the US Department of Agriculture Chemistry Research Unit (Gainesville, FL, USA).
The inflorescences were placed into a 15 cm diameter ¥
40 cm tall glass volatile collection chamber. Volatiles
were collected over three time periods: 9:00–12:00 am,
12:00 am–5:00 pm and 5:00 pm–7:00 am. The volatile traps
were extracted with 150 mL methylene chloride. Gas
chromatography-mass spectroscopy (GC/MS) analyses of
the collected volatiles were carried out on an HP-6890 gas
chromatograph coupled to an HP5973 mass spectrometer
(Agilent, Santa Clara, CA, USA). The GC/MS peaks of
interest were identified by comparing their mass spectral
data with data from three mass spectral libraries. Only
two inflorescences could be used because very limited
plant material from wild populations is available owing to
the endangered status of the species.
Results
Breeding system determination
Capsule formation occurred in four out of the seven treatments tested during both the 2006–2007 and 2007–2008
seasons (Figs 1c,3). There was no evidence that the agamoPlant Species Biology 24, 92–103
Fig. 3 Percentage capsule set among seven breeding system
treatments. Data from 2006 and 2007. Six plants and 30 flowers
per treatment were used per year. Percentages with the same
letter are not significantly different. Control (no treatment, open
pollination); agamospermy (emasculated flower, no pollination);
spontaneous autogamy (no treatment, pollen from the same
flower); induced autogamy (selfing with pollen from the same
flower); artificial geitonogamy (selfing with pollen from the
same plant, different flower); artificial xenogamy (pollen from
different population); induced xenogamy (pollen from a plant in
the same population).
spermy, spontaneous autogamy or open pollination
(control) treatments resulted in capsule formation (Fig. 3).
The induced geitonogamy (pollen from the same flower)
and artificial geitonogamy (pollen from the same plant, but
from different flowers) treatments resulted in the production of significantly fewer capsules (14.6 and 27.1% capsule
formation, respectively) than the artificial xenogamy
(pollen from a different population) and induced
xenogamy (pollen from a different plant in the same population) treatments (48.9 and 73.9%, respectively) (Fig. 3).
Capsule widths and lengths were significantly different
among the pollination treatments (Figs 4,5). During the
2006–2007 growing season, the length of the capsules
formed following induced autogamy was significantly
less than the length of the capsules produced following
the other treatments 240 and 300 days after pollination
(Fig. 5). In the 2007–2008 season, the capsule length of
capsules formed following both the induced autogamy
(pollen from the same flower) and artificial geitonogamy
treatments (pollen from the same plant, different flower)
were significantly less than the length of capsules formed
in the other treatments (Fig. 5). In addition, in the 2007–
2008 season, the width of the capsules formed from the
induced autogamy treatment was significantly less than
that in all other treatments throughout the year (Fig. 4).
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98 D . D U T R A E T A L .
Fig. 4 Effect of breeding system treatment on mean capsule
width (⫾ standard error). The interaction between the 2006–2007
and 2007–2008 flowering seasons was significant.
Viability and asymbiotic seed germination tests
We found no differences among treatments for either seed
viability or asymbiotic seed germination. In general, seed
germination was very low (induced autogamy, 2.9%; artificial geitonomy, 1.9%; artificial xenogamy, 4.3%; induced
xenogamy, 4.7%). This was unexpected considering the
high seed viability observed across treatments that
resulted in seed production, particularly induced autogamy (79.9%), artificial geitonogamy (67.6%), artificial
xenogamy (87.2%) and induced xenogamy (79.1%;
Table 3). However, capsules that originated from selfing
(induced autogamy and artificial geitonogamy) had significantly lower seed germination (2.9 and 1.9%, respectively) than seeds that originated from outcrossing
(induced xenogamy, 4.7%).
Pollinator identification
During the two flowering seasons (2007 and 2008), four
bee species were observed visiting the flowers of C. punctatum (Fig. 6). Apis mellifera was the most frequent visitor
in 2007 (53 visits in 46.5 h; Table 4). However, A. mellifera
Fig. 5 Effect of breeding system treatment on mean capsule
length (standard error). The interaction between the 2006–2007
and 2007–2008 flowering seasons was significant.
and Megachile xylocopoides were only observed during the
2007 flowering season (Table 4). Both Xylocopa micans and
Xylocopa virginica were frequent visitors to the flowers
during both flowering seasons (Table 4). Flowers were
observed without pollinia; however, no visitors were
observed carrying pollen to or from flowers.
Fragrance analysis
Nine fragrance compounds were identified as being
produced by C. punctatum flowers: benzaldehyde,
myrcene, benzyl alcohol, Z-b-ocimene, E-b-ocimene,
1,3,8-para-menthatriene, methyl salicylate, indole and
E,E-a-farnesene. The relative abundance of these compounds was recorded (Fig. 7).
Discussion
Breeding system
Our results show that C. punctatum is not capable of reproducing autogamously and requires a pollinator for
© 2009 The Authors
Journal compilation © 2009 The Society for the Study of Species Biology
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99
Table 3 Seed viability, germination and development stage percentages generated from Cyrtopodium punctatum pollination treatments in
the 2006–2007 flowering season
Pollination treatment
Control‡
Agamospermy‡
Spontaneous autogamy‡
Induced autogamy
Artificial geitonogamy
Artificial xenogamy
Induced xenogamy
Seed
viability (%)
Total
germination
1
0.0
0.0
0.0
79.9 ab
67.6 b
87.2 a
79.1 ab
0.0
0.0
0.0
2.9 bc
1.9 c
4.3 ab
4.7 a
0.0
0.0
0.0
17.9 ab
18.1 a
17.6 b
17.5 b
Seedling development stages (%)†
2
3
0.0
0.0
0.0
1.2 b
1.2 b
2.7 a
4.0 a
0.0
0.0
0.0
1.7 a
0.49 b
1.9 a
1.1 ab
4
0.0
0.0
0.0
0.35 a
0.44 a
0.55 a
0.29 a
† See Table 2 for descriptions of the seedling developmental stages. ‡ Treatments produced no capsules. Six plants were used and 30
flowers were used per treatment. Values with the same letter are not significantly different (a = 0.05).
Fig. 6 Bees observed visiting flowers. (a) Apis mellifera (honey bee). (b) Xylocopa virginica. (c) Xylocopa micans (female). (d) Megachile
xylocopoides. (e) X. micans (male).
capsule set. Given that no capsules were formed from
the agamospermy or spontaneous autogamy treatments
implemented in the field, we demonstrated that no spontaneous self-fertilization occurs. Although a few capsules
were formed from induced autogamy (selfing with pollen
from the same flower; 17.2%) and artificial geitonogamy
(selfing with pollen from the same plant, but from a difPlant Species Biology 24, 92–103
ferent flower, 27.2%), significantly more capsules were
produced following artificial and induced xenogamy
(48.9 and 73.9%, respectively; Fig. 3). Species within the
Orchidaceae are predominantly self-compatible; however,
species normally require an insect vector to facilitate
pollen movement (Dressler 1981). This is supported in C.
punctatum; spontaneous autogamy treatments resulted in
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100 D . D U T R A E T A L .
Fig. 7 Fragrance analysis. (a) Relative
amounts
of
fragrance
compounds
extracted from Cyrtopodium punctatum
inflorescences.
Table 4 Insect visitors to Cyrtopodium punctatum
Insect visitors
Xylocopa micans
Xylocopa virginica
Apis melifera
Megachile xylocopoides
2007
2008
28
15
53
2
17
2
0
0
Total number of observation hours for 2007 and 2008 were
46.5 h and 14.2 h, respectively.
no capsule formation, but both induced autogamy and
artificial geitonogamy treatments, where pollen was
manually moved to simulate the action of the pollinator,
resulted in capsule formation.
Other studies with epiphytic orchid species show a
degree of self-incompatibility and the need for pollinators
(Ackerman 1989; Jaimes & Ramírez 1999; Borba et al. 2001;
Lehnebach & Robertson 2004). Lehnebach and Robertson
(2004) and Borba et al. (2001) reported that capsules were
not produced following agamospermy and spontaneous
autogamy treatments on the epiphytic orchid species that
they examined, showing dependence on a pollinator.
Some degree of self-incompatibility was also found for
these species (Borba et al. 2001; Lehnebach & Robertson
2004). Significantly fewer capsules were formed from artificial selfing than from induced outcrossing. Similarly,
some degree of self-incompatibility exists in C. punctatum
because fewer capsules were formed from selfing than
outcrossing treatments.
Cyrtopodium punctatum uses deceit via aromatic compounds and visual signals to attract insect visitors. Our
fragrance analysis and compound identification showed a
floral bouquet strategy in which a diverse array of attractant compounds are produced. However, C. punctatum
flowers provide no food reward to floral visitors. There
are implications of having a deceit pollination strategy.
Neiland and Wilcock (1998) found that orchids that offer
food rewards (e.g. nectar) had double the probability of
setting capsule than non-rewarding species across all geographical areas. In North America, the rate of capsule
set was 19.5% for species that offer no nectar reward
compared with 49.3% for rewarding orchids (Neiland &
Wilcock 1998). A closely related species in Brazil, Cyrtopodium polyphyllum, also uses a deceit pollination system.
This plant mimics the yellow flowers of a rewardproducing unrelated species that occurs in the same
habitat. This species also has low capsule set as a result of
low pollinator visitation (Pansarin et al. 2008). Pollinator
limitation has also been observed in other deceptive
orchids, such as Serapias vomeracea and Pogonia japonica
(Matsui et al. 2001; Pellegrino et al. 2005).
Historically, populations of C. punctatum in Florida
were numerous and plants were abundant. In this situation, a deceit pollination system with low capsule production was viable because the probability of capsule
formation was higher owing to plant abundance. However, populations are now small and fragmented; thus,
the deceit pollination system may minimize future reproductive success for the species as the likelihood of capsules being produced is so low. The open pollination
treatment resulted in no capsule formation during both
the 2006–2007 and 2007–2008 growing seasons in the
population studied. However, two capsules were formed
on two different plants during the 2006–2007 season in
inflorescences not used in the breeding system study, suggesting that natural pollination does occur, albeit at very
low levels. The deceit pollination system alone may not be
the cause of the low capsule formation at the FPNWR. A
decrease in pollinator populations may also be affecting
natural capsule set. A more detailed study on the insect
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populations of the area should be conducted, particularly
because of the closeness of the populations to agricultural
fields that continually use pesticide applications.
The capsule size measurements (length and width)
taken during the 11-month maturation period in both
years indicated that the induced autogamy and artificial
geitogamy treatments resulted in capsules that were
relatively smaller than capsules produced from other
treatments (except for capsule length in 2006–2007).
However, capsule size may vary from year to year owing
to significant variation in environmental factors, such as
rainfall and temperature, which can influence capsule formation and maturation over time. Our research suggests
that breeding system studies should be conducted across
multiple growing seasons.
Seed viability and asymbiotic germination
Tetrazolium seed viability testing showed high seed
viability in all breeding system treatments that formed
capsules in C. punctatum. However, germination was very
low across all treatments. This may be because of suboptimal in vitro cultural conditions for asymbiotic germination. Light may inhibit germination of C. punctatum seeds
(Dutra et al. 2009). By sowing seeds and initially incubating them in the dark, germination could be significantly
improved in this species.
Pollinator identification
During the 2007 flowering season, honey bees (Apis
melifera) were the most common visitors to C. punctatum
flowers. However, these bees were too small to remove
pollinaria and efficiently act as pollinators. In the 2008
flowering season, honey bees were not seen visiting C.
punctatum flowers. Xylocopa bees (X. micans and X. virginica) were observed visiting the flowers, but removal
or deposition of pollinia was not observed during the
field observation period, despite being large enough to
fit inside the flowers. In Puerto Rico, C. punctatum is
reported to be pollinated by Xylocopa bees (Ackerman
1995). Pemberton and Liu (2008) observed two species of
Centris bees, visiting the flowers of C. punctatum in southeast Florida. However, these pollinator observations were
conducted at Fairchild Tropical Botanical Garden and in
Fort Lauderdale, Florida, in an artificial setting with
plants of unknown origins. Observations conducted in a
garden setting do not accurately reflect the real ecological
links because a mixture of exotic and native plants are
planted in unnatural arrangements causing insect pollinator densities to change. Pollination observations conducted in situ are more representative because it is where
natural conditions prevail and where future plant reintroductions will take place.
Plant Species Biology 24, 92–103
101
Fragrance analysis
The compounds identified in the fragrance analysis of C.
punctatum are considered relatively common pollinator
attractants produced by many other orchid species (Kaiser
1993). However, two compounds identified, indole and
methyl salicylate, can be associated with the Euglossini
pollination system (Williams & Whitten 1983). Although
Florida is located outside the range of Euglossini bees, C.
punctatum is reported to be pollinated by Euglossa bees in
part of its range (van der Pijl & Dodson 1966; Jeffrey et al.
1970; Luer 1972; Dressler 1993). An introduced species of
Euglossa (E. viridissima) has recently become naturalized in
southern Florida (Skov & Wiley 2005). Pemberton and
Wheeler (2006) extracted and identified compounds collected by E. viridissima in southern Florida. Although
methyl salicylate and indole have been detected in many
other Euglossini species in the tropics (Ramírez et al.
2002), these compounds were not found in the collection
storage organs of E. viridissima in southern Florida (see
Pemberton & Wheeler 2006). It is possible that even if E.
viridissima was found in the same location as C. punctatum,
it would not be attracted by the types of compounds that
the plant produces and would not affect pollination.
Conservation implications
Understanding a plant’s breeding system and pollinator
diversity can help conservation and management strategies because plant populations may be greatly impacted
by a reduction in pollinator visitations (Kearns et al. 1998;
Havens 1999). For example, Sahli and Conner (2006)
found that pollinator visitation and not pollinator effectiveness is the main driving factor in determining pollination importance. In a generalized pollination system, such
as that in C. punctatum, the pollinators that visit the plants
the most are most likely to be the ones responsible for
capsule formation and not simply infrequent visitors.
Xylocopa bees visited the plants during both seasons and
were responsible for most of the visits during 2008.
Although A. melifera was the most frequent visitor in 2007,
it did not visit the plants during the 2008 season.
Observations made during the present study and by
biologists over the past 10 years at the FPNWR indicate
that very few capsules were formed by C. punctatum. Pesticide use in nearby agricultural areas is another possible
direct cause of low capsule formation as pesticide use may
decrease pollinator populations at the study site. Similarly,
habitat degradation may affect insect populations. The
area has been impacted from hydrological changes that
have shortened the hydroperiod. Local insect population
dynamics should be studied for conservation purposes
and land management in areas were C. punctatum occurs.
Our study indicates that sexual reproduction in C. punctatum is severely depressed, suggesting that recruitment is
© 2009 The Authors
Journal compilation © 2009 The Society for the Study of Species Biology
102 D . D U T R A E T A L .
highly unlikely. Consequently, the long-term viability/
persistence of the existing population is at risk. In
response to a likely need for a reintroduction program,
additional studies aimed at determining the population
genetic diversity and structure of the existing populations
and developing efficient asymbiotic germination procedures for reintroduction are underway. Although genetic
diversity studies are being conducted, manual pollination
should be conducted within populations to ensure that
seeds are being produced for in situ recruitment and ex
situ propagation for future reintroductions.
Pemberton and Liu (2008) suggested that restoration of
C. punctatum could be aided by planting Brysonima lucida,
a rare species in the Malpighiaceae that is restricted to
southern Florida, which attracts Centris bees. However, B.
lucida is not naturally found growing in the big cypress
region where natural populations of C. punctatum are
found (Wunderlin & Hansen 2004). We suggest that local
recovery plans for the species be developed given the
high habitat variation (e.g. between Everglades National
Park vs FPNWR) and fragmentation found in Florida. Recommendations should not be made solely from the relatively few plants in cultivation and should be based on
in-depth studies that will likely affect the survival of the
species in the wild.
Acknowledgments
We thank Hans Alborn (USDA) for his help with the
fragrance analysis, Jim Wiley (Florida Department of Agriculture and Consumer Services) for his help with insect
identification and the Florida Panther National Wildlife
Refuge staff for logistical support in the field. Special
thanks to Nancy Philman, Timothy Johnson, Scott Stewart
and Philip Kauth. This project was supported by both the
US Fish and Wildlife Service and the American Orchid
Society.
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