ABSTRACT
South Florida is home to a number of native species of orchids. The Florida Panther National Wildlife Refuge has 27 known species, including Prosthechea cochleata, the clamshell orchid, which is listed as endangered on Florida's Regulated Plant Index. In a prior study done on this species in Mexico, P. cochleata was found to produce no floral fragrance at the particular study location. However, blooming orchids of this species at the University of Florida in Gainesville, were noted to be fragrant. In this paper, we document the presence of floral fragrance compounds from P. cochleata by using by gas chromatography mass spectrometry (GC/MS) analysis of headspace volatile collection. The orchids sampled were found to be consistently producing eight volatiles that are common in floral fragrances, including those of previous orchid species studied. By knowing the fragrance compounds produced, we can better understand the pollination biology of this endangered orchid. This information could be used to help future conservation efforts for P. cochelata by increasing pollination and subsequent seed capsule production.
Introduction
The fragrance produced by flowers is one of the primary attractants of pollinators to a flower. Orchids, one of the largest families of flowering plants in the world, specifically have a diverse range of pollination systems, including approximately 10,000 species that use deception to attract pollinators.Citation1,Citation2 Many orchid species that are bee-pollinated use fragrance as a dominant attraction method, along with flower color and structure as reinforcement of the signal.Citation3 A flower's fragrance is often a combination of compounds, which can belong to numerous chemical classes.Citation4 Typically, pollination by insects requires floral fragrances for pollinator attraction.Citation5 Studying the fragrance produced by flowers is an important component in understanding plant-pollinator interactions.
Floral fragrance analysis of Dendrophylax lindenii, the ghost orchid, an epiphytic orchid found in south Florida, has shown that (E)-β-ocimene is one of the most abundant volatile organic compound produced by this orchid, as well as the presence of α-pinene.Citation6 Three D. lindenii flowers were sampled, each with similar results. A study of another orchid, Epidendrum ciliare, found high fragrance compound variation between plants of the same species.Citation7
The purpose of this study was to identify the fragrance compounds from the clamshell orchid, Prosthechea cochleata (). In the United States, this native orchid (listed as endangered on Florida's Regulated Plant Index) is found growing epiphytically in southern Florida. According to Cancino and Damon,Citation8 flowers of P. cochleata found in Mexico did not produce any volatiles when flowers were sampled with the protocol developed by Heath and Manukian.Citation9 Our Florida data collected from using the same sampling procedure indicated that P. cochleata does produce volatiles, including α-pinene, (E)-β-ocimene, benzaldehyde, mesitylene, and nonanal. A fragrance study of the orchid genus Gongora found most of these volatiles present in the genus, as well as being present in a study from the genus Cypripedium.Citation10,Citation11 Floral fragrance is an essential component of pollination, and lack of pollination limits flowering plant reproduction. Understanding threats to plant reproductive success is an important key to conservation.
Figure 1. Flower of Prosthechea cochleata, the clamshell orchid, growing at the Florida Panther National Wildlife Refuge (Photograph by Larry W. Richardson).
Results
Analysis of the fragrance by GC/MS indicated several compounds repeatedly appearing in the floral volatiles of P. cochleata (). The samples from all four flowers presented similar volatile analyses. All of the chemical compounds listed were compared to those on Pherobase and prior floral fragrance publications, with many found in floral fragrances from other plants in the Orchidaceae family.Citation12 The most abundant volatiles collected were Pseudocumene, Nonanal, and Mesitylene, respectively.
Table 1. A list of the top eight compounds detected in the floral fragrance samples of Prosthechea cochleata. Samples include three flowers from the University of Florida greenhouse (UF 1–3), the flower from a wild plant at the Florida Panther National Wildlife Refuge (FPNWR), and an air control.
Differences in fragrance samples from 8:00 AM, 4:00 PM, and 12:00 AM were noted. According to the GC/MS analysis, the abundance was lowest during the 12:00 AM collection period, while the 8:00 AM and 4:00 PM samples resulted in higher abundances. This trend was also noted by human olfaction in the laboratory at the same time periods. There were also slight differences in the amount of certain volatiles between the three plants, most noticeably in the Pseudocumene.
Discussion
The results of this study contrast those of Cancino and Damon,Citation8 by showing several floral fragrance compounds detected in P. cochleata. The difference in volatile production between these flowers could be due to temperature, humidity, or geographical differences causing a change in fragrance production. It is also possible that flowers of a different age were used in the previous study in Mexico. A recent study on the floral volatiles of a terrestrial orchid, Gymnadenia odoratissima, determined that the floral compounds of these flowers differed by location, being stronger in lowlands than in the mountains.Citation13 The geographic region in which the plant is growing could be influencing the volatiles produced, as G. odoratissima produces differing compounds based on the location of the plants.
The differences in volatile percentage over the duration of the study, such as with Pseudocumene, could be explained by the overall fragrance changing over time. In a study by Ackerman,Citation14 it was found that several species of euglossine bees, also known as orchid bees, exhibited a fragrance preference between varying seasons. The collections were done as the flowers from each plant opened, which took place over the course of 8 to 10 weeks. As for the lower fragrance emission at night (12:00 AM) compared to 8:00 AM and 4:00 PM, this suggests that a pollinator would be active during the day, and that the flower wouldn't be as attractive to pollinators active in the evening. Pollination requires the floral fragrance emissions to be synchronized with the activity of insect pollinators, so that successful pollination will be maximized.Citation5
An interesting aspect of the reproductive biology of P. cochleata is that this flower is thought to be autogamous, or self-pollinating.Citation15 It takes energy for a flower to produce a fragrance, which seems counterproductive for a flower species that is autogamous to be producing fragrance volatiles.Citation2 Additionally, the fragrance declined during the evening hours, suggesting that it would be most attractive to a day-flying pollinator. However, some flowers such as Arabidopsis thaliana produce floral rewards, while being self-compatible.Citation16,Citation17 It is possible that this results in low amounts of cross-pollination, and reduces the level of inbreeding.Citation16
In a review of floral scents published in 2006, there were an estimated 417 species in the Orchidaceae from which fragrance had been analyzed.Citation12 Although this was a significantly higher number than the next family (Araceae- 55), it is still a small percentage of orchid species worldwide, as there are nearly 30,000 known orchids.Citation1 The review by Knudsen et al.Citation12 also noted the top five fragrance compounds found in plant species (α-pinene, (E)-β-ocimene, limonene, linalool, and myrcene), the first three of which were identified in P. cochleata. It is important to understand the fragrance aspect of P. cochleata reproductive biology, as it could lead to better conservation efforts for this endangered orchid. By knowing the composition of the floral fragrance, it could allow for better methods of pollinator attraction such as augmenting fragrance by introducing a blend of the compounds found being produced by the flowers. An increase in the pollinators would result in more seed capsule production, and elevate the number of flowering plants in a location.
Materials and methods
Fragrance collection
Volatiles were collected using a procedure originally described by Heath and Manukian,Citation9 and modified by Cancino and DamonCitation18 and Sadler et al.Citation6 Using this method, samples could be collected repeatedly from the same flower over several days, without removing the flower from the plant. A Reynolds® oven bag was used to cover the flower, and sealed around the inflorescence to prevent ambient air from entering the bag during sampling (). The oven bags were used once, and then decontaminated by washing with 75% ethanol and baking in an oven at approximately 93ºC for 30 minutes. This prevented any residual volatiles from a sample being detected in a subsequent sample. For each sample, volatiles were collected for 10 minutes. Dry charcoal filtered air was pushed into one end the oven bag and exited the chamber via a vacuum system. The air then passed through a volatile collection filter containing 50 mg of Tenax® Porous Polymer Adsorbent (Sigma-Aldrich, USA).
Figure 2. Image showing the collection of floral volatiles from the greenhouse grown Prosthechea cochleata. Photograph by Haleigh Ray.
Four individual plants were sampled by collecting volatiles from one flower per plant. The sampled plants included three growing in pots in a greenhouse at the University of Florida Entomology and Nematology Department (UF) in Alachua County, Florida, and one wild orchid growing in situ at the Florida Panther National Wildlife Refuge (FPNWR) in Collier County, Florida. The orchid in the FPNWR is found growing epiphytically, approximately 1.75 m above the ground. The collection equipment was elevated to accommodate this height. The beginning of flower sampling started on the first day that a new bloom was open, which was deemed Day 0.
The flowers from the UF greenhouse were sampled on days 0, 5, 10, and 15. Flowers were sampled twice, at 8:00 AM and 4:00 PM, on days 0 and 10; and three times, at 8:00 AM, 4:00 PM, and the following 12:00 AM EST, on days 5 and 15. Volatile collection occurred within ±15 minutes of the scheduled time. Sampling the flowers at different time points allowed us to detect any fluctuation in fragrance strength throughout the day. This resulted in 10 total floral samples for each of the three UF flowers.
The in situ P. cochleata orchid at FPNWR was sampled on two different days. The orchid selected in FPNWR was the P. cochleata that was determined to have opened the most recently. The sampling schedule on day 0 and day 5 was the same as sample collection from greenhouse flowers at UF, for a total of five floral samples collected. This method yielded 35 total floral samples collected from all of the flowers ().
Table 2. Sample schedule for each of three Prosthechea cochleata flowers from different plants at the Entomology and Nematology Department (UF), and the single flower at the Florida Panther National Wildlife Refuge (FPNWR). Additional samples for three controls were collected, the plant, substrate, and the air control (Control- Air*a, Control- Plant *p, Control- Substrate*s).
Three control samples (plant, substrate, air) were collected. One by sampling the plant excluding a flower (leaves, stems, pseudobulbs), a second by sampling either the plastic container and growing medium used for the greenhouse plants or part of the tree for the epiphytic orchid in FPNWR, and a third by sampling the ambient air in the place of collection. Peak volatiles collected from control samples were compared with the peak volatiles from the floral samples to identify and remove any peaks that do not originate from the flower. Ambient air control samples were collected at each sampling date, and plant and substrate samples were collected at 8:00 AM EST on the second sampling day for each plant. This resulted in six control samples for each UF plant, and four control samples for the FPNWR plant, for a total of 22 control samples ().
Identification of floral odor
After the floral and control samples were collected, they were labeled and sealed using Teflon thread seal tape and stored in a freezer (-80 ºC) until they were analyzed. The volatile compounds collected were analyzed at the United States Department of Agriculture (USDA) in Gainesville, Florida by gas chromatography mass spectrometry (GC/MS) [GC: Agilent 6890 with an HP-5MS capillary column of 30 m long, 0.25 mm inner diameter, and 0.25-µm film thickness; MS: Agilent 5973 mass selective detector, 70 eV, equipped with a thermal desorption cold trap injector (TCT) (CP4010; Chrompack, Bergen op Zoom, The Netherlands)]. Headspace volatiles collected on Tenax® TA were released from the adsorbent by heating in the TCT at 220°C for 8 minutes within a flow of helium gas. The desorbed compounds were collected in the TCT cold trap unit (SIL5CB-coated fused silica capillary) at –130°C. Flash heating of the cold trap unit injected the compounds into the capillary column of the gas chromatograph to which the cold trap unit was connected. The oven temperature of the GC was programmed to rise from 40°C (5-min hold) to 280°C at 15°C/min. The headspace volatiles were identified by comparing their mass spectra to those of the database (Wiley7N and Wiley275) and by comparing their retention times to those of authentic compounds. Volatiles were identified by comparison of mass spectra (a) with mass spectra libraries (NIST and Department of Chemical Ecology, Göteborg University, Sweden) and (b) with mass spectra and retention times of authentic standards. After the results were obtained, the volatile peaks were compared to each other to locate the common peaks in the flower samples. These volatiles were then referenced on Pherobase, an online database of pheromones and semiochemicals.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
We would like to thank the United States Fish and Wildlife Service (USFWS) for access to orchids from the Florida Panther National Wildlife Refuge to complete this project.
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