Ecology, Propagation and Conservation of Eulophia alta L., a Threatened Florida OrchidScott Stewart1, Tim Johnson1, Daniela Dutra1, Michael Kane1, Larry Richardson2

1University of Florida, Environmental Horticulture Department, PO Box 110675, Gainesville, FL 32611. E-mail: sstewart@ifas.ufl.edu2U.S. Fish and Wildlife Service, Florida Panther National Wildlife Refuge, 3860 Tollgate Boulevard, Suite 300, Naples, FL 34114.

Introduction

Nearly 250 species of orchids are native to the United States, and approximately 120 of those species are known from Florida. Orchids throughout Florida face many conservation obstacles such as habitat conversion to homesites or agricultural lands, habitat mismanagement, and illegal poaching for commercial trade. While no Florida orchid is federally-listed as endangered or threatened, many of the state’s orchid species face the immediate possibility of extinction if conservation and recovery plans are not developed and instituted. The decline of Florida native orchids may also negatively impact populations and distributions of the moth, bee, and butterfly pollinators common among these plants. This research is designed to study the current ecological status and propagation science of the Florida native terrestrial orchid Eulophia alta. At the current time, no information exists concerning the propagation, ecology, or conservation of this species.

Eulophia alta (wild coco; Fig. 1) is a terrestrial orchid that is considered locally common throughout central and southern Florida. The species is commercially-exploitable due to its tall, colorful, and many-flowered raceme that can bear up to 30 individual flowers in colors ranging from pale green and white (forma pallida), green and white (forma pelchatii), deep brick red, to pale rose (Brown, 2005).

Materials and Methods

StatusSurveys will be conducted to determine the ecological status of E. alta on the Florida Panther National Wildlife Refuge (Fig. 1; Fig. 3, a-b). These surveys will classify particular habitats where E. alta is known to historically occur on the FPNWR, as well as define new habitats where the species is presently found. Data concerning numbers of individual plants, flowering versus vegetative individuals, and reproductive status (i.e. seed set) will be recorded.

Pollination BiologyStudies to determine the breeding system of E. alta will be conducted following the methods outlined by Wong and Sun (1999) and Zettler et al. (1996). Briefly, E. alta will be tested for six mechanisms of pollination: 1) spontaneous autogamy, 2) induced autogamy, 3) artificial geitonogamy, 4) artificial xenogamy, 5) agamospermy, and 6) natural pollination. Data will be taken on seed capsule set, capsule fresh weight, average number of seeds per capsule, and seed viability in each pollination mechanism condition. These data will be analyzed using general linear model procedures and mean separation measures at α=0.05 (SAS, 1999).

Mycobiont Isolation and IdentificationThe isolation and identification of these root-inhabiting endomycorrhizal fungi will follow the methods outlined by Zettler (1997) and Stewart and Zettler (2002).

Seed GerminationTwo seed sources will be used to study the asymbiotic and symbiotic seed germination of E. alta. The first seed source (S73) originated from private land near Avon Park, Florida (Highlands County) and was collected on 15 March 2003. The second seed source (S128) originated from the Florida Panther National Wildlife Refuge (Collier County) and was collected on 13 December 2005.

Seed Germination—AsymbioticFive asymbiotic orchid seed germination media will be tested: 1) Knudson C, 2) P723, 3) Malmgren Modified Terrestrial Orchid Medium, 4) Vacin & Went Modified Orchid Medium, and 5) ½-Strength Murashige & Skoog (Fig. 3, c-f). Eight replications per media will be used. Data will be analyzed using general linear model procedures, least square means, and Waller-Duncan at α=0.05 (SAS, 1999). Seed germination counts will be arcsine transformed to normalize variation.

Seed Germination—SymbioticTwelve fungal mycobionts (Figure 2; Table 1) will be tested for their ability to support the germination of E. alta. Germination methods will follow those described by Stewart and Zettler (2002), modified by the use of oat meal agar (OMA): 3.0 g pulverized whole oats, 7.0 bacto-agar, 100 mg yeast extract, 1 L purified water (Fig. 3, c-f). Eight replications per mycobiont will be used. Data will be analyzed as previously outlined.

Objectives

3) To study the in situ growth and development, pollination biology, and mycobiont diversity of E. alta populations on the Florida Panther National Wildlife Refuge.

4) To collect and store mature seed of E. alta from populations throughout southern Florida.

5) To develop asymbiotic and symbiotic seed germination protocols for E. alta.

a b c

d

Florida Panther National Wildlife Refuge

Avon Park

Gainesville

Figure 1—a) E. alta flowers; b) E. alta leaves and typical habitat; c) E. alta rhizome and root system; d) E. alta collection and research sites

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d

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Figure 3—a) E. alta habitat on the FPNWR, wet prairie; b) E. alta habitat on the FPNWR, wet prairie boarded by cypress dome swamp; c) laminar flow hood at Gainesville laboratory; d) typical tools used in both asymbiotic and symbiotic orchid seed germination; e) sowing orchid seeds using inoculating loop; f) inoculating seed germination vessel with appropriate fungal mycobiont

Results

StatusSurveys throughout the FPNWR based on previous observations (L. Richardson, pers. comm.) have verified historic locations and yielded numerous new sites for E. alta. The species is now considered locally abundant on the FPNWR, with the highest concentrations of plants being along tramways and roadsides (Stewart, 2006). These sites are characterized by historic and/or current disturbance, which may indicate the ability of E. alta to quickly colonize and persist within disturbed sites. The species is also considered locally common throughout the many mesic pine flatwoods on the FPNWR (Stewart, 2006). Eulophia alta is considered common to locally abundant throughout its south Florida range, and many large groups of plants can easily be found on private and public lands bordering the FPNWR. Both the pale-colored (forma pallida) and white-green (forma pelchatii) color forms can be found scattered throughout the FPNWR.

Pollination BiologyBoth artificial geitonogamy and induced autogamy were found as possible pollination mechanisms for E. alta based on a preliminary study of greenhouse grown south Florida plants. All other pollination mechanisms tested failed to produce a seed capsule. Open pollination and artificial xenogamy were not tested in this preliminary study because all greenhouse plants were collected from one population on the FPNWR. Field testing of pollination mechanisms was not possible in 2005 because peak E. alta flowering coincided with the landfall of hurricane Wilma in southwestern Florida. Post hurricane inspection of pollination field sites revealed that the majority of flowers had been stripped from study plants by the high winds.

Mycobiont Isolation and IdentificationEleven distinct fungal mycobionts were isolated from the roots of south and central Florida E. alta plants (Fig. 2). Eight isolates originated from root samples collected on the FPNWR (Collier County), while three isolates originated from root samples collected on private land near Avon Park (Highlands County; Table 1). Each isolate is currently cataloged and stored in a collection located at the Plant Restoration, Conservation, and Biotechnology Program (Environmental Horticulture Department, Gainesville), and will be fully characterized and identified. All eleven isolates, plus an additional outgroup isolate, are currently being surveyed in symbiotic seed germination trials.

Seed Germination—Asymbiotic & SymbioticThe asymbiotic and symbiotic seed germination requirements of E. alta are currently being explored. Previous symbiotic germination attempts were unsuccessful, most likely due to the use of fungal mycobionts not originating from E. alta (S. Stewart, unpub. data). No previous studies have examined the asymbiotic seed germination requirements of this species.

Table 1—Eulophia alta fungal mycobionts (Ealt-XXX) and Spiranthes brevilabris (Sbrev-XXX) used in symbiotic seed germination trials.

Accession Number

Collection Location

Collection & Isolation Date

Tentative Identification

Ealt-385 Collier Co. 29 July 2005 UnknownEalt-386 Collier Co. 29 July 2005 Sclerotinia spp.Ealt-387 Collier Co. 29 July 2005 Epulorhiza spp.Ealt-388 Collier Co. 29 July 2005 UnknownEalt-389 Collier Co. 29 July 2005 UnknownEalt-390 Collier Co. 29 July 2005 Epulorhiza spp.Ealt-391 Collier Co. 29 July 2005 UnknownEalt-392 Collier Co. 29 July 2005 UnknownEalt-395 Highlands Co. 16 August 2005 UnknownEalt-396 Highlands Co. 16 August 2005 Ceratorhiza spp.Ealt-397 Highlands Co. 16 August 2005 Epulorhiza spp.

Sbrev-266 Levy Co. 30 April 1999 Epulorhiza spp.

Summary

Eulophia alta, wild coco, is considered locally abundant to common throughout southwestern Florida; however, its showy flowers make the species commercially-exploitable and subject to poaching. Habitat mismanagement and land development also threaten E. alta throughout Florida. In order to preserve E. alta as an independent organism in its natural habitat an integrated conservation plan must be developed (Fig. 4). Understanding the current ecological status, pollination biology, and seed germination requirements of the species will greatly aid in developing an integrated conservation that can be immediately enacted. Using populations of E. alta at the FPNWR study units, preliminary testing has shown the species to persist in both slightly disturbed and natural habitats and possess an induced autogamic and/or artificial geitongamic pollination mechanism. Currently, studies are underway to assess the asymbiotic and symbiotic seed germination requirements of the species. Given the conservation and commercial potential of E. alta, the development of an integrated conservation plan for the species remains a high priority.

Figure 4—Generalized integrated conservation plan for Eulophia alta in Florida.

Literature CitedBrown PM (2005) Wild orchids of Florida. University Press of Florida, Florida.

SAS Institute Inc. (1999) SAS Version 8.02. SAS Institute, North Carolina.

Stewart SL (2006) Orchid flora of the Florida Panther National Wildlife Refuge. U.S. Fish and Wildlife Service, Florida.

Stewart SL & Zettler LW (2002) Symbiotic germination of three semi-aquatic rein orchids (Habenaria repens, H. quinqueseta, H. macroceratitis) from Florida. Aquatic Botany 72:25-35.

Wong KC & Sun M (1999) Reproductive biology and conservation genetics of Goodyera procera (Orchidaceae) American Journal of Botany 86:1406-1413.

Zettler LW (1997) Terrestrial orchid conservation by symbiotic seed germination: techniques and perspectives. Selbyana 18:188-194.

Zettler LW, Ahuja NS & McInnis TM (1996) Insect pollination of the endangered monkey-face orchid (Platanthera integrilabia) in McMinn County, Tennessee—one last glimpse of a once common spectacle. Castanea 61:14-24.

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Figure 2—Fungal mycobionts isolated from Eulophia alta (a-k) and mycobiont outgroup from Spiranthes brevilabris (l).