SOWING SEEDS OF EPIPHYTES IN SITU. - Because of their minute size (1-10 mm), protocorms are very difficult to locate and recover in situ.

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1 Protocorms of an epiphytic orchid (Epidendrum amphistomum A. Richard) recovered in situ, and subsequent identification of associated mycorrhizal fungi using molecular markers. Laura L. Corey 1, Lawrence W. Zettler 1,*, Larry W. Richardson 2, April Y. Ross 1, and Lillian Moller-Jacobs 1 1 Orchid Recovery Program, Jacksonville, IL USA 2 Florida Panther National Wildlife Refuge, U.S. Fish & Wildlife Service, Naples, FL USA SOWING SEEDS OF EPIPHYTES IN SITU - Because of their minute size (1-10 mm), protocorms are very difficult to locate and recover in situ. - Based on work with terrestrials - Rasmussen and Whigham (1993) dust-like seeds Seeds housed in nylon mesh w/in 35 mm slide mounts 1

2 RECOVERING PROTOCORMS Seed packets retrieved > 6 months may harbor trapped protocorms and mycorrhizal fungi can be subsequently isolated and identified. To our knowledge, this technique has yet to be successfully applied to epiphytic orchids (arboreal substrates). OBJECTIVES 1) Devise a modified technique that allows for the recovery of epiphytic orchid protocorms in situ from arboreal substrates (tree limbs). 2) Identify the mycorrhizal fungi associated with protocorms captured in this manner using molecular markers. 2

3 In situ seed sowing Seed from dehiscing capsules of E. amphistomum and E. nocturnum placed directly into seed packets (ca ,000 seeds/packet) on 30 May Packets affixed to tree bark on 18 different sites within McBride s Pond. Each site was separated by at least 5 m, and on separate host tree. Two host tree species were selected: Pop Ash Fraxinus caroliniana Mill. Red Maple Acer rubrum L. Packets placed on 3 substrates: Exposed Bark (B), Moss (M), lichen (L). Packets were secured using standard gutter mesh and staple gun. 3

4 Seed viability and germination assessment Seed viability confirmed with asymbioitic germination. Seed packets remained in situ for 267 days. Seed packets were detached from trees, returned to the laboratory, and protocorms immediately photographed. 1 mm Recovery of E. amphistomum protocorms 1/18 packets of E. amphistomum contained protocorms 1 mm Nine protocorms from one seed packet after 267 days incubation in situ. Packet was previously affixed to a moss substrate on Pop Ash. Protocorms were green and 3 of 9 forming a leaf. 4

5 Identification of mycorrhizal fungi Random Amplified Polymorphic DNA (RAPD) UBC primer set #1 (10-mers) Randomly amplify Ceratorhiza is present in E. amphistomum protocorms Asym. in situ 5

6 Ceratorhiza is present in E. amphistomum protocorms Controls in situ ITS primers as modified by D. Lee Taylor ITS1 ITS2 18S 5.8S 28 S Primer ITSOF1 Primer ITSOF4 Conclusions from the pilot study First report documenting the capture of epiphytic orchid protocorms in situ using seed packets. Gutter mesh / staple gun appears to have merit. Support for the role of moss in epiphyte germination 1 mm Protocorms of Epidendrum amphistomum captured in situ were associated with Ceratorhiza 6

7 Challenges from the pilot study 1/18 packets of E. amphistomum contained protocorms 0 packets of E. nocturum contained protocorms Insufficient DNA sample for sequencing Ceratorhiza cannot be cultured from substrate Can we use Ceratorhiza to promote germination in situ? Repeat in situ germination Culture peletons recovered from new protocorms DNA isolation and ITS sequencing Bait new seed packets with cultured Ceratorhiza Culture peletons recovered from new protocorms 7

8 ACKNOWLEDGMENTS James (JJ) Sadler Jaclyn Smith Haleigh Ray John McCormick Alishia Jacks University of Florida Dr. Scott L. Stewart Andrew Stice Kankakee Community College Student-Faculty Research Committee 8