Seed Germination of Habenaria repens (Orchidaceae) in situ Beyond its Range, and its Potential for Assisted Migration Imposed by Climate Change Author(s) :Brian G. Keel, Lawrence W. Zettler, and Beth A. Kaplin Source: Castanea, 76(1):43-54. 2011. Published By: Southern Appalachian Botanical Society DOI: http://dx.doi.org/10.2179/09-054.1 URL: http://www.bioone.org/doi/full/10.2179/09-054.1 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. CASTANEA 76(1): 43–54. MARCH 2011 Seed Germination of Habenaria repens (Orchidaceae) in situ Beyond its Range, and its Potential for Assisted Migration Imposed by Climate Change Brian G. Keel,1* Lawrence W. Zettler,2 and Beth A. Kaplin1 1Antioch University New England, 40 Avon St., Keene, New Hampshire 03431-3516 2Orchid Recovery Program, Illinois College, 1101 West College Avenue, Jacksonville, Illinois 62650 ABSTRACT All orchids require free-living, mycorrhizal fungi to complete their life cycles in nature and consequently, orchid conservation must take into account both organisms. In light of climate change now underway, orchids and other plants must be capable of migrating to higher latitudes, either on their own or with human intervention (5assisted migration). In this paper, we describe the symbiotic germination of a common terrestrial orchid, Habenaria repens, in situ using seeds from a southern ecotype (Florida) placed at latitudes at and above the species’ current natural range. To recover fungi in situ, 500 nylon packets containing 25,000– 50,000 seeds were buried at 5 field sites within the Atlantic coastal plain in North Carolina, Maryland, and Virginia. After ca. 5 months, a total of 10 leafless seedlings (protocorms) were recovered from two North Carolina sites, one harboring an extant H. repens population, the other an extirpated population. These protocorms yielded mycorrhizal fungi assignable to the anamorphic genus Epulorhiza. The physiological significance of each isolate was confirmed after H. repens seeds germinated in vitro following fungal inoculation (5symbiotic germina- tion). This study demonstrates that seeds of H. repens from a southern ecotype (Florida) are indeed capable of germinating at higher latitudes where fungi already persist. Consequently, the mycotrophic demands of orchids like H. repens might be met by assisted migration involving seed release alone. INTRODUCTION In nature, orchids re- contact the orchid seed and infect the embryo quire free-living, mycorrhizal fungi to prompt forming intracellular coils (pelotons, Peterson seed germination and seedling development et al. 2004). These pelotons are subsequently to circumvent the lack of nutrient reserves digested by the embryo in a controlled (endosperm) in their minute, dust-like seeds manner, releasing water, carbohydrates, vi- (Clements 1988). After release from a capsule, tamins, minerals and hormones, and prompt- copious numbers of orchid seeds become ing development. During the course of the airborne and may be carried considerable orchid’s life, fungi may be digested off and on distances on wind currents (Rasmussen 1995) into adulthood (Rasmussen 2002), supple- until they eventually become affixed to a menting (or replacing) photosynthesis (Had- substrate (e.g., tree limb, leaf litter) harboring ley and Pegg 1989, Rasmussen 1995, Zettler a diverse mycoflora. Hyphae from certain 2005). Moreover, different taxonomic groups groups of fungi, especially basidiomycetes of of fungi may be utilized along the way that Rhizoctonia sensu lato (e.g., Ceratorhiza, Epu- are different from those that initially prompt- lorhiza; Zelmer and Currah 1995), physically ed seedling development (5fungal succession, Dijk et al. 1997, Zelmer et al. 1996). This *email address: [email protected] process of ‘‘feeding’’ on fungi (mycotrophy) is Received October 19, 2009; Accepted June 11, 2010. apparently unique to the Orchidaceae and 43 44 CASTANEA VOL.76 may even have contributed to the family’s spawn seedlings themselves due to the absence evolutionary success (Rasmussen 1995, 2002; of suitable mycorrhizal fungi in the immediate Taylor et al. 2003). However, the reliance on area (Rasmussen 1995). In this paper, we mycotrophy renders the Orchidaceae espe- describe the symbiotic germination of H. repens cially vulnerable, compared to other angio- in situ using seeds from Florida placed at sperms, because without fungi orchids could latitudes at the northern boundary and be- not complete their life cycles in situ. In light of yond the species’ natural range (North Car- climate change now underway, orchid con- olina, Maryland, Virginia). In addition, we servation must take into account orchid food provide a description of the mycorrhizal fungi (5fungi) as well as the orchid. capable of prompting seedling development in Habenaria repens Nuttall, is a semi-aquatic these northern latitudes. These two studies terrestrial orchid native to the West Indies, were undertaken to understand what factors Central America, Mexico, and coastal plain need consideration in planning for the assisted of the southeastern United States (Williams migration of orchids in general and H. repens and Williams 1983, Brown 2002). The species in particular. is apparently ephemeral and may utilize a broad range of fungi (5‘‘generalist strategy,’’ METHODS Rasmussen and Rasmussen 2007) based on in Seed Collection, Storage and In Situ Sowing vitro germination studies (Keel 2007, Stewart Seed collection and storage followed the and Zettler 2002). Photoperiod experiments techniques outlined by Zettler (1997). Seeds conducted in conjunction with in vitro symbi- were obtained from yellowing, mature cap- otic seed germination (Keel 2007) suggest this sules just prior to dehiscence from four species has the potential to migrate farther sources, all originally obtained from Florida north, taking advantage of longer day (Figure 1). Immediately after collection, cap- lengths. Thus, the current northern limit of sules were placed over Drierite (CaSO4) desic- its range (east central North Carolina) may be cant at ca. 22uC up to 91 days. Seeds were restricted by cooler temperatures and/or the removed from capsules assisted by a sterile absence of suitable fungi needed for germi- scalpel, added to sterile vials, and promptly nation, not by limits imposed by photoperiod. stored at 27to210uC in darkness (L:D 0:24 If cooler temperatures primarily limit the hrs) until use. northern extent of H. repens, and not mycor- To obtain mycorrhizal fungi in situ, seeds rhizal fungi, it is conceivable that this terres- served as ‘‘baits’’ (Masuhara and Kutsuya trial orchid may be capable of migrating 1994, Rasmussen and Whigham 1993, van farther north given climate change. Under the der Kinderen 1995, Zelmer et al. 1996, Zettler worst-case climate change scenario (for this et al. 2005). Briefly, ca. 50–100 seeds were study we chose to focus on climate change placed in retrievable nylon mesh packets driven latitudinal migration and not address (95 mm pore size, #FR65-2222M Carolina other issues such as sea-level rise) projected Biological Supply Co., Burlington, North for the next 50–100 years (5.8uC increase), Carolina) which were then folded and insert- plants will need to expand their northern ed into plastic 35 mm slide mounts. Staples range by ca. 580 km (Hansen et al. 2001, were used to secure the packets within the Davis 1989). For H. repens, the northern limit frame. A total of 500 packets were construct- would extend into southern Pennsylvania ed, and each packet was selected at random (Keel 2007). As a species with lightweight, for eventual placement in situ. A small hole wind-dispersed seeds, H. repens might be capa- was drilled through one or two corners of each ble of extending its northern range naturally slide mount and the packets were tied with or via assisted migration (5intentional intro- 5 kg test nylon fishing line. Packets were tied duction by humans beyond its natural range, in two rows of 5 packets with 15 cm between Keel 2005). The latter is a distinct possibility packets within the rows and 30 cm between given the ease by which this species can be the rows. Packets were selected at random for artificially cultivated (Stewart and Zettler the groups of 10. Each group of 10 packets 2002). This goal would be meaningless, how- was placed in a plastic sandwich bag, and 10 ever, if introduced seedlings were unable to bags of packets were selected at random for 2011 KEEL ET AL.: HABENARIA REPENS AND ASSISTED MIGRATION 45 Figure 1. Map of SE United States showing the current range of Habenaria repens, the original origin of seeds utilized in this study, and the 5 sites sampled for the presence of mycorrhizal fungi. Only one of the 5 sites (WF Site, Cumberland Co., North Carolina) harbored an existing population of H. repens. Dark line is boundary of coastal plain. each of the research areas. At each field site completely covered by soil. The knife was two rows of packets were sown parallel to the wiped clean and surface-disinfected with 70% shore line with the first row ca. 2.5 cm from EtOH between site visits. Packets remained the water’s edge (Figure 2). The 5 field sites underground for ca. 5 months. were located within the Atlantic coastal plain of North Carolina, Maryland, and Virginia Fungal Isolation and Identification (Figure 1 and Table 1).