Mycorrhiza (2012) 22:393–402 DOI 10.1007/s00572-011-0414-y ORIGINAL PAPER Is rarity of pinedrops (Pterospora andromedea) in eastern North America linked to rarity of its unique fungal symbiont? Christina Hazard & Erik A. Lilleskov & Thomas R. Horton Received: 8 July 2011 /Accepted: 27 September 2011 /Published online: 12 October 2011 # Springer-Verlag 2011 Abstract Like other myco-heterotrophic plants, Ptero- Keywords Myco-heterotrophy. Pterospora andromedea spora andromedea (pinedrops) is dependent upon its (pinedrops) . Rhizopogon . Fungal specificity. Rarity . specific fungal symbionts for survival. The rarity of Distribution pinedrops fungal symbiont was investigated in the eastern United States where pinedrops are rare. Wild populations of eastern pinedrops were sampled, and the plant haplotypes Introduction and fungal symbionts were characterized with molecular techniques; these data were compared to those from the Mycorrhizal symbioses are often generalized as mutually West with phylogenetic analyses. The frequency of the beneficial for the plant and fungal symbionts, but this is fungal symbiont in eastern white pine forests was assessed not always correct in that mycorrhizal symbioses can using a laboratory soil bioassay and in situ pinedrops seed fall along a continuum from mutualism to parasitism baiting. Only one plant haplotype and fungal symbiont was (Johnson et al. 1997; Jones and Smith 2004). An extreme detected. The plant haplotype was not unique to the East. example in this mutualism-parasitism continuum is the The fungal symbiont appears to be a new species within the parasitic mycorrhizal relationship between many non- genus Rhizopogon, closely related to the western sym- photosynthetic plants and their fungal symbionts. These bionts. This fungal species was not frequent in soils with or non-photosynthetic or myco-heterotrophic plants (Leake without pinedrops, but was less frequent in the latter and in 1994), develop a unique relationship with their mycorrhizal comparison to the fungal symbionts in western forests. fungi to acquire fixed carbon from photosynthetic hosts Seed baiting resulted in few germinants, suggesting that via a shared mycorrhizal fungus (Björkman 1960; mycelial networks produced by the eastern fungal symbiont McKendrick et al. 2000). were rare. Results suggest that eastern pinedrops rarity is The myco-heterotrophic strategy evolved independently influenced by the distribution and rarity of its fungal and repeatedly, resulting in over 400 achlorophyllous myco- symbiont. heterotrophic species (Leake 1994; Merckx and Freudenstein 2010). Several researchers have used molecular techniques Electronic supplementary material The online version of this article to demonstrate the associations of many myco-heterotrophic (doi:10.1007/s00572-011-0414-y) contains supplementary material, plants with a narrow range of closely related species of which is available to authorized users. : fungi, even over large geographical distances (Cullings et al. C. Hazard (*) T. R. Horton 1996; Taylor and Bruns 1999a; Kretzer et al. 2000; State University of New York-Environmental Bidartondo and Bruns 2001, 2002; Taylor et al. 2002; Science and Forestry, 1 Forestry Drive, Yagame et al. 2008; Bougoure et al. 2009; Barrett et al. Syracuse, NY 13210, USA 2010). The specificity of myco-heterotrophic plants for their e-mail: [email protected] fungal host follows patterns of specificity seen with other parasite-host systems, such as plant rusts and other plant E. A. Lilleskov USDA Forest Service, Northern Research Station, pathogens (Thompson 1994), and contrasts with the majority Houghton 49931, USA of autotrophic plants which can associate with a diverse 394 Mycorrhiza (2012) 22:393–402 range of mycorrhizal fungal species (Trappe 1977;Molinaet The research on pinedrops and its fungal symbionts al. 1992, and see Bruns et al. 2002a). has been focused in the western United States where The myco-heterotrophic plant pinedrops (Pterospora the plant and its fungal symbionts are common (Cullings et al. andromedea Nutt.; Ericaceae, Monotropoideae) forms 1996; Bruns and Read 2000; Bidartondo and Bruns typical monotropoid mycorrhizae with a narrow range 2001, 2002, 2005; Kjøller and Bruns 2003). To conserve of basidiomycete ectomycorrhizal fungi in the genus and manage for eastern pinedrops a better understanding Rhizopogon subgenus Amylopogon (Cullings et al. 1996; of this plant and its fungal symbionts are needed. Here we Bidartondo and Bruns 2001). Fungi in the genus Rhizopogon use molecular techniques to identify eastern pinedrops are disturbance tolerant, mammal dispersed, and form truffle- haplotypes and fungal symbionts from plant and fungal like hypogeous fungi that are otherwise almost exclusively tissue collected from pinedrops in the field. These data associated with Pinaceae (Molina et al. 1992;Molinaand are compared to those from the West with phylogenetic Trappe 1994). Five plastid DNA haplotypes of pinedrops analyses. We further investigate the fungal symbionts have been identified and these haplotypes show preference with regards to their frequency in eastern forests using for either R. salebrosus or R. arctostaphyli, even when they soil bioassay and in situ seed sowing methods. We co-occur (Bidartondo and Bruns 2002). In the Sierra predicted that the fungal symbionts of eastern pinedrops National Forest where pinedrops is common, these fungal are less frequent than their counterparts in western symbionts are common and found frequently in the soil forests. spore bank (Kjøller and Bruns 2003). Soil spore banks are analogous to soil seed banks, referring to resistant spores that are found in soils awaiting germination cues following Materials and methods disturbance (Baar et al. 1999). Like other myco-heterotrophic plants, pinedrops produce Field sites and experimental plots large quantities of wind dispersed dust-like seeds that contain reduced endosperms (Leake 1994; McKendrick et Field sites were located within mixed deciduous al. 2000). An in vitro germination study showed that conifer forests that were dominated by Pinus strobus. pinedrops seeds would only germinate when stimulated by Two types of sites were used: (1) sites with an active a diffusible chemical produced by its fungal symbiont, or a population of pinedrops (i.e. inflorescences observed closely related species (Bruns and Read 2000). An in situ within the last 3 years) and (2) sites in which pinedrops seed germination study showed further fungal specificity have never been reported; hereafter referred to as requirements in that only the pinedrops seeds associated pinedrops (PD) and non-pinedrops (NPD) sites, respec- with the mycorrhizal fungi of their maternal seed source (i.e. tively. Due to the rarity of pinedrops in the eastern United R. arctostaphyli or R. salebrosus) could develop into States only a limited number of study sites were mycorrhizal seedlings (Bidartondo and Bruns 2005). These available. Sites were established in New York and methods for sowing seeds that bait for the respective fungal Michigan with three PD and NPD sites in each state. symbiont can also be used for assessing symbionts’ presence The New York sites were all located in the only known and distribution. The biology of pinedrops clearly shows that location with active population in the state, in Letchworth the fungal symbiont plays a crucial role in survival, State Park (77° 55′N by 42° 40′W), Livingston county recruitment and distribution of this plant species. (Livingston-1, -2 and -3). The Michigan sites were located Pinedrops is endemic to North America where it is found in Emmet (84° 15′Nby45°40′W), Mecosta (85° 30′Nby43° in mixed conifer/deciduous forests. The species is limited to 35′W), and Keweenaw (88° 15′Nby47°20′W), each with one either western or eastern forests, with no occurrences in the PD and NPD site. In New York, the PD sites were all middle of the continent. Western occurrences extend separated by ∼1 km and in Michigan by at least 249 km. In throughout the Sierra Nevada and Rocky Mountain ranges both states, NPD sites were located between 1 and 1.6 km from Canada to Mexico, and eastern occurrences extend from a NPD site. throughout the Great Lake states across Canada to New Each site was defined by a 30×100 m sampling area; England and New York (Bakshi 1959). Although western from which five randomly selected 5×5 m plots were pinedrops is common, the species appears to be relatively established, hereafter referred to as NPD plots. In addition, rare and in decline in the East (Schori 2002). Specifically, four and five 5×5 m plots containing flowering pinedrops in Ontario Canada pinedrops are now recorded in only four were established at the Emmet and Keweenaw PD sites, counties. Only two towns in Vermont now hold one respectively. At the Livingston-1 PD site, remnant inflor- occurrence each. Only three locations in New York have escences from the previous year were used to establish recent records of the plants, all of which are in the same three additional 5×5 m plots. Plots containing flowering or county. remnant inflorescences of pinedrops are hereafter referred Mycorrhiza (2012) 22:393–402 395 to as PD plots. PD plots were not established at the Field soil bioassay with pine Livingston-2 and -3 PD site or the Mecosta PD site as flowering or remnant inflorescences of pinedrops were From each soil core, two pots (Ray Leach Tubes - 3.8 cm absent during the time frame of this study. Pinedrops do not diameter by 21 cm deep, Stuewe & Sons, Inc., Corvallis, necessarily flower every year, remaining underground and OR, USA) were prepared, containing either 0.5% or 50% undetected even though they are present at a site (Schori field soil, for a total of 144 pots (72 soil cores×2 dilutions= 2002). See Fig. 1 for a schematic of the sampling design 144 pots). The field soils were diluted with extra field soil and Table 1 for a summary of the number and types of from their respective site that was pooled, mixed 1:1 with study sites and plots used for the field soil bioassay with sand, and then sterilized (autoclaved twice).