Page  129 ï~~ 2011 THE MICHIGAN BOTANIST 129 THE MYCORRHIZAL SYSTEM OF PTEROSPORA ANDROMEDEA (PINE-DROPS) IN WEST MICHIGAN INFERRED FROM DNA SEQUENCE DATA Jianhua Li*, Jeffrey Corajod, Holly Vander Stel, and Austin Homkes Department of Biology Hope College 35 E 12th St., Schaap Science Center, Holland, MI 49423 ABSTRACT Pterospora andromedea is a mycoheterotrophic plant with a disjunct distribution between western and eastern North America and obtains carbon and nutrients indirectly from photosynthetic plants via an ectomycorrhizal fungal bridge. In this study, we used DNA sequence data to determine the organisms involved in the system in West Michigan. Our results suggest that at least two photosynthetic plants (Tsuga and Acer) are the potential carbon source of the system and that Pterospora is specifically associated with an unidentified species of subgenus Amylopogon of Rhizopogon. Previous studies have shown that seed germination of Pterospora relies on chemical cues from the fungus, implying a dominant role of the fungus in the system. Our field observations suggest that repeated branching of Pterospora roots increases the mass production of the fungal mycelia and lead us to speculate that Pterospora may be a mutualistic partner, not a parasite or exploiter, in the mycorrhizal system. KEYWORDS: Pterospora, nrDNA ITS, rbcL, mutualism, mycorrhizal, subgenus Amylopogon, Rhizopogon. Pterospora andromedea Torr. (pine-drops) is a mycoheterotrophic plant relying on fungal host for germination, growth, and development (Bakshi 1959). Molecular studies have shown its close relationship with other mycoheterotrophic plants in Ericaceae such as Monotropa, Allotropa, and Sarcodes (Cullings 1994; Kron et al. 2002). Pine-drops show a disjunct distribution between the eastern and western North America with an extension in the west to northern Mexico (Bakshi 1959). Albeit with a wide distribution, no more than a dozen plants have been seen in one season of any area investigated (Bakshi 1959). This is also true in Michigan where there are 43 occurrences each with a single or small number of individuals, and Pterospora has been listed as a State S2 threatened species in Michigan (Higman and Penskar 1999). Prior to Bakshi (1959) it had not been clear whether Pterospora is parasitic on the roots of pine trees (Coulter and Nelson 1909; Piper and Beattie 1914; Hutchinson 1926; Hylander 1939; Fernald 1950) or saprophytic (MacDougal and Lloyd 1899; Jepson 1901; Henderson 1919; Rendle 1925; Copland 1941; Benson 1957). Since there is no chlorophyll in Pterospora and its roots have not been found to be organically connected with the roots of photosynthetic vascular *Author for correspondence. E-mail:, Phone: 616-395-7460, Fax: 616-395-7125.

Page  130 ï~~ 130 THE MICHIGAN BOTANIST Vol. 50 130 THE MICHIGAN BOTANIST Vol. 50 FIGURE 1. A. Partially excavated root ball and stems of Pterospora. B. Close-up image (40 x) of lateral roots of root ball. plants, Bakshi (1959) concluded that Pterospora has to obtain nutrients from fungal mycelia that form a mat completely surrounding the roots of Pterospora (Robertson and Robertson 1982; Massicotte et al. 2005). Physiological studies using radioactive elements have shown the movement of materials between parasitic plants (e.g., Monotropa and Sarcodes) and photosynthetic vascular plants via a fungal bridge (Bj rkman 1960; Furman 1966; Furman and Trappe 1971; Vreeland et al. 1981). These studies also suggest a more diverse list of host species for the mycorrhizal fungi than has been traditionally speculated. For example, Acer as well as Quercus may be involved in the mycorrhizal system of Monotropa (Furman and Trappe 1971), while Abies concolor, Pinus jeffreyi, and Populus tremuloides have been documented in the mycorrhizal system of Sarcodes sanguinea (Vreeland et al. 1981). Roots of Pterospora produce short branches repeatedly generating a globular

Page  131 ï~~ 2011 THE MICHIGAN BOTANIST 131 root ball (Fig. 1), which is penetrated through by, but does not seem to have organic connections with, the roots of photosynthetic vascular plants (Bakshi 1959). It is the fungi that function as a pipeline for material movement between Pterospora and photosynthetic vascular plants (Vreeland et al. 1981). Generally, plants have a diverse group of fungal associates to secure the connections (Malloch et al. 1980; Borowicz and Juliano 1991). However, molecular data have demonstrated that Pterospora in the western North America is specifically associated with the Rhizopogon subcaerulensis group of the genus Rhizopogon (Cullings et al. 1996), which is an ectomycorrhizal fungus (Molina and Trappe 1994). However, little information is available for the mycorrhizal system of Pterospora in eastern North America since Cullings et al.'s study (1996) did not include any plants from the east and a later study (Bidartondo and Bruns 2001) used only one sample from Qu6bec, Canada. In vitro experiments have suggested that mycorrhizal fungal associations are important for the seed germination of Pterospora (Bruns and Read 2000). Therefore, gaining knowledge of the specific association of Pterospora and mycorrhizal fungi helps determine factors affecting continual survival of Pterospora in Michigan. Specific objectives in this study were dual: (i) to determine what photosynthetic plants may be involved in the three way system of Pterospora, and (ii) to examine whether populations of Pterospora in West Michigan are specifically associated with Rhizopogon species and whether the associated species are the same as those in the Pacific West. MATERIALS AND METHODS Study site. We conducted field collecting in Betts Creek of Mecosta County, MI, in early July of 2011, and found less than 10 individuals of Pterospora. To minimize potential damage to the population, we limited our collecting to only two plants: the first with two stems and the other with a single stem. We collected one stem of the first plant as a voucher specimen (Li, J., J. Corajod, H. Vander Stel, A. Homkes, S. Ross 20117701, stored at HCHM) and several short roots branching off of the root ball of both plants (Fig. 1). Roots of other vascular plants that were penetrating through the pinedrop root ball were also collected. The roots were washed thoroughly in the lab with distilled water before DNA extraction. Molecular techniques. The genomic DNA of the root samples was extracted with a Plant DNeasy Mini Kit (Qiagen, CA). Polymerase chain reactions (PCR) were performed to amplify the internal transcribed spacer (ITS) regions of the nrDNA from the root samples of Pterospora using universal primers ITS1 and ITS4 (White et al. 1990). Both ITS and rbcL genes were amplified from the root samples of other vascular plants using primer pairs ITS1/ITS4 and rbcL5/rbcL3 respectively (Jiao and Li 2007). The PCR products were cleaned up using a RapidTip protocol (Diffinity Genomics, Inc., NY). The rbcL gene was sequenced directly from the PCR product using BigDye fluorescent chemistry (Applied Biosystems, CA) and analyzed in a GA 3130 Sequencer (Life Technologies, Carlbad) at Hope College. Because the universal primers for the ITS region may amplify the region from both plants and mycorrhizal fungi, we carried out cloning using the pGEM-T cloning kit (Promega Corporation, WI) to separate sequences from plants and fungi. Clones with inserts were sequenced. All DNA sequences were edited using Sequencher (version 5, Gene Codes Inc., Ann Arbor). We used the nucleotide Blast function in the NCBI's website to identify sequence identity. All ITS sequences of Rhizopogon in the GenBank database were downloaded and aligned with our new sequences using MUSCLE program (Edgar 2004). Gomphidius, Suillus, and Truncolumella were included in the dataset to root the tree, as they may be the closest relative of Rhizopogon (Grubisha et al. 2002; Binder and Hibbett 2006). Phylogenetic relationships were estimated using maximum parsimony methods as implemented in MEGA (Tamura et al. 2011) with close-neighbor-exchange (CNI) search on 5000 initial random trees. Nonparametric bootstrapping of 500 replicates was used

Page  132 ï~~ 132 THE MICHIGAN BOTANIST Vol. 50 TABLE 1. Affinity of sequences from roots of photosynthetic plants (Pp) and Pterospora (Pt) as determined by blasting analysis in the GenBank. Numbers 1 and 2 after Pp and Pt indicate plants 1 and 2, respectively, and the second numbers represent root samples from all around the root ball of Pterospora. Numbers in parentheses indicate number of clones sequenced. NA, no amplifications tried. Sample rbcL ITS Pp 1A Tsuga (hemlock) NA Pp 2.1 (4) Acer saccharum (sugar maple) Agrocybe (Basidiomycetes), Sordariomycetes, Rhizopogon, Acer saccharum Pt 1.2 (3) NA Rhizopogon Pt 1.3 (3) NA Rhizopogon Pt 1.8 (2) NA Rhizopogon Pterospora Pt 1.10 (3) NA Rhizopogon Pt 1.11 (1) NA Rhizopogon Pt 2.2 (4) NA Rhizopogon Pt 2.4 (1) NA Rhizopogon Pt 2.6 (5) NA Rhizopogon to measure relative support for individual clades (Felsenstein 1985) and the tree search options were as in the MP analysis except for the initial random trees set to 100. RESULTS Genomic DNA was successfully extracted from eight root samples of Pterospora and two root samples of other vascular plants. BLAST analysis of the plastid rbcL gene from the roots of other vascular plants suggested that they were from either Tsuga or Acer. A root sample of other vascular plants contained ITS sequences of Acer saccharum, Agrocybe, Sordariomycetes, and Rhizopogon (Table 1). The ITS sequences from all Pterospora root samples were found to be from Rhizopogon except for one that contained both Rhizopogon and Pterospora (Table 1). There were 105 sequences and 996 sites in the aligned ITS data set, 420 of which were parsimony informative. Phylogenetic analyses generated 67 most parsimonious trees of 2108 steps, a consistency index of 0.59, and a retention index of 0.77. Sequences from the mycorrhizal fungi of both individuals of Pterospora formed a well-supported lade (bootstrap support = 93%), which was in the lade containing species of subgenus Amylopogon sensu Grubisha (bs=97%), but its relationship with other species was not resolved (Fig. 2). All sequences are from Rhizopogon samples in the Pacific West except for Queb3000, which is an environmental sample from Qu6bec, Canada.

Page  133 ï~~ 2011 THE MICHIGAN BOTANIST 133 2011 THE MICHIGAN BOTANIST 133 FIGURE 2. Strict consensus of 67 most parsimonious trees produced from ITS sequences of Rhizopogon fungi and close relatives (CI=0.59, RI=0.77). Numbers at branches are bootstrap percentages of 500 replicates greater than 50%.

Page  134 ï~~ 134 THE MICHIGAN BOTANIST Vol. 50 DISCUSSION Several photosynthetic plants may be the ultimate source of carbon for the mycorrhizal system of Pterospora andromedea. The plant grows in woods containing conifers (e.g. pines, hemlock, spruce) and broad-leaved deciduous trees (e.g., aspen, maple, and birch) (Higman and Penskar 1999). Although no study has been done on nutritional movement between photosynthetic plants and Pterospora, results from other mycoheterotropic systems of Ericaceae (Bj rkman 1960; Furman 1966; Furman and Trappe 1971; Vreeland et al. 1981) indicate that several photosynthetic plants are involved in the system providing carbon source, and imply that the mycorrhizal system of Pterospora may also have more than one photosynthetic plant participating. Our results support the implication; DNA sequences we obtained from roots penetrating root ball of Pterospora show close affinity with those of Tsuga canadensis and Acer saccharum, both naturally occurring together with Pterospora. Interestingly, we did not get sequences from pines, another possible carbon source of Pterospora hinted by the common name of the plant. This is probably due to our limited sampling of pine-drops in the population. Like the populations of Pterospora in the Pacific West (Cullings et al. 1996; Bidartondo and Bruns 2001; Bidartondo and Bruns 2002), Pterospora in West Michigan shows an extreme mycorrhizal specificity with subgenus Amylopogon of Rhizopogon. Mycorrhizal mutualistic association is nearly universal in terrestrial plant systems and has important impact on ecology and evolution of organisms involved (Malloch et al. 1980; Molina and Trappe 1994). In general, mutualistic associations of fungi and plants are nonspecific (Hadley 1970): one plant with many fungal associates and vice versa. However, when present, the specific association is generally one-sided with one plant having multiple fungal associates (Malloch et al. 1980; Borowicz and Juliano 1991). Monotropes are a rare example of the evolution of mycorrhizal associations to an extreme specificity; all populations of Pterospora in the Pacific West are associated only with species of subgenus Amylopogon in Rhizopogon (Cullings et al. 1996). In the root of Acer saccharum, a potential carbon source of Pterospora, there are several fungal associates including Agrocybe, Sordariomycetes, and Rhizopogon. However, only subgenus Amylopogon of Rhizopogon is found in the root samples of Pterospora (Table 1). Interestingly, while all sequences of Rhizopogon from the two Pterospora plants form a well-supported lade with an existing sequence in the GenBank (Qu6bec, Canada) (Fig. 2), they differ from other species of subgenus Amylopogon from the west. Unfortunately, subgenus Amylopogon (ca. 16 spp. in North America) has rarely been collected from the east (Smith 1951, 1966, 1968; Smith and Zeller 1966). Therefore, it is unclear whether or not the species associated with Pterospora in West Michigan and Qu6bec represents a new species within subgenus Amylopogon. We did not find direct connections of Pterospora with roots of photosynthetic plants, supporting the conclusion that Pterospora obtains carbon from the mycelia of Rhizopogon (Bakshi 1959). Pterospora has been considered as a parasite (Bakshi 1959) or an exploiter of fungi (Bidartondo and Bruns 2002) be

Page  135 ï~~ 2011 THE MICHIGAN BOTANIST 135 cause it is not obvious that Pterospora may be beneficial to the mycorrhizal fungi. Pterospora produces thousands of winged seeds per plant (Bakshi 1959) and should therefore have ample opportunities to form associations with numerous fungi in the soil. Nonetheless, the association of Pterospora with subgenus Amylopogon is extremely specific (Bidardonto and Bruns 2001, 2002; Fig. 1). Seeds of Pterospora do not contain fungal hyphae and their germination has to be stimulated by chemical cues released from the fungi (Bruns and Read 2000), indicating that fungi might be the dominant player in the unique association. Fungal mycelia form a mantle enclosing roots of Pterospora and may stimulate the frequent branching of the roots to form a root ball. We noted in the field that the root ball apparently increases the production of fungal mass, which may be advantageous for fungal growth, development, and reproduction. Therefore, Pterospora may not be a fungal exploiter or parasite, but a partner of a mutualistic system. The survival of Pterospora depends on the specific species of Rhizopogon and its symbiotic relationship with photosynthetic plants. Therefore, any protection plan for the threatened species in Michigan (Higman and Penskar 1999) should consider the protection of the entire system including the photosynthetic plants and false truffles as well as pine-drops. ACKNOWLEDGMENTS We thank the Michigan Botanical Foundation for supporting the project, Steve Ross for help in the field, Department of Natural Resources of Michigan for granting collection permit, Mike Penskar for providing locality information of Pterospora in West Michigan, and Dr. Garrett and anonymous reviewers for their constructive comments on the manuscript. LITERATURE CITED Bakshi, T.S. 1959. Ecology and morphology of Pterospora andromedea. Botanical Gazette 120: 203-217. Benson, L. 1957. Plant Classification. D.C. Heath and Co. Boston. Bidartondo, M.I. and Bruns, T.D. 2001. Extreme specificity in epiparasitic Monotropoideae (Ericaceae): widespread phylogenetic and geographic structure. Molecular Ecology 10: 2285-2295. Bidartondo, M.I. and Bruns, T.D. 2002. Fine-level mycorrhizal specificity in the Monotropoideae (Ericaceae): specificity for fungal species groups. Molecular Ecology 11: 557-569. Binder, M. and Hibbett D.S. 2006. Molecular systematics and biological diversification of Boletales. Mycologia 98: 971-981. Bjirkman, E. 1960. Monotropa hypopithys L.-an epiparasite on tree roots. Physiologia Plantarum 13: 308-327. Borowicz, V.A. and Juliano, S.A. 1991. Specificity in host-fungus associations: do mutualists differ from antagonists? Evolutionary Ecology 5: 385-392. Bruns, T.D. and Read, D.J. 2000. In vitro germination of nonphotosynthetic, myco-heterotrophic plants stimulated by fungi isolated from the adult plants. New Phytologist 148: 335-342. Copland, H.F. 1941. Further studies on the Monotropoideae. Mondrono 6: 97-119. Coulter, J.M. and Nelson, A. 1909. A new manual of botany of the Central Rocky mountains (Vacular Plants). American Book Co. New York. Cullings, K.W. 1994. Molecular phylogeny of the Monotropoideae (Ericaceae) with a note on the placement of the Pyroloideae. Journal of Evolutionary Biology 7: 501-516. Cullings, K.W., Szaro, T.M., Bruns, T.D. 1996. Evolution of extreme specialization within a lineage of ectomycorrhizal epiparasites. Nature 379: 63-66.

Page  136 ï~~ 136 THE MICHIGAN BOTANIST Vol. 50 Edgar, R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792-1797. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791. Fernald, M.L. 1950. Gray's manual of botany. American Book Co. New York. Furman, T.E. 1966. Symbiotic relationships of Monotropa. American Journal of Botany 53: 627. Furman, T.E. and Trappe, J.M. 1971. Phylogeny and ecology of mycotropic achlorophyllous angiosperms. The Quarterly Review of Biology 46: 219-225. Grubisha, L.C., Trappe, J.M., Molina, R., and Spatafora, J.W. 2002. Biology of the ectomycorrhizal genus Rhizopogon. VI. Re-examination of infrageneric relationships inferred from phylogenetic analyses of ITS sequences. Mycologia 94: 607-619. Hadley, G. 1970. Non-specificity of symbiotic infection in orchid mycorrhizae. New Phytologist 69: 1015-1023. Henderson, M.W. 1919. A comparative study of the structure and saprophytism of the Pyrolaceae and the Monotropaceae with reference to their derivation from the Ericaceae. Contributions from the Botanical Laboratory of the University of Pennsylvania 5: 42-109. Higman, P.J. and Penskar, M.R. 1999. Special plant abstract for Pterospora andromedea (pine drops). Michigan Natural Features Inventory ( /mnfi). Hutchinson, J. 1926. The families of flowering plants. Macmillan and Co., Ltd. London. Hylander, C.J. 1939. The world of plant life. Macmillan and Co. New York. Jepson, W.L. 1901. A flora of western middle California. Encina Publishing Co. Berkeley. Jiao, Z. and Li, J. 2007. Phylogeny of intercontinental disjunct Gelsemiaceae inferred from chloroplast and nuclear DNA sequences. Systematic Botany 32: 617-627. Kron, K., Judd, W.S., Stevens, P.F., Crayn, D.M., Andergerg, A.A., Gadek, P.A., Quinn, C.J., and Luteyn, J. L. 2002. Phylogenetic classification of Ericaceae: molecular and morphological evidence. Botanical Review 68: 335-423. MacDougal, D.T. and Lloyd, F.E. 1899. Symbiotic saprphytism. Annals of Botany 13: 1-48. Malloch, D.W., Pirozynski, K.A., and Raven, P.H. 1980. Ecological and evolutionary significance of mycorrhizal symbioses in vascular plants (A review). Proceedings of the National Academy of Sciences of U.S.A. 77: 2113-2118. Massicotte, H.B., Melville, L.H., and Peterson, R.L. 2005. Structural featues of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea. Mycorrhiza 15: 101-110. Molina, R. and Trappe, J.M. 1994. Biology of the ectomycorrhizal genus, Rhizopogon. New Phytologist 126: 653-675. Piper, C.V. and Beattie, R.K. 1914. Flora of Southeastern Washington and adjacent Idaho. New Era Printing Co. Lancaster. Rendle, A.B. 1925. The classification of flowering plants. Cambridge University Press. Cambridge. Robertson, D.C. and Robertson, J.A. 1982. Ultrastructure of Pterospora andromedea Nuttall and Sarcodes sanguinea Torrey mycorrhizas. New Phytologist 92: 539-55 1. Smith, A.H. 1951. Puffballs and their allies in Michigan. University of Michigan Press. Ann Arbor. Smith, A.H. 1966. New and noteworthy higher fungi from Michigan. The Michigan Botanist 5: 18-27. Smith, A.H. 1968. Further studies on Rhizopogon. I. J. Elisha Mitchell Science Society 84: 274-280. Smith, A.H. and Zeller, S.M. 1966. A preliminary account of the North American species of Rhizopogon. Memoirs of the New York Botanical Garden 14: 1-178. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. 2011. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution 28: 2731-2739. Vreeland, P., Kleiner, E.F., and Vreeland, H. 1981. Mycorrhizal symbiosis of Sarcodes sanguinea. Environmental and Experimental Botany 21: 15-25. White, T.J., Bruns, T., Lee, S., and Tayler, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322. In: Innis, M., Gelfand, D., Sninsky, J., and White, T. PCR Protocols: A Guide to Methods and Applications. Academic Press: San Diego, CA.