1Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 37996. 2Current address - Department of Biology, Box 125, 402 East College Street, Bridgewater College, Bridgewater, VA 22812. 3Applied Biology and Biomedical Engineering, Rose-Hulman Institute of Technology, Terre Haute, IN 47803. *Corresponding author - elickey@bridgewater.edu. The Mushroom TWiG: A Marvelous Mycological Menagerie in the Mountains Edgar B. Lickey1,2,*, Shannon M. Tieken3, Karen W. Hughes1, and Ronald H. Petersen1 Abstract - We present an update on efforts to catalog the basidiomycete taxa, particularly the mushroom-forming fungi, of Great Smoky Mountains National Park (GSMNP) for the All Taxa Biodiversity Inventory. The goals of this project are to: 1) collect, identify, and voucher specimens with the help of visiting mycologists and volunteers; 2) extract DNA, amplify and sequence the nrITS region for barcoding, and deposit these sequences on GenBank; and 3) create species web pages for general public use. At present (April 2006), approximately 2000 specimens comprising about 770 species have been collected. As many as 45% are new Park records, and several may represent species new to science. DNA has been extracted from about 1000 specimens, and the nuclear ribosomal ITS region has been amplified and sequenced for about 500 of those. A surprising amount of genetic heterogeneity has been found, in part due to population migration patterns in response to glacial cycles. Studies with Artomyces pyxidatus support this hypothesis, showing distinct contributions from Central America and a second unidentified refugium. Introduction The southern Appalachian Mountain region, of which Great Smoky Mountains National Park (GSMNP) serves as the major protective refuge, is rich in fl oristic diversity and is an area of high endemism (Estill and Cruzan 2001). This is probably due to the wide variety of habitat types as well as population-migration patterns caused by successive glacial periods. However, little is known about how the mycota of this region compares to others because intensive fungal inventories are uncommon in North America. While several mycologists visited and collected in the southern Appalachians in the late 1800s and early 1900s, reports of early forays in the area of what is now GSMNP are very scarce (Petersen 1979). Among the earliest was Rev. Moses Ashley Curtis who traversed the mountains in the late 1850s, but reported finding few macrofungi (Petersen 1979). In September 1916, Charles Kauffman spent three weeks in and around Elkmont and reported 283 species of fungi, including two new species (Kauffman 1917). It was not until 1919, when Dr. Lexemuel Ray Hesler joined the faculty at the University of Tennessee - Knoxville, that regular forays were made to the GSMNP area. A preliminary checklist was published in 1937 and included 73 The Great Smoky Mountains National Park All Taxa Biodiversity Inventory: A Search for Species in Our Own Backyard 2007 Southeastern Naturalist Special Issue 1:73–82 74 Southeastern Naturalist Special Issue 1 752 fungal taxa (Helser 1937). Following a number of years of collecting by Hesler and a host of visiting mycologists, including forays associated with the 1939 Mycological Society of America meeting, the checklist grew to 1975 taxa by 1962, but this list was not published. Taxa were continually added to the Park list subsequent to formal forays associated with the Hesler Symposium in 1968, the 2nd International Mycological Congress in 1977, and periodic informal forays by students, faculty, and visiting mycologists. Updated taxonomic treatments of large genera such as Hygrophorus Fr. (Hesler and Smith 1963), Entoloma (Fr.) Kummer emend. Donk (Hesler 1967), and Lactarius Pers. ex S.F. Gray (Hesler and Smith 1979) added even more taxa to the list, for a total of nearly 2200 when it was published in its most recent form as a National Park Service management report (Petersen 1979). During the establishment of the All Taxa Biodiversity Inventory program in GSMNP in 1997, a fungal taxonomic working group (TWiG) was formed marking the start of the current period of intensive collecting. The original organizational effort was the product of collaboration between Amy Rossman, Lorelei Norvell, Rod Tulloss, the North American Mycological Association (NAMA), and the Asheville Mushroom Club. This endeavor was further bolstered by a National Science Foundation grant awarded in 2004 to K.W. Hughes and R.H. Petersen to concentrate on the Homobasidiomycete mycota (mushrooms and their allies). The goals of this grant, in addition to updating the GSMNP checklist, include gathering sequence data, based on the nuclear ribosomal inter-transcribed spacer region (nrDNA ITS), which will be made public on the GenBank database for “barcoding,” and the creation of species webpages for public use. The timing of this grant conveniently coincided with the joint Mycological Society of America - North American Mycological Association meeting in Asheville, NC in July, 2004, bringing together many mycologists, both amateur and professional. To take advantage of such a convergence of mycological expertise, two “mycoblitzes” (intensive collecting of fungi over a four-day period) were executed, funded in large part by the NSF grant. Since biodiversity also includes infraspecific genetic diversity, the preliminary results of an ongoing study with populations of Artomyces pyxidatus (Pers.: Fr.) Jülich (also known as Clavicorona pyxidata (Pers.: Fr.) Doty) will also be presented. This work is a continuation of studies by Tieken (2002) and Lickey et al. (2002), which showed the existence of two major nrDNA ITS haplotypes in North America, one predominantly northeastern and one southwestern Central American, with a putative introgression zone in the southeastern United States. Methods Collections Collecting trips were made at least once a week in the summer and fall months, and at least once a month in the winter and spring. Locations were chosen to give a broad spectrum of geography (i.e., east and west ends) and 2007 E.B. Lickey, S.M. Tieken, K.W. Hughes, and R.H. Petersen 75 habitat types (e.g., high and low elevation, cove hardwood and oak-pine forests, etc.) at approximately the same time of year. Fresh basidiomata were collected and stored in either fishing tackle boxes or wrapped in wax paper or aluminum foil for transport back to the lab. In the lab, collections were assigned a field number, and notes were made concerning location (state, county, trail or area, and GPS coordinates), date, and collector. Descriptive notes such as substrate, size, stature, vestiture, color (standards based on Kornerup and Wanscher 1967), odor, and taste were sometimes included. Photographs of each collection were taken in natural light on an 18% gray card using Kodak Elitechrome 100 ASA film. An approximately 100–200-mg tissue sample of one basidioma was frozen in foil for later DNA extraction. Cultures were made for some species if their basidiomata were too small or too few to sacrifice voucher material. In this case, either tissue or spores were placed directly on malt extract agar (15 g malt extract, 20 g agar, 1 L distilled H2O) to obtain a dikaryotic mycelium which was then grown in potato dextrose broth (24 g in 1 L distilled H2O) to yield enough tissue for DNA extraction. The rest of the collection was placed in a food dehydrator for 24–48 hrs and later was identified, databased, and deposited in the University of Tennessee Herbarium (TENN). For population studies involving Artomyces pyxidatus haplotype distribution, multiple collections were made in Cataloochee Cove, NC, Greenbrier Cove, TN, and near Sugarlands Visitor Center, TN. Identifications Collections were cursorily identified using an initial set of keys such as Largent and Baroni (1988) and Bessette et al. (1997), followed by more specific keys (e.g., published monographs) to more accurately identify collections to species. Microscopic characters were examined using typical techniques with small amounts of tissue and/or thin, hand-sliced sections mounted on slides in 3% aqueous KOH. Iodine-based Melzer’s reagent (see Largent et al. 1988) was also used where tests for amyloidity were needed. Collections representing taxonomically difficult genera were sent to experts in those groups for identification. Molecular analysis DNA was extracted from frozen, liquid culture, or dried herbarium tissue using a modified CTAB method (Hughes et al. 1999) or Qiagen DNeasy Plant Mini kit. The nuclear ribosomal internal transcribed spacer region (ITS1 - 5.8S - ITS2) was PCR-amplified using primers ITS1f (Gardes and Bruns 1993) and ITS4 (White et al. 1990) or ITS4b (Gardes and Bruns 1993), and the large subunit (LSU) was amplified using primers ITS3 (White et al. 1990) and LR7 (Moncalvo et al. 2000). Amplification reactions for ITS used the following parameters: 94 °C for 4 min; 35 cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min; 72 °C for 4 min. The same parameters were used for the LSU region, except that the 72 °C extension step was increased to 2 min. PCR products were cleaned using ExoSAP-IT (United States Biochemical), and sequenced with ABI Prism Big Dye Terminator v. 3.1 using primers ITS5 and ITS4 (White et al. 1990) for the ITS region and LR5 (Moncalvo et al. 2000) for the LSU region. Sequencing reaction parameters were 25 cycles of 96 °C for 10 sec, 50 °C for 5 sec, and 60 °C for 4 min, and products 76 Southeastern Naturalist Special Issue 1 were electrophoresed on an ABI 3100 automated sequencer (University of Tennessee Molecular Biology Research Facility). Resulting electropherograms were viewed and edited using Sequencher 4.1.2 (GeneCodes), and GenBank BLAST searches were done to determine presence or absence of similar sequences in that database. When electropherograms were unreadable due to apparent insertion-deletion events that could not be resolved with sequencing with different primers, PCR products were cloned and screened using the pGEM-T vector system and JM109 competent cells following manufacturer’s directions (Promega). Restriction enzymes CfoI and BsaJI (Promega) were used in the haplotype analysis of Artomyces pyxidatus populations following Tieken (2002) and Lickey et al. (2002). Sequencing as described above was sometimes used to verify presence or absence of restriction sites. Results Collections A comparison of the numbers of species included in GSMNP checklists since 1937 of all fungi, Homobasidiomycetes, and agarics and allies is presented in Figure 1. Since the start of the fungal TWIG of the ATBI in 1998, and as of April 2006, approximately 1900 collections have been made, representing about 770 species and bringing the total number of fungi known to occur in the Park to over 2500 species (Fig. 1). Of those 770 species, 347 (45%) are new records to GSMNP, and possibly as many as 25 are species new to science, according to experts in selected fungal groups (pers. comm. with: Bart Buyck, Museum National D’Histoire Naturelle, Paris, France [Russula Pers. spp.]; Urmas Koljag, University of Tartu, Estonia [Tomentella Pat. spp.]; Karl-Henrik Larsson, University of Göteborg, Sweden [Corticium Fr. spp.]; Brandon Matheny, Clark University, Worcester, MA [Inocybe (Fr.) Fr. spp.]; and Rod Tulloss, New York Botanical Garden, New York, NY [Amanita Pers. spp.]). However, a large backlog of collections awaits further study for identification. Molecular analysis DNA has been extracted from more than 1000 collections, and the nrDNA ITS region has been amplified and sequenced for more than 500 collections. An unexpectedly high proportion of collections were apparently heterozygous for insertion-deletion mutations (indels). A few examples are included in Table 1. For most of these, sequences had to be obtained through cloning procedures. Collections of Artomyces pyxidatus revealed that both the northeastern and southwestern haplotypes identified by Lickey et al. (2002) and Tieken (2002) are present in the three low-elevation coves. The northeastern haplotype is present in much higher frequencies in all three populations, and apparent heterozygotes have been identified (Tieken 2002). Discussion It is difficult at this time to estimate how many basidiomycete species will be found, much less exist undetected, in GSMNP. The rate of new discoveries 2007 E.B. Lickey, S.M. Tieken, K.W. Hughes, and R.H. Petersen 77 versus accounts of species known from the Park has been outstanding; approximately 45% of the species collected are new Park records. In other words, for every account of a 1979-checklist species we are finding at least one new record. In addition, large areas of GSMNP, such as the “Northshore” area in North Carolina, remain unsurveyed due to their remoteness and inaccessibility. On the other hand, only about 400 of the species collected since the start of the ATBI were included on the 1979 checklist which listed almost 1600 basidiomycetes (Petersen 1979). These apparently low re-collection numbers may be due to several factors. First, the species that have not been re-collected could be rare or not fruit for long periods of time and by chance have not been re-encountered, or they might possibly be extirpated. Second, there may be a better understanding of the taxonomy of some groups where segregate species that were previously recognized have been synonymized, while others have only recently been recognized as distinct species. Third, recent collections may Figure 1. A comparison of the numbers of species of all fungi (Total), Homobasidiomycetes, and agarics and their allies included in the three previous checklists (Hesler 1937; Hesler, unpubl. data; Petersen 1979) and the current list as of 12 March, 2006. *The number of agarics for 2006 is an estimate. Table 1. Numbers of nrDNA ITS sequences for representative mushroom groups and the percentage exhibiting indel heterozygosities. Mushroom group # nrITS sequences % indel heterozygous Hygrophorus s.l. 40 58.0% Cortinarius 48 31.0% Lactarius 40 25.0% Saprobes 309 28.5% 78 Southeastern Naturalist Special Issue 1 have been identified as a different species than what they were identified as on the 1979 checklist either because of misidentification or differences in character interpretations. Finally, as seen in Table 2, less than half the number of species of the largest, but often taxonomically difficult, agaric genera included on Petersen’s 1979 checklist have been re-collected. While some collections of Cortinarius Fr., Entoloma and Mycena S.F. Gray have been made and await identification by experts in these groups, active collecting has been somewhat limited in favor of other more easily identifiable taxa. The ephemerality and phenology of most mushroom taxa are not fully understood, but they can have a large effect on fl oristic studies. In a 21-year study of a 1500-m2 plot in a Swiss forest, Straatsma et al. (2001) found that of the more than 400 species of mushrooms, only eight were observed in every year while an average of 17 were recorded as new occurrences in the last five years of sampling. The numbers of species they reported per year ranged from 18 in 1989 to 194 in 1992, indicating that productiveness can change drastically with conditions (Straatsma et al. 2001). As with the present study, rarity of some taxa and the transient nature of others due to habitat and environmental changes over time may account for some of the disparity in the numbers collected from year to year. Even though fruit bodies are the most visible evidence we have of a mushroom species’ occurrence, fruiting is only a small part of the life cycle and is often tied to specific environmental conditions. Because of this, some species may not be seen if they fruited at times when we are not in the field, if they lie dormant for long periods of time waiting for favorable fruiting conditions, or even if they forgo sexual reproduction altogether. An example from the present study involves Collybia (Fr.) Staude subgenus Collybia section Collybia (Halling 1983), a group of small mushrooms that often grow on the remains of other mushrooms and referred to here as the “microcollybias.” While sequencing the nrDNA ITS region for a collection of Lactarius griseus Peck, a second sequence was isolated that apparently belongs to a microcollybia. It is likely that the Lactarius fruit body was colonized by the Collybia with no evidence of fruiting, but based on sequence comparisons with known microcollybia species, it is apparently undescribed. Had we not incorporated molecular data in our study, this species might still have gone Table 2. Number of species of the largest genera listed in Petersen’s 1979 checklist compared with the numbers that have been re-collected and those that are Park records. 1979 list Re-collectedA New Park records Cortinarius 103 # 1 Entoloma s.l. 101 # 1 Lactarius 86 34 15 Russula 74 14 15B Mycena 73 # 2 Hygrophorus s.l. 55 15 4B Inocybe 43 6 13B Amanita 34 17 16B A# indicates that less than 1/3 the number of the 1979 checklist have been re-collected. BSpecies putatively new to science have been identified, but their number is not included with the new Park records. 2007 E.B. Lickey, S.M. Tieken, K.W. Hughes, and R.H. Petersen 79 undetected. Likewise, environmental sampling, such as that described by O’Brien et al. (2005), will undoubtedly uncover many more species that have been overlooked because their fruiting has yet to be observed. The numbers of taxa included in the growing checklist may be underestimated due to the potential existence of cryptic species—those that are morphologically identical, but in reality represent distinct biological species. There are a number of mushroom “morphospecies,” such as Xeromphalina campanella (Batsch: Fr.) Kühner & Maire (Johnson 1997), Marasmius androsaceus (L.: Fr.) Fr. (Gordon 1994), Gymnopus subnudus (Ellis: Peck) Halling (Murphy and Miller 1997), which may represent more than one “biological species” (Petersen 1995). These studies, based on crosses between collections, have shown that intersterile populations coexist in the southern Appalachians and further draw attention to possible intersterility substructuring in morphological species that are assumed to have intercontinental distributions. Many genera, for example the genus Cortinarius, may harbor cryptic species, as over half the species recorded from GSMNP are based on European names (Table 2). While mating studies are not feasible at present in mycorrhizal species such as Cortinarius, molecular data, particularly nrDNA ITS sequences, may shed some light on the degree of genetic differentiation. Lack of or limited amounts of gene fl ow among geographically separated populations have been demonstrated using molecular data in several morphospecies including: Artomyces pyxidatus (Lickey et al. 2002) Lentinellus castoreus (Secretan apud Fr.) Kühner & R. Maire and L. ursinus (Fr.) Kühner (Hughes and Petersen 2004) and Flammulina velutipes (Fr.) Karsten (Methven et al. 2000). With little or no gene fl ow, European and North American taxa may have diverged genetically. However, the degree of genetic divergence needed to identify North American taxa as different from their European counterpart will remain unresolved until many more collections are made and compared to topotype collections from Europe. For some fungi, the number of species may be overestimated due to differences in taxonomic interpretations among researchers, leading some to recognize multiple taxa where others would simply recognize one. For example, there are 101 Entoloma s.l. species included in the 1979 checklist. In his monograph, Hesler (1967) described 59 of those species, 54 of which are based on type material collected in GSMNP, and of those, 32 are known only from the type collection. These 32 taxa may not have been encountered again because they are extremely rare, exhibit infrequent fruiting, or might simply have been avoided because of the difficulty in identifying species in this very taxonomically challenging group. On the other hand, it is possible that some of these may represent aberrations or are simply part of the range of natural variability, genetic or plastic. Again, more collections and more complete genetic study are warranted to determine the true extent of species boundaries. One genus that has recently received such attention is Amanita, being studied by Rod Tulloss, one of the original ATBI Fungal TWIG coordinators. Of 80 Southeastern Naturalist Special Issue 1 the 34 species included on the 1979 checklist, only half of those have been recollected, while at least 16 new Park records have been found along with eight species putatively new to science (a periodically updated list can be found at Tulloss’ website - http://pluto.njcc.com/~ret/amanita/key.dir/am_gsmnp. html). Genetic data in the form of nrDNA ITS sequences are being gathered in conjunction with morphological studies to aid in species identification and delimitation. At the rate of collecting new records and putative new species of Amanita, and with respect to the number already reported from the Park and the number species known from the southern Appalachian region, Tulloss (2005 version of website) believes as many as 200 species of Amanita may inhabit GSMNP. In the interest of gathering data for barcoding efforts, a large number of nrDNA ITS sequences are being accumulated and will be deposited in the publicly accessible GenBank sequence database after identifications have been verified. Not surprisingly, GenBank BLAST searches on some of the sequences completed so far have yielded few matches of ≥97%, an arbitrary but conservative upper estimate of infraspecific ITS sequence divergence (O’Brien et al. 2005). Only about a third of our sequences have such conspecific counterparts, illustrating how incomplete the database is, but also emphasizing the need for depositing sequences of correctly identified species. There are also a number (≈5%) of our sequences that matched (≥97%) entries listed as unidentified, uncultured basidiomycete from environmental samples, and as more sequences are generated from identifiable specimens, names may be attached to these unknown environmental samples. We have also found that the amount of genetic diversity in the Park is higher than we had expected. Multiple nrDNA ITS haplotypes have been found for many different taxa and they often appear as heterozygotes (Table 1). One explanation for this phenomenon is that present-day populations are infl uenced by past events, such as glacial cycles. Phylogeographic patterns associated with glacial cycles have been used to explain population genetic patterns observed in fungi such as Artomyces pyxidatus (Lickey et al. 2002, Tieken 2002); Lentinellus ursinus and L. castoreus (Hughes and Petersen 2004); and Rhodocollybia butyracea (Bull.: Fr.) Lennox, Gymnopus biformis (Peck) Halling, and Flammulina velutipes (K. Hughes, unpubl. data); as well as in some plants such as Liriodendron tulipifera L. (Parks et al. 1994, Sewell et al. 1996). It is hypothesized that during the last glacial maximum, populations of plants (and presumably fungi) tolerated ecological conditions far south of their present ranges in what have been termed refugia (Delcourt and Delcourt 1981). We posit, as did Parks et al. (1994), Sewell et al. (1996), and several others, that populations separated in geographically isolated refugia accumulated mutations creating new and unique haplotypes. As the climate ameliorated and glaciers receded, populations expanded and reconnected on the way to their present-day ranges, and these divergent haplotypes introgressed. In the case of Artomyces pyxidatus, both the northeastern and southwestern haplotypes are present in the GSMNP, with the northeastern haplotype maintained in higher frequencies. The occurrence of these apparent hybrid haplotypes indicates the presence of interbreeding and introgression. 2007 E.B. Lickey, S.M. Tieken, K.W. Hughes, and R.H. Petersen 81 This same phenomenon may be similar in the many different species of fungi where multiple haplotypes are being found. While great strides have been made in cataloging the mycota of the GSMNP, much work remains to be done. We are continually finding new Park records and seem to be a long way from reaching the asymptote of the number of fungal species occurring in the Park. In this regard, herbarium work is just as important as continued field surveys to identify both new Park records as well as species new to science. More also needs to be done to study the amazing amount of genetic diversity that this region harbors. Species webpages, our public-outreach medium, are still in development, but will soon provide species descriptions, collection locations, and links to nrDNA ITS sequences deposited in GenBank. Acknowledgments The authors thank two anonymous reviewers for their comments and suggestions. The following people are thanked for their collections and expert identifications: Cathie Aime, Joe Ammirati, Rich Baird, Meredith Blackwell, Bart Buyck, Julieta Carranza, Joaquin Cifuentes, Roy Halling, Rick Kerrigan, Jean Lodge, Juan Mata, Brandon Matheny, Coleman McCleneghan, Andy Methven, Andy Miller, Lorelei Norvell, Clark Ovrebo, Amy Rossman, Somsak Sivichai, Rod Tulloss, and Annamieke Verbeken, as well as Vince Hustad, Sean Jones, Huzefa Raja, and Matt Woods. Others that helped in lab and/or field include Rob Shepard, Pamela Pokorny, Jason Smith, Beth Helmbrecht, Stacy Huskins, Kim Kennard, and Jessica Hite. We also thank Pat Cox for the organization of and invitation to contribute to this special ATBI Symposium. Funding for this project was provided by NSF DEB grants 9521526 to R.H. Petersen and K.W. Hughes and 0338699 to K.W. Hughes and R.H. Petersen, as well as the Hesler Endowment Fund. Literature Cited Bessette, A.E., A.R. Bessette, and D.W. Fischer. 1997. 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