Journal of Heredity Advance Access originally published online on March 23, 2005
Journal of Heredity 2005 96(4):396-403; doi:10.1093/jhered/esi045
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A First Assessment of Genetic Variation Among Morchella esculenta (Morel) Populations
From the Department of Biology, Grinnell College, Grinnell, IA 50112
Harmony J. Dalgleish is currently at the Division of Biology, Kansas State University, Manhattan, KS 66506
Address correspondence to Kathryn M. Jacobson at the address above.
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Abstract |
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Habitat loss and fragmentation have serious consequences for species diversity as well as genetic diversity within a species. As the most sought-after culinary fungus in the Midwest United States, morels (Morchella esculenta and related species) demand the attention of conservationists interested in preserving biological and genetic diversity. Little is known about the natural history of M. esculenta, which is critical information for understanding population dynamics as well as the impacts of habitat fragmentation and harvesting. We report initial results from our long-term studies of genetic variability among fruiting bodies at the Conard Environmental Research Area at Grinnell College, Grinnell, Iowa. Using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), a technique that has been successfully used to examine intrapopulation structure and detect clonal populations in numerous fungi, we found substantially higher levels of genetic polymorphism among 57 fruiting bodies than has been previously reported. Though laboratory studies indicate that the inbreeding potential for this fungus is high, we found little evidence for inbreeding, with only two pairs of the randomly chosen isolates having identical genotypes at the 34 loci examined. This work highlights the importance of further attempts to resolve important aspects of the morel life cycle regarding heterokaryosis and inbreeding potential.
Human land use change is cited as one of the greatest threats to ecological systems worldwide (Vitousek 1994). Land use change can result in both habitat loss and fragmentation, which reduces the size and increases the isolation of habitats and populations (Saunders et al. 2001). Habitat fragmentation can have serious consequences for species diversity as well as genetic diversity within a species by decreasing the number of outcrossing mating events (Young and Brown 1999). Conversion to agriculture has claimed much of the native habitats of the Midwest United States, with some states sustaining losses of 99.9% (Smith 1998). Though studies of the effects of habitat loss and fragmentation on fungal species are few, several studies have demonstrated deleterious effects of both habitat loss and fragmentation (Bergland and Jonsson 2001; Högberg and Stenlid 1999; Komonen et al. 2000). Indeed, Bergland and Jonsson (2001) cite continued habitat loss and fragmentation as the greatest threat to endangered fungal species in Scandinavia. Of particular interest for harvested species, such as morels, is the maintenance of sustainable populations in the face of habitat loss and widespread collection (Nantel et al. 1996). As the most sought-after culinary fungus in the Midwestern United States, morels (Morchella esculenta L. and related species) demand the attention of conservationists interested in preserving biological and genetic diversity (Molina et al. 2001).
To date, we know little about the natural history of M. esculenta (see the review by Gessner [1995]), critical information for understanding their population dynamics as well as the impacts of habitat fragmentation and harvest (Molina et al. 2001). In two recent independent studies, M. esculenta formed ectomycorrhizal associations with trees (Dahlstrom et al. 2000; Harbin and Volk 1999), resolving a long-standing debate about the ecological role of this fungus. It remains unknown whether morels are annual or perennial, although the ability of the fungus to form sclerotia (a hard mass of fungal cells) in the soil strongly suggests that they might be perennial (Buscot 1989; Miller et al. 1994). The mating systems of Morchella species are unknown (Buscot 1993), and whether heterokaryosis (mating of two compatible strains) followed by meiosis occurs in natural populations of morels is also unknown (Volk and Leonard 1989, 1990). Most knowledge of variation in fruiting patterns focuses on annual climatic variation (Tiffany et al. 1998), and to date there have been no studies published that examine the distribution, longevity, or persistence of morel populations, nor how extensive harvesting patterns and habitat fragmentation in the Midwest might be affecting their viability. We have begun a long-term investigation of morel populations at Grinnell College's 365 acre Conard Environmental Research Area (CERA) in central Iowa, where morel harvesting by others is forbidden. We are tracking interannual variation in environmental factors affecting fruiting, fruiting phenology, fruiting body abundance, distribution, and genetic variability of M. esculenta.
Differentiating between species of morels has been the principal focus of genetic studies of morels (Buscot et al. 1996; Gessner et al. 1987; O'Donnell et al. 1997; Royse and May 1990; Wipf et al. 1996, 1999; Yoon et al. 1990). While aimed specifically at resolving taxonomic distinctions between Morchella species, two studies have examined genetic variation within and between morel populations. Gessner et al. (1987) examined genetic variation in two Illinois populations using allozymes. In one of the populations, the 10 fruiting bodies were homozygous at the 16 loci examined, while 75% of these loci were heterozygous (but only dimorphic) in the second population. Similarly, Yoon et al. (1990) found high levels of genetic similarity among and within two Wisconsin and one Illinois population (Nei's unbiased genetic identity values 0.935–0.980), reflecting low levels of allozyme variation between populations. Furthermore, within two of the populations (n = 34) there was very little variation, with 60% of the loci being homozygous, while the third population (n = 54) was more variable, with 75% of the 20 loci examined having 2–4 alleles.
In this article we report results from our initial studies of genetic variability among fruiting bodies located at three sites separated by distances of 100–900 m. Based on the results of Gessner et al. (1987) and Yoon et al. (1990), we hypothesized that fungi at the three CERA sites would exhibit little variation both within and between sites. A number of studies (Hervey et al. 1978; King and Jacobson 2000; Volk and Leonard 1989) have shown that large proportions of morel spores (98–100%) from a single fruiting body are compatible with one another and can form heterokaryons under laboratory conditions. Assuming that heterokaryons are similarly formed under natural conditions, we would expect potentially high levels of inbreeding, manifested as low levels of genetic polymorphism, and nonrandom genetic population structure. Given that no studies to date have examined the scale at which morels function as individuals and populations, but that Yoon et al. (1990) suggest genetic drift accounted for the genetic differences they observed between geographically distant populations, we hypothesized that geographic distances would predict genetic variation among the three sites.
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Materials and Methods |
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Study Sites
The 365 acre Conard Environmental Research Area (CERA) of Grinnell College is located in southern Jasper County in central Iowa. It encompasses areas of restored tallgrass prairie and second-growth forest dominated by elms, oak savannas, and oak-hickory forests. In 1999 we mapped and collected all of the M. esculenta fruiting bodies at three sites. Site 2, comprising an area of 1824 m2, is located at the western side of CERA in biannually burned bur oak savanna. Sites 7 (1875 m2) and 8 (200 m2) are 100 m apart and separated by a creek. They are located 800 m and 900 m, respectively, to the east of site 2 in second-growth forest comprised mainly of elm, hickory, wild cherry, and bur oak. We dried the fruiting bodies at 60°C: 147 from site 2, 159 from site 7, and 19 from site 8. DNA was isolated from all fruiting bodies from site 8, and 19 randomly chosen fruiting bodies each from sites 2 and 7, for a total sample size of 57 fruiting bodies.
Molecular Methods
DNA was extracted from small blocks of dried fruiting body stalk tissue (5 mm x 5 mm) and quantified as described previously (Jacobson et al. 1993). Random amplified polymorphic DNA (RAPD) reactions (25 µl) consisted of 5 ng genomic DNA, 25 mM MgCl2, 250 µM each dATP, dTTP, dGTP, and dCTP, 0.5 units Taq DNA polymerase (Promega), and 40–50 ng of each 10-mer (Operon Technologies, Alameda, CA). Amplifications were performed in an MJ Research PTC-150 minicycler with an initial 5 min denaturation cycle at 95°C, followed by 45 cycles of 37°C for 1 min (annealing), 36°C for 1 min (polymerization), and 93°C for 1 min (denaturation). Amplified fragments were separated by gel electrophoresis in 0.7% agarose with 1% Synergel (Diversified Biotech, Boston MA) and 1x TAE. A 100 bp ladder (Promega, Madison, WI) was used, with each gel as a size standard. Gels were run for 3 h at 100 mV, stained with 1% ethidium bromide, and photographed with Polaroid 667 film. In addition to running water controls with each reaction setup, all reactions were performed twice to confirm that the RAPD markers scored were reproducible (Hadrys et al. 1992; Perez et al. 1998). The dataset analyzed used two primers: OPA-4 (5'-AATCGGGGCTG-3') and OPA-8 (5'-GTGACGTAGG-3').
The entire dataset of 57 fruiting bodies was used to create a similarity matrix in NTSYS (Rohlf 1988) using the Jaccard algorithm: a/n – d. This similarity matrix was then used to create a phenogram using the unweighted pair group method with arithmetic mean (UPGMA). The cophenetic correlation statistic was determined using the COPH function, and the goodness-of-fit of the phenogram for the similarity matrix was calculated using MXCOMP. In the same manner, similarity matrixes and phenograms for the 19 fruiting bodies from each site were subsequently created to more accurately assess intrasite relationships.
A drawback of RAPD markers is that dominance precludes the use of F statistic-based analyses of population structure. Instead, we used analysis of molecular variance (AMOVA; Excoffier et al. 1992), a statistical program that has been used effectively to obtain a first assessment of genetic variation in other organisms using dominant RAPD markers (e.g., Buso et al. 1998; Gabrielsen and Brochman 1998; Jacobson and Lester 2003). To compare the amount of genetic variation within and between populations, we created a distance matrix using the Euclidean distance algorithm (Liao and Hsiao 1998) and analyzed it using AMOVA. AMOVA partitions the total genetic variation into specified hierarchical groupings. We conducted our AMOVA using three hierarchical levels: variation within sites 2, 7, and 8; between sites; and between two locations separated by more than 800 m: east (sites 7 and 8) and west CERA (site 2).
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Results |
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Variation Within Sites
The two primers yielded 34 unambiguous RAPD loci from the 57 fruiting bodies, of which 76–85% were variable within each site (Table 1). The phenogram using the entire dataset (Figure 1) (r = 0.70, poor fit [Rohlf 1988]) illustrates the considerable variation detected among the 57 fruiting bodies. Likewise, AMOVA revealed that 86% of the total variation is accounted for within sites (Table 2).
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Because the phenogram for the entire dataset was a poor fit, we examined relationships among morels from each site using site-specific phenograms (Figures 2–4). Fits of the data for each of the three sites (n = 19) were very good (r = 0.84–0.86). Sites primarily contained fruiting bodies with unique genotypes (see also Table 1), and only two pairs of fruiting bodies had identical genotypes (Figures 2 and 3). Fruiting bodies 24 and 25 were located approximately 0.25 m from one another in site 2, whereas fruiting bodies 712 and 713 were approximately 8.00 m apart in site 7. Patterns of genetic similarity did not correspond with geographic location within any of the sites. Specifically, randomly chosen fruiting bodies located within 25 m2 were not genetically more similar than those located further away within a site.
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Finally, all estimates of genetic diversity within sites approach values of one (Table 1), further illustrating the genetically diverse structure of morels at each site.
Variation Between Sites
Fruiting bodies from the three sites do not form three site-specific genetic clusters in the phenogram for the entire dataset (Figure 1). However, the highest levels of genetic similarity among fruiting bodies (>70%) are between those from a single site. (The exception is 27, which is 76% similar to two fruiting bodies from site 8). Similarly, results from the AMOVA show that the between-site differentiation among the three sites is highly significant (P < .001) (Table 2). In contrast, however, AMOVA revealed no significant genetic differentiation among morels from the eastern (sites 7 and 8) and western locations (site 2) of CERA.
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Discussion |
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Genetic polymorphism among the 57 M. esculenta sporocarps used in this study is substantially higher than has been previously reported for this species. The Illinois and Wisconsin populations examined by Gessner et al. (1987) and Yoon et al. (1990) possessed little or no allozyme variation at the 16–20 loci examined. The use of RAPD markers that randomly sample polymorphisms throughout the genome may alone account for the higher levels of variation that we observed. RAPD markers have been successfully used to examine intrapopulation structure and detect clonal populations in numerous fungi (e.g., Bertault et al. 1998, 2001; Gandeboeuf et al. 1997; Selosse et al. 1996; Smith et al. 1992), whereas allozymes are more highly conserved (Weising et al. 1995).
Results from the hierarchical AMOVA reveal significant differentiation among the three sites despite the relatively small distances separating them (100–900 m), suggesting that they are appropriately termed genetic populations. While the phenogram for all 57 isolates does not reveal obvious site-specific clusters, the highest levels of genetic similarity (>70%) are among isolates from the same site (with one exception). This regional genetic population structure is most likely the result of more frequent interbreeding between isolates within a site than between isolates from different sites (Gryta et al. 2000). Furthermore, the fact that the hierarchical AMOVA did not reveal any significant genetic differentiation between the eastern and western locations at CERA suggests that gene flow is occurring among the three populations at similar rates. Thus it appears that spores are equally effective dispersal agents over distances of 100–900 m.
The CERA populations reveal little evidence of inbreeding: only two pairs of the randomly chosen isolates had identical genotypes for the markers examined. Contrary to our hypothesis, our data provide no evidence that these populations are inbred or clonal. While spores from a single fruiting body may be capable of forming stable heterokaryons (Volk and Leonard 1989) and haploid sclerotia may form fruiting bodies (Ower et al. 1986) that subsequently undergo meiosis and form spores, our preliminary analysis reveals no evidence that either phenomenon affects the genetic structure of these morel populations.
It is entirely possible, however, that the low levels of variation detected in other populations might be the result of inbreeding. While previous laboratory-based studies have provided convincing evidence that M. esculenta has high inbreeding potential, this may simply be a reproductive strategy aiding dispersal of individual pioneers in new habitats, where finding both a suitable mycorrhizal tree host as well as a compatible mating type in order to produce a reproductive heterokaryon would be highly improbable. This strategy has been documented in mycorrhizal Basidiomycetes (e.g., Jacobson and Miller 1994; Tommerup 1990a,b), but not yet in Ascomycetes. It has been suggested that morels appear in burn sites as short-lived pioneers, and such a reproductive strategy might thus be expected. It is possible that two of the populations sampled by Yoon et al. (1990), as well as those sampled by Gessner et al. (1987), were composed of a few pioneering individuals experiencing only minimal levels of intrapopulation gene flow, whereas the CERA populations (and possibly the third population of Yoon et al. [1990]), while equally capable of selfing (King and Jacobson 2000), are composed of established perennial individuals that are interbreeding on a regular basis. Further studies of CERA populations will address this hypothesis.
Despite heavy harvesting pressure and continued habitat fragmentation in the Midwest, there is little public concern about the viability of morel populations. While fruiting bodies do not always continue to fruit in the same location from year to year, this has been attributed to changes in habitat (e.g., death of elm trees, lack of burning) rather than excessive harvesting. Similarly, differences in fruiting body numbers from year to year are assumed to be a function of the climate. In reality, there has been no definitive study of the status of M. esculenta populations and whether they are being affected by harvesting and/or changes in habitat. The discrepancies between our study and previous work (Gessner et al. 1987; Yoon et al. 1990) highlight the importance of further attempts to resolve important aspects of the morel life cycle regarding heterokaryosis and inbreeding potential, and to increase the number of populations in which genetic variation has been examined using genetic markers that detect genetic variation at appropriate levels within and between populations.
Long-term population studies of other choice edible fungi are currently under way in Europe (black truffles [Bertault et al. 1998, 2001]) and in the Pacific Northwest (chanterelles [Dunham et al. 2003]) and will provide important comparative results, allowing us to more effectively identify the characteristics of fungal species susceptible to anthropogenic forces. Regarding morels, the initial results presented here, in addition to studies by Tiffany et al. (1998), lead us to hypothesize that this species has life-history characteristics that allow metapopulations to exist in burned savannas or elm forests; yet well-established populations persist in mature oak-hickory forests for multiple years. We propose that the mycorrhizal status of the species, as well as its ability to form sclerotia, may confer a perennial life history as has been extensively documented for Basidiomycete species (Dahlberg and Stenlid 1990; de la Bastide et al. 1994; Gryta et al. 2000, Selosse et al. 1998). Further, we hypothesize that the high inbreeding potential of M. esculenta may be a critical factor allowing the species to easily colonize new isolated habitats with suitable mycorrhizal hosts, as has been similarly documented for secondarily homothallic mycorrhizal Basidiomycete species (Jacobson and Miller 1994; Tommerup 1990a,b). We further propose that the unknown mating system (Buscot 1993) may also facilitate outcrossing, resulting in high levels of genetic variation at established sites such as those at CERA.
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Acknowledgments |
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We thank Thomas Volk, Peter Jacobson, and two anonymous reviewers for critical reviews of this article. This study was supported by grants from the Iowa Academy of Science (to H.J.D. and K.M.J.), a Woodrow Wilson Career Enhancement Fellowship (to K.M.J.), and Grinnell College.
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Footnotes |
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Corresponding Editor: Reid Palmer
Received June 4, 2004
Accepted October 20, 2004
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-
Bergland H and Jonsson BG, 2001. Predictability of plant and fungus species richness of old-growth boreal forest islands. J Veg Sci 12:857–866.
Bertault G, Raymond M, Berthomieu A, and Callot G, 1998. Trifling variation in truffles. Nature 394:734.[CrossRef]
Bertault G, Rousset F, Fernandez D, Berthomieu A, Hochberg ME, Callot G, and Raymond M, 2001. Population genetics and dynamics of the black truffle in a man-made truffle field. Heredity 86:451–458.[CrossRef][Medline]
Buscot F, 1989. Field observations on the growth and development of Morchella rotunda and Mitrophora semilibera in relation to forest soil temperature. Can J Bot 67:589–593.
Buscot F, 1993. Mycelial differentiation of Morchella esculenta in pure culture. Mycol Res 97:136–140.
Buscot F, Wipf D, Di Battista C, Munch J-C, Botton B, and Martin F, 1996. DNA polymorphism in morels: PCR/RFLP analysis of the ribosomal DNA spacers and microsatellite-primed PCR. Mycol Res 100:63–71.
Buso GSC, Rangel PH, and Ferreira ME, 1998. Analysis of genetic variability of South American wild rice populations (Oryza glumaepatula) with isozymes and RAPD markers. Mol Ecol 7:107–117.[CrossRef]
Dahlberg A and Stenlid J, 1990. Population structure an dynamics in Suillus bovinus as indicated by spatial distribution of fungal clones. New Phytol 115:487–493.[CrossRef]
Dahlstrom J, Smith JE, and Weber NS, 2000. Mycorrhiza-like interaction by Morchella with species of the Pinaceae in pure culture synthesis. Mycorrhiza 9:279–285.
de la Bastide PY, Kropp BR, and Piché Y, 1994. Spatial distribution and temporal persistence of discrete genotypes of the ectomycorrhizal fungus Laccaria bicolor (Maire) Orton. N Phytol 127:547–556.
Dunham SM, Kretzer A, and Pfrender ME, 2003. Characterization of Pacific golden chanterelle (Cantharellus formosus) genet size using co-dominant microsatellite markers. Mol Ecol 12:1607–1618.[CrossRef][Medline]
Excoffier L, Smouse PE, and Quattro JM, 1992. Analysis of molecular variance inferred from metric differences among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131:479–491.[Abstract]
Gabrielsen TM and Brochman C, 1998. Sex after all: high levels of diversity detected in the arctic clonal plant Saxifraga cernua using RAPD markers. Mol Ecol 7:1701–1708.[CrossRef]
Gandeboeuf D, Dupré C, Roeckel-Drevet P, Nicolas P, and Chevalier G, 1997. Grouping and identification of Tuber species using RAPD markers. Can J Bot 75:35–45.
Gessner RV, 1995. Genetics and systematics of North American populations of Morchella. Can J Bot 73:S967–S972.
Gessner RV, Romano MA, and Schultz RW, 1987. Allelic variation and segregation in Morchella deliciosa and M. esculenta. Mycologia 79:683–687.
Gryta H, Debaud J-C, and Marmeisse R, 2000. Population dynamics of the symbiotic mushroom Hebeloma cylindrosporum: mycelial persistence and inbreeding. Heredity 84:294–302.
Hadrys H, Balick M, and Schierwater B, 1992. Applications of randomly amplified polymorphic DNA (RAPD) in molecular ecology. Mol Ecol 1:55–63.[Medline]
Harbin M and Volk TJ, 1999. The association of Morchella with plant roots In: International Botanical Congress/Mycological Society of America Meeting Abstracts. Lawrence, KS: Mycological Society of America; 559.
Hervey A, Bistis G, and Leong I, 1978. Cultural studies of single ascospore isolates of Morchella esculenta. Mycologia 70:1268–1275.
Högberg N and Stenlid J, 1999. Population genetics of Fomitopsis rosea—a wood decay fungus of the old-growth European taiga. Mol Ecol 8:703–710.[CrossRef]
Jacobson KM and Lester E, 2003. A first assessment of genetic variation in Welwitschia mirabilis. Hook. J Hered 94:212–217.
Jacobson KM and Miller OK, 1994. Post-meiotic mitosis in the basidia of Suillus granulatus: implications for population structure and dispersal biology. Mycologia 86:510–515.
Jacobson KM, Miller OK, and Turner BJ, 1993. RAPD markers are superior to somatic compatibility tests for discriminating genotypes in natural populations of the ectomycorrhizal fungus Suillus granulatus. Proc Natl Acad Sci USA 90:9159–9163.
King HJ and Jacobson KM, 2000. Genetic diversity within and between populations of morel mushrooms (Morchella esculenta) In: Proceedings of the Iowa Academy of Science Meeting, Des Moines, April 22, 2000. Cedar Falls: Iowa Academy of Science.
Komonen A, Penttilä R, Lindgren M, and Hanski I, 2000. Forest fragmentation truncates a food chain based on an old-growth forest bracket fungus. Oikos 90:119–126.[CrossRef]
Liao LC and Hsiao JY, 1998. Relationship between population genetic structure and riparian habitat as revealed by RAPD analysis of the rheophyte Acornis gramineus Soland. (Araceae) in Taiwan. Mol Ecol 7:1275–1281.[CrossRef]
Miller SL, Torres P, and McClean TM, 1994. Persistence of basidiospores and sclerotia of ectomycorrhizal fungi and Morchella in soil. Mycologia 86:89–95.
Molina RD, Pilz D, Smith J, Dunham S, Dreisbach T, O'Dell T, and Castellano M, 2001. Conservation and management of forest fungi in the Pacific Northwestern United States: an integrated ecosystem approach In: Fungal conservation (Moore D, Nauta M, and Rotheroe M, eds). Cambridge: Cambridge University Press; 19–63.
Nantel P, Gagnon D, and Nault A, 1996. Population viability analysis of American ginseng and wild leek harvested in a stochastic environment. Conserv Biol 10:608–621.[CrossRef]
O'Donnell K, Cigelnik E, Weber NS, and Trappe JM, 1997. Phylogenetic relationships among ascomycetous truffles and the true and false morels inferred from 18S and 28S ribosomal DNA sequence analysis. Mycologia 89:48–65.
Ower R, Mills G, and Malachowski J, 1986. Cultivation of Morchella U.S. Patent no. 4,594,809.
Perez T, Albornoz J, and Dominguez A, 1998. An evaluation of RAPD fragment reproducibility and nature. Mol Ecol 7:1347–1357.[CrossRef][Medline]
Rohlf FJ, 1988. NTSYS-pc, numerical taxonomy and multivariate analysis system, version 1.40 Setauket, NY: Exeter.
Royse DJ and May B, 1990. Interspecific allozyme variation among Morchella spp. and its inferences for systematics in the genus. Biochem Syst Ecol 18:475–479.[CrossRef]
Saunders DA, Hobbs RJ, and Margules CR, 2001. Biological consequences of ecosystem fragmentation: a review. Conserv Biol 5:18–32.[CrossRef]
Selosse M-A, Costa G, di Battista C, le Tacon F, and Martin F, 1996. Segregation and recombination of ribosomal haplotypes in the ectomycorrhizal basidiomycete monitored by PCR and heteroduplex analysis. Curr Genet 30:332–337.[CrossRef][Medline]
Selosse M-A, Jacquot D, Bouchard D, Martin F, and le Tacon F, 1998. Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete Laccaria bicolor in European forest plantations. Mol Ecol 7:561–573.[CrossRef]
Smith DD, 1998. Iowa prairie: original extent and loss, preservation and recovery attempts. J Iowa Acad Sci 105:94–108.
Smith ML, Bruhn JN, and Anderson JB, 1992. The fungus Armillaria bulbosa is among the largest and oldest living organisms. Nature 356:428–431.[CrossRef]
Tiffany LH, Knaphus G, and Huffman DM, 1998. Distribution and ecology of the morels and false morels of Iowa. J Iowa Acad Sci 105:1–15.
Tommerup IC, 1990a. Laccaria species ectomycorrhizal with eucalyps: why does the ecology of bisporic species differ from quadrisporic species? In: Proceedings of the Eighth North American Conference on Mycorrhizae (Allen M and Williams SL, eds). Laramie: University of Wyoming; 284.
Tommerup IC, 1990b. Significance of post-meiotic mitoses in basidiomes of ectomycorrhizal fungi In: Proceedings of the Eighth North American Conference on Mycorrhizae (Allen M and Williams SL, eds). Laramie: University of Wyoming; 282.
Vitousek P, 1994. Beyond global warming: ecology and global change. Ecology 75:1861–1876.[CrossRef]
Volk TJ and Leonard TJ, 1989. Experimental studies on the morel. I. Heterokaryon formation between monoascosporous strains of Morchella. Mycologia 81:523–531.
Volk TJ and Leonard TJ, 1990. Cytology of the life-cycle of Morchella. Mycol Res 94:399–406.
Weising K, Nyborn H, Wolff K, and Meyer W, 1995. DNA fingerprinting in plants and fungi Ann Arbor, MI: CRC Press.
Wipf D, Fribourg A, Munch J-C, Botton B, and Buscot F, 1999. Diversity of the internal transcribed spacer of rDNA in morels. Can J Microbiol 45:769–778.[CrossRef]
Wipf D, Munch J-C, Botton B, and Buscot F, 1996. DNA polymorphism in morels: complete sequences of the internal transcribed spacer of genes coding for rRNA in Morchella esculenta (yellow morel) and Morchella elata (black morel). Appl Environ Microbiol 62:3541–3543.[Abstract]
Yoon C-S, Gessner RV, and Romano MA, 1990. Population genetics and systematics of the Morchella esculenta complex. Mycologia 82:227–235.
Young AG and Brown AHD, 1999. Paternal bottlenecks in fragmented populations of the grassland daisy Rutidosis leptorrhynchoides. Genet Res Camb 73:111–117.[CrossRef]
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