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Significant diversity and potential problems associated with inferring population structure within the Cenococcum geophilum species complex

     Department of Plant Pathology and Microbiology, University of California, Riverside, California 92521

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Cenococcum geophilum is perhaps the most widely distributed and most recognized ectomycorrhizal fungus with a host range of more than 200 tree species from 40 genera of both angiosperms and gymnosperms. We conducted a phylogenetic analysis on a large collection of isolates (n = 74) from North America and Europe based on glyceraldehyde 3-phosphate dehydrogenase (gpd). A subset of isolates (n = 22) also was analyzed with the more conservative LSU-rDNA locus. Significant nucleotide diversity was detected ( 20%) in the gpd region and the LSU-rDNA analysis supported that the C. geophilum isolates studied were monophyletic but distinct from two isolates, Am5–1 and N2–10, which previously were used in population genetic studies of this species. These results suggest that Am5–1 and N2–10 are likely two undescribed species or even genera. Our results suggest that C. geophilum sensu lato is a species complex and support previous molecular, physiological and morphological studies that have shown significant diversity in C. geophilum. This study also revealed that caution is advised when conducting population genetic studies in C. geophilum due to the possibility of pooling unrelated isolates. This potential problem also has implications for other fungal taxa because cryptic species routinely have been found in recent years based on molecular data.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Cenococcum geophilum is one of the most commonly encountered soil fungi and is found in diverse habitats throughout northern temperate regions (Trappe 1964). C. geophilum is thought to play a significant role in many ecosystems because it forms ectomycorrhizal (EM) associations with a diverse array of gymnosperm and angiosperm hosts (LoBuglio 1999). This mutualism allows the uptake of mineral nutrients, organic nutrients and water for the host, and in exchange the fungi receive photosynthates (Smith and Read 1997). C. geophilum is one of the few mycorrhizal species that routinely is identified based on the morphology of the colonized roots where it produces a dark black mantle with emanating stellate hyphae (Trappe 1964). This fungus also is isolated easily and cultured in vitro directly from sclerotia found in the soil. Given the wide host range and distribution, ease of experimental manipulation and potential ecological importance, a considerable amount of research has been conducted with this species. Most studies have found considerable cultural and physiological variation among isolates of C. geophilum sensu lato collected from the same environment as well as from diverse geographic regions (e.g. LoBuglio 1999).

C. geophilum is also one of the many fungal species in which the production of sexual or asexual spores is not known to occur. However Fernández-Toirán and & Aacute;gueda (2007) have claimed to have found cleistothecia of C. geophilum based on similar morphology between the cleistothecia and sclerotia. No single ascospore cultures or molecular methods were used to confirm the identification so this finding remains to be substantiated. The only known means of reproduction for C. geophilum are the production of mitotically derived sclerotia that can serve as dispersal and survival structures. However recent population genetic analyses have revealed considerable genotypic diversity within and among populations of this fungus (Jany et al 2002, LoBuglio and Taylor 2002, Panaccione et al 2001, Wu et al 2005). Based on restriction fragment length polymorphism (RFLP) analysis of the entire rDNA region, it has been suggested that C. geophilum is either a heterogeneous species or is a species complex (LoBuglio et al 1991). For example LoBuglio et al (1991) detected 32 unique genotypes out of 71 isolates collected from broad host and geographic ranges. However some of this variation was attributed to a Group-I intron (CgSSU intron) found within the 3' end of the small subunit (SSU) of rDNA (LoBuglio 1999). A phylogenetic analysis on the same isolates was conducted with the ITS-rDNA region. Shinohara et al (1999) found up to 4% sequence divergence among the isolates and concluded that C. geophilum was in fact a "single taxonomic entity, possibly a single species" that was extremely adaptable and widespread.

We recently detected phylogenetically distinct lineages or cryptic species of C. geophilum at the spatial scale of a single soil sample in an oak-woodland of California based on the analyses of a glyceraldehyde 3-phosphate dehydrogenase (gpd) gene, ITS-rDNA, a group I intron located in the 3' end of the SSU-rDNA and a portion of the mitochondrial SSU-rDNA (Douhan and Rizzo 2005). Moreover C. geophilum isolates from Oregon, Alaska and Maryland also clustered within the California lineages, suggesting this "species complex" has a wide geographic distribution. These results help explain the large amount of physiological, phenotypic and genetic differences reported among isolates of C. geophilum from similar as well as diverse geographic regions (LoBuglio 1999). However the ecological role that these cryptic species play remains to be determined.

The objectives of this study were to broaden our views of C. geophilum diversity by examining widely distributed isolates from North America and Europe. We chose to use the gpd locus because we have found that it is highly variable and easy to PCR amplify. This locus also shows significant congruence with other loci that we have tested (Douhan and Rizzo 2005, Douhan unpubl). We hypothesized that the gpd locus would reveal even more cryptic diversity than we previously have found from isolates mostly collected from a single environment (Douhan and Rizzo 2005).

	MATERIALS AND METHODS

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Fungal isolates.— – Both dried hyphal material and cultures of C. geophilum were used. Information regarding the C. geophilum isolates is provided (TABLE I). For cultured material freshly transferred hyphal tips were transferred to potato-dextrose agar plates and incubated 1–3 mo to allow enough growth for DNA extraction. The hyphae were scraped off the agar plates, freeze-dried, ground and the DNA was isolated with a slightly modified phenol:chloroform extraction procedure of Lee and Taylor (1990). DNA from the dried material provided by K. LoBuglio (Harvard University Herbaria, USA) was extracted in a similar fashion.

For each reaction 2.5 µL was separated on a 1.5% agarose gel, stained with SYBR Green I nucleic acid stain and viewed under UV light. PCR products were cleaned with ExoSap-IT (USB, Cleveland, Ohio) following the manufacturer’s instructions. The gpd region was sequenced in both directions whereas the LSU-rDNA was sequenced only in one direction with LR3 using Big Dye^® Terminator v3.1 chemistry (Applied Biosystems, Foster City, California). Sequencing was performed at the Core Instrumentation Facility (CIF) of the University of California at Riverside’s Institute of Integrative Genome Biology. The sequences were edited with Sequencher (version 4.6, Gene Codes Corp., Ann Arbor, Michigan), aligned with Clustal X (version 1.81) (Thompson et al 1997) and visually edited in MacClade version 4 (Maddison and Maddison 2001). For LSU-rDNA care was taken to use only sequences that had strong chromatograms because only a single read was done.

Six analytical methods were used to test for recombination within the gpd region before phylogenetic analysis with RDP (Recombination Detection Program, beta version 2.6: htpp://darwin.uvigo.es). The specific recombination tests that were used included RDP (Martin and Rybicki 2000), GENECOV (Padidam et al 1999), Bootscanning (Salminene et al 1995), MaxChi (Maynard Smith 1992), Chimaera (Posada and Crandall 2001) and SiScan (Gibbs et al 1997). Default settings in RDP were used for each test and = 0.05 was used to test for significance.

We previously identified a 42–44 bp indel from some of our C. geophilum isolates from lineage II (Douhan and Rizzo 2005). The inclusion of worldwide samples increased the size of the indel to 42–48 bp. This region was deleted from isolates that had the "extra" bases as well as approximately 50 bp adjacent to the indel because the alignment was ambiguous. Moreover this was also a region identified as possibly recombinant (see RESULTS). Maximum parsimony (MP) analysis was conducted with the heuristic search procedure with 1000 random-addition sequence replicates and tree-bisection-reconnection branch swapping were conducted with PAUP* version 4.0 beta 10 (Swofford 2002). Confidence in tree topology was examined with bootstrap with 10 000 replicates using the "fast" stepwise addition procedure. The LSU-rDNA tree was rooted with Am5–1, whereas the gpd was midway rooted because the divergent sequences found in isolates Am5–1 and N2–10 could not be aligned unambiguously (see RESULTS) to root the tree and no other sequences in GenBank were related closely enough to make a reliable alignment.

	RESULTS

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Testing for recombination in the gpd locus.— – The gpd locus was tested for recombination to ensure that it was a proper locus to infer phylogenetic relationships among the C. geophilum isolates in this study, because preliminary analysis found substantial nucleotide variation in this region. The gpd region for isolates Am5–1 and N2–10 were not included in the analysis because they were significantly divergent from the rest of the gpd sequences and could not be aligned unambiguously. Out of the six tests used to detect recombination only the SiScan method detected any potential recombination in gpd sequences. However this method might be sensitive to evolutionary rate variation along the length of an alignment and prone to reporting false positives (Worobey et al 2002). Nevertheless we removed the region where SiScan revealed potential recombination, which was adjacent to the indel. Moreover this putative recombinant region also was difficult to align, which justified deleting it from the final alignment. Analyses run on the dataset after removal of this problematic region showed no evidence of recombination. This was the dataset that was used to estimate the phylogeny of our isolates based on gpd.

Phylogenetic analyses.— – MP analysis of the gpd region (362 bp) for 74 isolates produced a tree that was highly diverse with 290 constant sites, 19 uninformative sites and 53 informative sites (FIG. 1). Two isolates were sequenced twice from independent cultures (I-3 = I-3A and N3–4 = 03–4-II). All new sequences have been deposited in GenBank (accession Nos. EU306912 [GenBank] –EU306956 [GenBank] ). The gpd sequences for the isolates from Douhan and Rizzo (2005) have been deposited in GenBank.

View larger version (19K): [in this window] [in a new window]   FIG. 1. Maximum parsimony analysis of Cenococcum geophilum isolates based on glyceraldehyde 3-phosphate dehydrogenase gene. Bootstrap support of 50% and above are indicated above nodes based on 10 000 replicates.

MP analysis of the LSU-rDNA region (590 bp) for a subset of the isolates (n = 22) produced a well resolved tree with 476 constant sites, 84 uninformative sites and 30 informative sites (FIG. 2). Isolates Am5–1 and N2–10 with the divergent gpd sequences clustered apart from the rest of the isolates with a bootstrap support of 99%, and no additional support was found for subclusters among the rest of the isolates. This supports the hypothesis that all isolates except Am5–1 and N2–10 represent a monophyletic lineage.

View larger version (8K): [in this window] [in a new window]   FIG. 2. Maximum parsimony analysis of Cenococcum geophilum isolates based on LSU-rDNA. Bootstrap support of 50% and above are indicated above nodes based on 10 000 replicates.

	DISCUSSION

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Isolate Am5–1 was isolated and identified as C. geophilum based on colony morphology from a beech forest in France (Jany et al 2002). However randomly amplified polymorphic DNA (RAPD) analysis placed it on a long branch by itself away from many of the other isolates. Our LSU-rDNA analysis suggests that this taxon does not belong to the C. geophilum species complex at all. BLAST of this isolate for the LSU matched 99% to Melinomyces bicolor (AY394885 [GenBank] ), a fungus that produces black ectomycorrhizal with hardwoods and pines (Mitchell and Gibson 2006). However the colony morphology on PDA is dark gray whereas C. geophilum produces dark brown to black colonies. Thus the identification of this isolate remains unknown because likely there is insufficient resolution in LSU for species identification. In contrast a BLAST of this isolate for the gpd region identified a sequence that closely matches (94%) another fungus, Helicoma irregulare (DQ128090 [GenBank] ). The sequences are different but a comparison to the alignment among these two sequences and the others clearly demonstrates their similarity (data not shown). Descriptions of Helicoma species have similar morphological characters as C. geophilum, such as dark melanized hyphae and subterranean growth characteristics in culture (Goos 1986). Therefore it is possible that Am5–1 might have been derived from a Helicoma ancestor and lost its ability to make spores. However this is purely speculation and additional studies are needed to test this hypothesis.

BLAST with the N2–10 LSU did not have any significant hits (<88%) and the gpd sequence was informative only in the broad sense in that a portion of sequence aligned with some Loculoascomycetes. Thus the identification of this isolate is also not possible. Of interest, the closest LSU hits were from Helicoma related species and Tsui and Berbee (2006) found that the closest relative of one species, H. isiola, was C. geophilum based on analysis of SSU and LSU sequences. N2–10 was used in a population genetic structure study based on amplified fragment length polymorphism (AFLP) in which significant genotypic diversity was found (Panaccione et al 2001). However, on inspection of some of the figures from Panaccione et al (2001), N2–10 has a unique RFLP-ITS pattern compared to the rest of the isolates, is on a long branch by itself in a phenogram based on AFLP data and has an ITS fragment that is a different size than the rest of the isolates that did not posses the Group I intron in the 3' end of the small subunit of rDNA. These findings along with our current results demonstrate the potential problems associated with pooling isolates that might not be related realistically when inferring population structure. This also was supported by a multigene analysis of 10 loci in which the acceptance or rejection of random mating based on gametic disequilibrium analyses was highly depended on species concept in C. geophilum (Douhan et al 2007).

For putative asexual fungi that lack spores and spore-bearing structures, traditional species concepts based on morphology might not be adequate to properly identify a taxon to species. For the fungi the ITS region, including partial LSU-rDNA, ITS-1, 5.8s, ITS-2 and partial SSU-rDNA, has been the marker of choice for differentiating fungi at the species level and a substantial public database has grown, namely GenBank (Bruns and Shefferson 2004, Bidartondo and Gardes 2005, O’Brien et al 2005). However there are examples of closely related fungi, such as the Phialocephala fortinii complex, where ITS phylogenies alone do not resolve species adequately (Grünig et al 2004). There are also examples of distinct species based on biological and ecological data where ITS sequences are identical or nearly identical among species such as in the mushroom genus Armillaria (Anderson and Stasovski 1992) and between the ascomycete species Ceratocystis polonica and C. laricicola (Harrington and Rizzo 1999).

For C. geophilum Shinohara et al (1999) published a previous ITS phylogeny of many of the same isolates and found only approximately 4% variability compared to almost 20% in gpd region in this study, and they suggested that C. geophilum was a cohesive species because intraspecific diversity in other fungi also has been reported (e.g. Shinohara et al 1999). However divergent lineages of C. geophilum were found that occupied the same soil core (Douhan and Rizzo 2005). If these organisms were functioning as a cohesive "biological" species we would not expect so much divergence at this scale because they potentially could interact with one another. This highlights the utility of using fine scale and macro-scale sampling when trying to understand species barriers within fungi, especially those in which any type of cytoplasmic exchange of genetic material is not known to occur. A similar pattern in bolete parasites (Hypomyces spp.) also has been observed (Douhan and Rizzo 2003). Within California isolates from divergent AFLP clades, which also correspond to ITS types, can be found at local scales but the same AFLP types also could be found separated by more than 600 km (Douhan and Rizzo 2003). If interbreeding were occurring we would expect more homogenized banding patterns from cohesive species.

Species concepts and the type of analysis are important when inferring how a biological organism reproduces and spreads. For the fungi and especially for putative asexual species that lack significant morphological differences, multigene genealogies have become a popular approach. Taylor et al (2000) advocated using the analyses of multiple genes as a criterion to identify species within the fungi, which they term the genealogical concordance phylogenetic species recognition (GCPSR). They suggest using multiple genes to determine the transition from concordance to conflict among taxa, which can be used to determine species boundaries and potential recombination within a phylogenetic species. Phylogenetic species for many morphospecies within various fungal genera have been identified with this approach with some examples including Fusarium (Skovgaard et al 2002), Stachybotrys (Cruse et al 2002), Coccidioides and some of its close relatives (Koufopanou et al 2001). However this approach can be vulnerable to sampling bias. For example we previously analyzed four loci in a local population of C. geophilum (Douhan and Rizzo 2005). Incongruence in the datasets was apparent only when isolates from outside the sampling location were included in the analysis. Therefore we ask whether the local population is not recombining and whether the history of recombination is evident only in this lineage due to past events. Moreover inclusion of many more isolates from broader geographic regions revealed much more diversity than found previously (Douhan and Rizzo 2005) and also blurred the phylogenetic relationships among C. geophilum isolates. Douhan and Rizzo (2005) found three well supported lineages of C. geophilum, whereas in the present study no support could be found for deep phylogenetic relationships and primarily only terminal nodes had any support.

C. geophilum sensu lato clearly is widespread geographically and ecologically successful, which is amazing given its inability to produce any type of spore for dispersal. However recognizing C. geophilum as an actual species complex helps to explain the apparent success of this ubiquitous mycorrhizal fungus. A detailed understanding of this species complex awaits further study. Multigene genealogy studies of C. geophilum populations sampled throughout its known range likely will be needed to understand this species complex. Detailed biological studies then may reveal associated phenotypic differences (morphological, physiological) among phylogenetic species within C. geophilum that might lead to a better understanding of the ecology of mycorrhizal symbiosis.

	ACKNOWLEDGMENTS

 
Financial support of the Agricultural Experiment Station, University of California at Riverside, is gratefully acknowledged. We thank Jim Trappe, Danniel Panaccione, Francis Martin and Susana Concalves for C. geophilum cultures, Darlene Southworth for sclerotia samples, Kathy LoBuglio for freeze dried mycelium, and Randolph Currah and anonymous reviewers for helpful comments and suggestions.

	FOOTNOTES

 
Accepted for publication July 30, 2007.

¹ Corresponding author. Fax: 951-827-4132; E-mail: gdouhan{at}ucr.edu

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Anderson JB, Stasovski E. 1992. Molecular phylogeny of northern hemisphere species of Armillaria. Mycologia 84:505–516.[CrossRef]

Berbee ML, Pirseyedi M, Hubbard S. 1999. Cochliobolus phylogenetics and the origin of known, highly virulent pathogens, inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences. Mycologia 91:964–977.[CrossRef]

Bidartondo MI, Gardes M. 2005. Fungal diversity in molecular terms: profiling, identification and quantification in the environment. In: Dighton J, White JF, Oudemans P, eds. The fungal community—its organization and role in the ecosystem. 3rd ed. CRC Press. p 215–239.

Bruns TB, Shefferson RP. 2004. Evolutionary studies of ectomycorrhizal fungi: recent advances and future directions. Can J Bot 82:1122–1132.[CrossRef]

Cruse M, Telerant R, Gallagher T, Lee T, Taylor JW. 2002. Cryptic species in Stachybotrys chartarum. Mycologia 94: 814–822.[Abstract/Free Full Text]

Douhan GW, Rizzo DM. 2003. Host-parasite relationships among bolete infecting Hypomyces species. Mycol Res 107:1342–1349.[CrossRef][Medline]

———, ———. 2005. Phylogenetic divergence in a local population of the ectomycorrhizal fungus Cenococcum geophilum. New Phytol 166:263–271.[CrossRef][Medline]

Douhna GW, Martin DP, Rizzo DM. 2007. Using the putative asexual fungus Cenococcum geophilum as a model to test how species concepts influence recombination ayalyses using sequence data from multiple loci. Curr Gen 52:191–201.[CrossRef][Medline]

Fernández-Toirán LM, &Aacute;gueda B. 2007. Fruitbodies of Cenococcum geophilum. Mycotaxon 100:109–114.

Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for Basidiomycetes application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118.[Medline]

Gibbs MJ, Armstrong JS, Gibbs AJ. 2000. Sister-scanning: a Monte Carlo procedure for assessing signals in recombinant sequences. Bioinformatics 16:573–582.[Abstract/Free Full Text]

Goos RD. 1986. A review of the anamorphic genus Helicoma. Mycologia 78:744–761.[CrossRef]

Grünig CR, McDonald BA, Siebert TN, et al. 2004. Evidence for subdivision and the root-endophyte Phialocephala fortinii into cryptic species and recombination within species. Fung Gen Biol 41:676–687.[CrossRef]

Harrington TC, Rizzo DM. 1999. Defining species in the fungi. In: Worrall JJ, ed. Structure and dynamics of fungal populations. Dordrecht, The Netherlands: Kluwer Academic Press. p 43–71.

Hopple J, Vilgayls R. 1994. Phylogenetic relationships among coprinoid taxa and allies based on data from restriction site mapping of nuclear rDNA. Mycologia 86:96–107.[CrossRef]

Jany JL, Garbaye J, Martin F. 2002. Cenococcum geophilum populations show a high degree of genetic diversity in beech forests. New Phytol 154:651–659.[CrossRef]

Koufopanou V, Burt A, Szaro T, Taylor JW. 2001. Gene genealogies, cryptic species, and molecular evolution in the human pathogen Coccidioides immitis and relatives (Ascomycota, Onygenales). Mol Biol Evol 18:1246–1258.[Abstract/Free Full Text]

Lee SB, Taylor JW. 1990. Isolation of DNA from fungal mycelia and single spores. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods, applications. San Diego: Academic Press. p 282–287.

LoBuglio KF. 1999. Ectomycorrhizal fungi: key genera in profile., Cairney JWG, Chambers SM, eds. Cenococcum. Berlin: Springer-Verlag. p 287–309.

———, Rogers SO, Wang CJK. 1991. Variation in ribosomal DNA among isolates of the mycorrhizal fungus Cenococcum geophilum. Can J Bot 69:2331–2343.[CrossRef]

———, Taylor JW. 2002. Recombination and genetic differentiation in the mycorrhizal fungus Cenococcum geophilum Fr. Mycologia 94:772–780.[Abstract/Free Full Text]

Maddison DR, Maddison WP. 2001. MacClade 4: analysis of phylogeny and character evolution. version 4.03. Sunderland, Massachusetts: Sinauer Associates.

Martin D, Rybicki E. 2000. RDP: detection of recombination amongst aligned sequences. Bioinformatics 16:562–563.[Abstract/Free Full Text]

Maynard Smith J. 1992. Analyzing the mosaic structure of genes. J Mol Evol 34:126–129.[Medline]

Mitchell DT, Gibson BR. 2006. Ericoid mycorrhizal associations: ability to adapt to a broad range of habitats. Mycologist 20:2–9.[CrossRef]

O’Brien HE, Parrent JL, Jackson JA, Moncalvo J-M, Vilgalys R. 2005. Fungal community analysis by large-scale sequencing of environmental samples. Appl Environ Microbiol 71:5544–5550.[Abstract/Free Full Text]

Padidam M, Sawyer S, Fauquet CM. 1999. Possible emergence of new geminiviruses by frequent recombination. Virology 265:218–225.[CrossRef][Medline]

Panaccione DG, Sheets NL, Miller SP, Cumming JR. 2001. Diversity of Cenococcum geophilum isolates from serpentine and non-serpentine soils. Mycologia 93:645–652.[CrossRef]

Posada D, Crandall KA. 2001. Evaluation of methods for detecting recombination from DNA sequences: computer simulations. Proc Nat Acad Sc 98:13757–13762.[Abstract/Free Full Text]

Salminen MO, Carr JK, Burke DA, McCutchan FE. 1995. Identification of breakpoints in intergenotypic recombinants of HIV type 1 by bootscanning. AIDS Res Hum Retrovir 11:1423–1425.[Medline]

Shinohara ML, LoBuglio KF, Rogers SO. 1999. Comparison of ribosomal DNA ITS regions among geographic isolates of Cenococcum geophilum. Curr Gen 35:527–535.[CrossRef][Medline]

Skovgaard K, Bodker L, Rosendahl S. 2002. Population structure and pathogenicity of members of the Fusarium oxysporum complex isolated from soil and root necrosis of pea (Pisum sativum L.). FEMS Microbiol Ecol 42:367–374.[Medline]

Smith SE, Read DJ. 1993. Mycorrhizal symbiosis. 2nd ed. San Diego: Academic Press. 605 p.

Swofford DL. 2002. PAUP*: phylogenetic analysis using parsimony (*and other methods). version 4.0b10. Sunderland, Massachusetts: Sinauer Associates.

Taylor JW, Jacobson DJ, Kroken S, et al. 2000. Phylogenetic species recognition and species concepts in fungi. Fung Gen Biol 31:21–32.[CrossRef]

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The Clustal X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nuc Acid Res 24:4876–4882.

Trappe JM. 1964. Mycorrhizal hosts and distribution of Cenococcum graniforme. Lloydia 27:100–106.

Tsui CKM, Berbee ML. 2006. Phylogenetic relationships and convergence of helicosporuous fungi inferred from ribosomal DNA sequences. Mol Phylogenet Evol 39: 587–597.[CrossRef][Medline]

Worobey M, Rambaut A, Pybus OG, et al. 2002. Questioning the evidence for genetic recombination in the 1918 "Spanish flu" virus. Science 296:5566.

Wu BY, Nara K, Hogetsu T. 2005. Genetic structure of Cenococcum geophilum populations in primary successional volcanic deserts on Mount Fuji as revealed by microsatellite markers. New Phytol 165:285–293.[CrossRef][Medline]

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Douhan, G. W. Articles by Douhan, L. I. Search for Related Content PubMed Articles by Douhan, G. W. Articles by Douhan, L. I. Agricola Articles by Douhan, G. W. Articles by Douhan, L. I.