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2 Oregon State University, Department of Botany and Plant Pathology, 2082 Cordley Hall, Corvallis, Oregon 97331-2902 USA; 3 University of California at Berkeley, Department of Environmental Science, Policy and Management, 111 Koshland Hall, Berkeley, California 94720-3102 USA; and 4 University of California at Berkeley, Department of Plant and Microbial Biology, 111 Koshland Hall, Berkeley, California 94720-3102 USA
Received for publication August 24, 1999. Accepted for publication February 17, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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180 km apart. To identify the fungal symbionts associated with these plants, we first analyzed restriction fragment length polymorphisms (RFLPs) of the internal transcribed spacer region (ITS) of the fungal nuclear ribosomal repeat. Fungal ITS-RFLPs were successfully produced from 57 of the 76 plants sampled, and all symbionts shared the same DNA fragment pattern. The morphology of S. sanguinea mycorrhizae was consistent with that expected from a Rhizopogon species in section Amylopogon. To confirm and refine this identification, a total of six fungal ITS sequences were determined from S. sanguinea mycorrhizae. These sequences were analyzed together with eight existing and eight newly determined ITS sequences from Rhizopogon section Amylopogon. The newly determined sequences include an ITS sequence from the fungal symbiont of pine drops (Pterospora andromedea, Monotropoideae, Ericaceae), a plant that was previously reported to be exclusively associated with the Rhizopogon subcaerulescens group. When these sequences were analyzed together, the Sarcodes symbionts grouped tightly with several collections of R. ellenae including the holotype, one collection of R. idahoensis, and one collection of R. semireticulatus. A different lineage comprised collections of R. subgelatinosus, R. subcaerulescens, another collection of R. semireticulatus, and the Pterospora symbiont. We conclude that S. sanguinea associates exclusively with a single species in the R. ellenae species complex throughout our sampling range. These results indicate a much higher level of specificity in S. sanguinea than was previously reported and confirm the emerging pattern that nonphotosynthetic, monotropoid plants generally associate very specifically with a narrow range of ectomycorrhizal fungi.
Key Words: monotropoid mycorrhizae • Pterospora andromedea • Rhizopogon • Sarcodes sanguinea • specificity (host)
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Like other members of the Monotropoideae, it is nonphotosynthetic and must receive all of its carbon from fungal root symbionts. The roots of S. sanguinea are coralloid, fleshy, and surrounded by a fungal mantle except for the tips, which are uncolonized. Although the importance of fungal root symbionts for the nutrition of monotropoid plants has been recognized for a long time (e.g., Kamienski, 1881
; Francke, 1934
), the fungi involved are just starting to be identified through the use of molecular techniques. Cullings, Szaro, and Bruns (1996)
sampled several monotropoid taxa mostly in the western United States and used partial sequence analysis as well as oligonucleotide probing of the mitochondrial large subunit rDNA to identify the fungal symbionts. The picture that emerged from these studies was that in contrast to photosynthetic mycorrhizal plants, which typically associate with large numbers of distantly related fungi, most monotropes appear to associate with specific groups of closely related ectomycorrhizal fungi: Monotropa uniflora was found associated with taxa in the Russulaceae, while Pterospora andromedea was found only in symbiosis with the Rhizopogon subcaerulescens species group. Recently, Lefevre, Carter, and Molina (1998)
found Allotropa virgata to be specifically associated with Tricholoma magnivelare. This pattern of specificity is also observed in nonphotosynthetic orchids (Taylor and Bruns, 1997
), and so it appears that specific fungal associations are the norm in nonphotosynthetic mycorrhizal plants.
Sarcodes sanguinea so far appeared to be the only exception to this pattern. Cullings, Szaro, and Bruns (1996)
reported that it associated with at least three distantly related lineages of fungi within the Hymenomycetes. Furthermore, only 12 individual plants were sampled; thus the diversity of associates could have been underestimated. None of the symbionts of S. sanguinea was identified to species level, and only one fell within a lineage known to be ectomycorrhizal at the time of publication. For these reasons we decided to investigate the mycorrhizal associates of S. sanguinea more closely.
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MATERIALS AND METHODS |
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In the spring of 1995, 1996, and 1997, we sampled a total of 64 S. sanguinea rootballs for direct identification of the fungal symbiont based on polymerase chain reaction (PCR) with fungus-specific primers. The plants originated from 16 populations in two areas of the Sierra Nevada of California (Table 1, Fig. 1). Plants sampled within a population were 5–100 m apart, and populations were 5–180 km apart. Twelve additional plants were sampled in the spring of 1997 at the "high-density site" and at one other site (Table 1) for cultivation of the fungal symbiont from S. sanguinea roots (and subsequent identification of the cultured mycelium).
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) that had been supplemented with 50 mg chloramphenicol, 50 mg streptomycin, 50 mg ampicillin, and 1 mg benomyl per litre. Fast growing and conidial isolates (primarily Mucor) were removed and discarded. After 4 wk of growth, cultures were transferred to modified Hagem's without antibiotics.
Molecular methods
Genomic DNA was extracted from mycorrhizae, fruitbodies, or mycelium using the method of Gardes and Bruns (1993)
. PCR reactions contained 10 mmol/L Tris/HCl pH 8.3, 50 mmol/L KCl, 2.5 mmol/L MgCl2, 0.1 mg/mL gelatin, 200 µmol/L of each of the four deoxyribonucleotide triphosphates, 0.5 µmol/L of each of two different primers, 25 U/mL Taq polymerase, and empirical amounts of genomic DNA. Reaction conditions were: denaturing at 94°C for 35 sec, annealing at 53°C for 55 sec, and polymerization at 72°C initially for 45 sec but increasing by 4 sec on every cycle (35 cycles total). PCR primers used were either ITS5 (general) or ITS-1f (fungus-specific) in combination with either ITS4 (general) or ITS-4b (basidiomycete-specific). For sequencing, the internal primers ITS2 and ITS3 were used as well. For primer sequences see White et al. (1990)
and Gardes and Bruns (1993)
.
ITS-RFLPs were produced as described before (Gardes and Bruns, 1996
). In brief, the internal transcribed spacer (ITS) of the nuclear ribosomal repeat was amplified by PCR and subsequently digested with the restriction enzymes AluI and HinfI; DpnII and CfoI were also used occasionally. Restriction fragments were then separated on agarose gels (1% ultrapure agarose from GibcoBRL, Grand Island, New York, and 2% NuSieve agarose from FMC BioProducts, Rockland, Maine, USA) and visualized with ethidium bromide.
PCR products were prepared for sequencing using a QIAquick PCR purification kit (QIAGEN, Valencia, California, USA). Nucleotide sequences were determined by the cyclic reaction termination method using fluorescence labeled dideoxyribonucleotide triphosphates. Sequencing reactions and the processing of reaction products were performed following instructions for the ABI PRISMTM Dye Terminator Cycle Sequencing Core Kit (PE Applied Biosystems, Foster City, California, USA). Electrophoresis and data collection were done on an ABI Model 377 DNA Sequencer (Perkin-Elmer Corporation). DNA Sequencing Analysis (version 2.1.2) and Sequence Navigator (version 1.0.1) were used for processing the raw data.
Phylogenetic analysis
Phylogenetic analyses were performed in PAUP*. ITS sequences were aligned manually using the PAUP editor and a color font. Sequences immediately adjacent to the priming sites of the sequencing primers were of low quality in some of the sequences, and the respective areas from the 18S, 5.8S and 28S genes were excluded from the analysis. Within the areas included in the active data set, most single-basepair alignment gaps were parsimony uninformative and were treated as missing data. Presence or absence of one parsimony informative single-bp alignment gap and several 2–4 bp alignment gaps was weighted equal to one substitution (=1 step). Technically, this was done by replacing one dash per alignment gap with a new character state "I." The longest alignment gap observed was 6 bp long, and was coded for by inserting the character state "I" twice, giving it a total weight of two steps. This recoding method attempts to conserve some of the information provided by alignment gaps, without weighting the insertion or deletion of every single nucleotide equal to a substitution. This downweighting of the alignment gaps seems justified, because several of the larger insertions (deletions) were found to represent duplications (losses) of two and four nucleotide motifs likely stemming from single insertion (deletion) events. The alignment with the recoded gaps can be viewed at http://ajbsupp.botany.org/v87/kretzer.txt.
Most parsimonious trees were retrieved through ten heuristic searches with random sequence addition. Bootstrap values are based on 500 replicates with five random sequence additions each.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We were able to directly amplify fungal ITS regions from 48 out of 64 S. sanguinea plants (Table 1). Amplification success and failure were strongly correlated with the presence or absence of a well-developed mantle as described above. Since the primer pair ITS-1f/ITS4 amplifies a shorter fragment than ITS-1f/ITS-4b, it was often successful when the latter failed. When the obtained PCR products were digested with the restriction enzymes AluI or HinfI, identical banding patterns were obtained across all samples, a selection of which is shown in Fig. 2.
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Similarity of the fungal ITS-RFLPs from S. sanguinea mycorrhizae to those obtained previously from Rhizopogon subcaerulescens (data not shown) as well as morphological characteristics (white mantle staining violaceous with age; occasional presence of rhizomorphs) led us to believe that the S. sanguinea root symbiont might be a taxon in Rhizopogon section Amylopogon. To address this hypothesis, we determined the nucleotide sequences of the fungal ITS region from four different S. sanguinea rootballs by direct sequencing of PCR-products obtained with the fungus-specific primer pairs ITS1f/4b or ITS1f/4. In addition, we also sequenced the ITS region from two fungal cultures that had been isolated from two different S. sanguinea plants (for details see Materials and Methods and Table 2). These sequences were aligned with previously published sequences from a wide range of Rhizopogon species (Grubisha, 1998
) and proved to be most similar to the ITS sequences of Rhizopogon ellenae AHS66137 and T17476 (data not shown). Rhizopogon ellenae is a species within the monophyletic section Amylopogon (Grubisha, 1998
). To increase the resolution within this section, we sequenced the ITS region from eight additional taxa given in Table 2. Collections with "SNF" collection numbers are Amylopogon fruitbodies collected in 1994 and 1995 in the "Sierra National Forest" of California (southeast of Shaver Lake). To determine genetic diversity among those collections, we first produced ITS-RFLPs with the restriction enzymes HinfI, AluI and CfoI, and subsequently sequenced one representative of every RFLP type. Collections with "HDT" collection numbers were obtained from the Harry Thiers Herbarium at San Francisco State University. Finally, we also sequenced the ITS region from one fungal symbiont of Pterospora andromedea. The complete dataset has been posted at the following website: http://ajbsupp.botany.org/v87/kretzer.txt. It comprises 22 taxa from section Amylopogon and 539 characters, of which 26 were parsimony informative.
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DISCUSSION |
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, and personal communication) has monitored a population of S. sanguinea over 17 yr and reports that most plants flowered only once during this period. However, in a certain number of cases (varying from 0 to 35% between years and averaging 12%) an inflorescence will appear in the very same location as in the previous year. We hypothesize that a small percentage of plants flower in two subsequent years and that the roots of those plants have an older appearance in the second year. Because those plants failed to yield PCR products, direct molecular evidence is missing to support the fact that they were colonized by the same fungal species as were the younger roots. Two other lines of evidence, however, suggest that the same fungus was present. (1) Remnant patches of a fungal mantle present on the aging roots did exhibit the pinkish to purplish color characteristic of aging mycorrhizae formed by section Amylopogon. (2) In a few cases we were able to culture the fungal symbiont from such aging roots, and it exhibited the same cultural and ITS-RFLP characteristics as did all the other symbionts. We conclude that all S. sanguinea plants that we sampled were associated with the same mycorrhizal fungus that is a species in the R. ellenae species complex.
These findings are in conflict with a previous report by Cullings, Szaro, and Bruns (1996)
who found S. sanguinea associated with fungi from at least three different lineages of Hymenomycetes. There are two possible reasons for this discrepancy: (1) Cullings, Szaro, and Bruns (1996)
included samples from a somewhat broader geographic range in the Sierra Nevada of California and also sampled at least some remote and unmanaged habitats (K. Cullings, personal communication), while all our samples were taken in two areas of the southern and central Sierra Nevada within a few hundred metres of roads. Geographic mosaics are a common feature of many parasites; often what appears to be a generalist at a broad geographic scale is composed of several geographically defined host-specific populations (Thompson, 1994
). (2) Cullings, Szaro, and Bruns (1996)
may have overestimated diversity through PCR chimeras or may have inadvertently amplified saprobic fungi associated with older roots. In either case, however, S. sanguinea can no longer be considered the generalist that it earlier appeared to be (Cullings, Szaro, and Bruns, 1996
). Instead, it appears restricted to associations with a single species of Rhizopogon within at least two large areas of its range and most of its habitats. This removes S. sanguinea as the only current exception to the emerging pattern that nonphotosynthetic mycorrhizal plants exhibit high levels of specificity. It also changes the way we view the evolution of specialization in the Monotropoideae. Initially it appeared that there was a gradual shift toward specialization (Cullings, Szaro, and Bruns, 1996
); now there is no clear evidence for a member of the Monotropoideae that is a true generalist.
ITS sequences collected and analyzed in this study have greatly refined our understanding of species groups within section Amylopogon. We have therefore revisited the P. andromedea symbiont that Cullings, Szaro, and Bruns (1996)
identified as a species in the "R. subcaerulescens group" based on sequence analysis and oligonucleotide probing of the mitochondrial large subunit rDNA. Cullings, Szaro, and Bruns (1996)
also report having sequenced the ITS region of six P. andromedea symbionts, but only two partial ITS sequences have been published in Cullings (1993)
. We therefore sequenced the complete ITS region of one P. andromedea symbiont and included it in the current analysis. Our sequence, as well as Cullings' two partial sequences (data not shown), groups tightly with species group 1 in the current analysis and is clearly distinct from the "R. ellenae species complex" to which the Sarcodes symbiont belongs (Fig. 3). However, because Cullings, Szaro, and Bruns (1996)
report RFLP variation as well as 0.3–2% sequence variation in the ITS region of P. andromedea symbionts, complete ITS sequences should be generated from the symbionts of more P. andromedea plants.
Although our work has contributed to a better understanding of species groups in Rhizopogon section Amylopogon, taxonomy of the group remains unsettling. Current taxonomy of Rhizopogon is based mostly on the morphological species concepts of Smith and Zeller (1966)
, which often turn out to be in conflict with genetic species concepts. From other studies we know for example that different paratype collections of Rhizopogon vinicolor Smith represent two very distinct genetic species (Kretzer, unpublished data). On the other hand, several collections representing different species according to the current morphological species concepts are often indistinguishable by ITS sequence data. The latter is also true for several collections in what we call the "Rhizopogon ellenae species complex." We have chosen that name, because R. ellenae is the only holotype sequence currently known to fall within this group of taxa that may represent one or more reproductively isolated species.
In conclusion, this study has shown that S. sanguinea specializes at least regionally on a single fungal symbiont within the R. ellenae species complex. Furthermore, based on the analysis of fungal ITS sequences from three P. andromedea plants (two partial sequences are not being shown), the emerging picture is that the two monotropoid species P. andromedea and S. sanguinea specialize on two closely related but distinct Rhizopogon species from section Amylopogon. Since both monotropoid plant species often co-occur within metres of each other, specificity of their fungal associations is apparently not governed by habitat or local availability but rather by direct plant–fungus interaction.
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FOOTNOTES |
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5 These authors have contributed equally to this study.
6 Author for reprint requests.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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———, T. M. Szaro, and T. D. Bruns. 1996 Evolution of extreme specialization within a lineage of ectomycorrhizal epiparasites. Nature 379: 63–66[CrossRef][ISI]
Francke, H.-L. 1934 Beiträge zur Kenntnis der Mykorrhiza von Monotropa hypopitys L. Analyse und Synthese der Symbiose. Flora (Jena) 129: 1–52
Gardes, M., and T. D. Bruns. 1993 ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113–118[Medline]
———, and ———. 1996 Community structure of ectomycorrhizal fungi in a Pinus muricata forest: above- and below-ground views. Canadian Journal of Botany 74: 1572–1583[CrossRef]
Grubisha, L. 1998 Systematics of the genus Rhizopogon inferred from nuclear ribosomal DNA large subunit and internal transcribed spacer sequences. Master's thesis, Oregon State University, Corvallis, Oregon, USA
Kamienski, F. 1881 Die Vegetationsorgane der Monotropa hypopitys L. Botanische Zeitung 29: 458
Lefevre, C. K., C. M. Carter, and R. Molina. 1998 Morphological and molecular evidence of specificity between Allotropa virgata and Tricholoma magnivelare. Poster presentation at the Second International Conference on Mycorrhiza in Uppsala, Sweden. Program and Abstracts: 107
Luoma, D. 1996 Fifteen years amongst the snow plants. Oral presentation at the First International Conference on Mycorrhizae in Berkeley, California. Program and Abstracts: 79
Smith, A. H., and S. M. Zeller. 1966 A preliminary account of the North American species of Rhizopogon. Memoirs of the New York Botanical Garden 14: 1–178
Stevens, R. B. [ed.] 1981 Mycology Guidebook, 2nd print. University of Washington Press, Seattle, Washington, USA
Taylor, D. L., and T. D. Bruns. 1997 Independent specialized invasion of ectomycorrhizal mutualism by two nonphotosynthetic orchids. Proceedings of the National Academy of Sciences, (USA) 94: 4510–4515[CrossRef]
Thompson, J. N. 1994 The coevolutionary process. University of Chicago Press, Chicago, Illinois, USA
Wallace, G. D. 1975 Studies of the Monotropoideae (Ericaceae): taxonomy and distribution. Wasmann Journal of Biology 33: 1–88
White, T. J., T. D. Bruns, S. Lee, and J. W. Taylor. 1990 Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White [eds.], PCR protocols; a guide to methods and applications, 315–322. Academic Press, San Diego, California, USA
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