Mycologia
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DOI: 10.3852/mycologia.98.4.535
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Mycologia, 98(4), 2006, pp. 535-540.
© 2006 by The Mycological Society of America

Monotropa uniflora plants of eastern Massachusetts form mycorrhizae with a diversity of russulacean fungi


S. Yang 1
D.H. Pfister

     Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

Plant species in the subfamily Monotropoideae are mycoheterotrophs; they obtain fixed carbon from photosynthetic plants via a shared mycorrhizal network. Previous findings show mycoheterotrophic plants exhibit a high level of specificity to their mycorrhizal fungi. In this study we explore the association of mycorrhizal fungi and Monotropa uniflora (Monotropoideae: Ericaceae) in eastern North America. We collected M. uniflora roots and nearby basidiomycete sporocarps from four sites within a 100 km2 area in eastern Massachusetts. We analyzed DNA sequences of the internal transcribed spacer region (ITS) from the fungal nuclear ribosomal gene to assess the genetic diversity of fungi associating with M. uniflora roots. In this analysis we included 20 ITS sequences from Russula sporocarps collected nearby, 44 sequences of Russula or Lactarius species from GenBank and 12 GenBank sequences of fungi isolated from M. uniflora roots in previous studies. We found that all 56 sampled M. uniflora mycorrhizal fungi were members of the Russulaceae, confirming previous research. The analysis showed that most of the diversity of mycorrhizal fungi spreads across the genus Russula. ITS sequences of the mycorrhizal fungi consisted of 20 different phylotypes: 18 of the genus Russula and two of Lactarius, based on GenBank searches. Of the sampled plants, 57% associated with only three of the 20 mycorrhizal fungi detected in roots, and of the 25 sporocarp phylotypes collected three, were associated with M. uniflora. Furthermore the results indicate that the number of different fungal phylotypes associating with M. uniflora of eastern North America is higher than that of western North America but patterns of fungal species abundance might be similar between mycorrhizae from the two locations.

Key words: epiparasite, internal transcribed spacer (ITS), monotropoid, mycoheterotroph, mycorrhiza, symbiosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
A mycoheterotroph (or epiparasite) is an achlorophyllous, nonphotosynthetic plant that obtains fixed carbon from photosynthetic plants via mycorrhizal fungi (Björkman 1960Go, Leake 1994Go). Björkman (1960)Go showed a connection between Monotropa hypopithys and trees when he injected radioactive glucose and phosphorus into the phloem of pine and spruce trees under which the mycoheterotroph grew. He found that the radioactivity passed from tree to M. hypopithys and physical separation from trees was severely detrimental to M. hypopithys growth. Björkman called this obligate interaction "epiparasitism" because the mycoheterotroph indirectly parasitizes trees. Bidartondo and Bruns (2001)Go suggest this indirect connection might make the mycoheterotroph successful because it can "cheat" the photosynthetic symbiont; the photosynthetic host cannot select against the mycoheterotroph without selecting against its own mutualist mycorrhizal fungi.

Among the mycoheterotrophs studied to date, all have specific associations with certain ectomycorrhizal (Cullings et al 1996Go, Bidartondo and Bruns 2001Go, Bidartondo and Bruns 2002Go) or arbuscular mycorrhizal fungi (Bidartondo et al 2002Go). Results of Bidartondo and Bruns 2002Go indicate that evolution of members of the Monotropoideae is tightly coupled to that of their mycorrhizal symbionts.

Studies of M. uniflora from various locations in eastern and western North America, Eurasia and Japan have shown that it specifically associates with fungi of the family Russulaceae (Basidiomycota) (Cullings et al 1996Go, Bidartondo and Bruns 2001Go, Young et al 2002Go). Bidartondo and Bruns (2001)Go sampled a total of 35 M. uniflora plants from Nova Scotia, Oregon, Virginia, Vermont and Japan and identified Russula brevipes, R. paludosa, R. cremoricolor, R. postiana, R. integra, and Lactarius theiogalus as mycorrhizal associates. Young et al (2002)Go sampled a total of 15 M. uniflora plants from three sites in British Columbia and found three mycorrhizal fungi, two sequences of which clustered with the hypogeous genera Martellia and Gymnomyces (both Russulaceae) in phylogenetic analysis. These previous studies hinted at an intriguing geographic variation in the species diversity of mycorrhizal fungi associated with the mycoheterotroph; diversity was lower in western North American populations than those in eastern North America (Bidartondo and Bruns 2001Go, Bidartondo 2005Go, Bidartondo and Bruns 2005Go).

M. uniflora is distributed widely in New England (Seymour 1989Go), where few of the plants have been sampled for mycorrhizal associates. To further explore the identity and diversity of mycorrhizal fungi associated with M. uniflora in the plant’s eastern North American range, we selected four sites in eastern Massachusetts, in the eastern mixed forest biome. We used sequencing, GenBank searches and phylogenetic analysis of sequences to study the association of eastern M. uniflora plants with members of the Russulaceae.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Selection and description of field sites.— – Four field sites were located in four towns in eastern Massachusetts. The Concord Field Station (Bedford) and Estabrook Woods (Concord) have a range of evergreen and deciduous tree genera, including Pinus, Tsuga, Acer, Betula, Quercus and Fagus. Whipple Hill (Lexington) is on a large rock outcropping with a mosaic of habitats, including grassland, wetlands, bare rock, low shrub stands and forest, including a Carya stand. Flint’s Pond woods (Lincoln) surrounds the Lincoln reservoir and has large stands of Betula and Fagus. The four field sites occur within an area of approximately 10 km x 10 km. Within each site, the minimum and maximum distances between any two samples were approximately 1 m and 1 km, respectively.

Sampling M. uniflora roots and fungal sporocarps.— – Throughout Jul and Aug 2003, we sampled the highly branched and tightly interwoven roots of M. uniflora. We sampled these root balls by locating a flowering M. uniflora plant and carefully following the shoot to the roots. We collected soil samples containing a portion of the tight M. uniflora root ball.

We stored the soil samples for a maximum of 2 d at 4 C until root harvesting. Root harvesting involved soaking the soil samples in tap water to loosen the soil around the roots, separating and cleaning the roots. We harvested at least four root tips from each M. uniflora root ball, storing them separately in 1.5 mL tubes at –80 C until extraction.

As Russula and other fungi fruited, we collected sporocarps within a radius approximately 5 m of sampled M. uniflora individuals to cursorily survey what fungi were available to M. uniflora plants in an area. Immediately upon return from a field site we made spore prints from the collected mushrooms and took notes on mushroom characters. We harvested 1–4 approximately 0.5 cm3 pieces of sporocarp tissue from each mushroom and stored the pieces separately at –80 C for DNA extraction. We dried the rest of the specimens in a heat-desiccator at least 48 h. Specimens used for DNA sequencing are in the Farlow Herbarium (TABLE IGo).


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  		TABLE I. GenBank accession numbers of sequences generated in this study (A–KK), and Farlow Herbarium (FH) collection numbers for sporocarps sequenced, followed by GenBank accession numbers of sequences from other studies included in our analysis

 
Molecular methods and analyses.— – We extracted DNA from two root tips per M. uniflora individual and from one sample of every sporocarp, following the protocol of Gardes and Bruns (1993)Go, except without ß-mercaptoethanol. We amplified the ITS region of root fungus and sporocarp DNA for sequencing. The ITS region, located in the ribosomal RNA gene, is good to use for fungal identification because it has sufficient variation to place unknowns at the species level or to species group (White et al 1990Go, Horton and Bruns 2001Go). We used the basidiomycete-specific primer pair ITS1F and ITS4B (Gardes and Bruns 1993Go) and amplified the ITS region in 25 µL reactions with the following final concentrations as recommended by the manufacturer of Taq polymerase (Invitrogen). We used a GeneAmp PCR System 9600 (Perkin Elmer) to run the program designed by Gardes and Bruns (1993)Go.

We sequenced a total of 56 mycorrhizal fungi and 25 sporocarp samples. We used a QIAquick PCR Purification Kit (QIAGEN) to clean the ITS1F-ITS4B PCR products for cycle sequencing. Each cycle-sequencing reaction contained 1.5 µL cleaned PCR product, 2.0 µL BigDye v. 3 (Applied Biosystems), 1.6 µL 1 µM primer, 2.9 µL purified H2O. We used a DNA Engine thermal cycler (MJ Research Inc.) for cycle-sequencing reactions with the program recommended by the BigDye manufacturer. We purified cycle-sequencing reactions by ethanol precipitation and performed electrophoresis in an ABI-Prism 3100 automated DNA sequencer (Applied Biosystems). If either or both of the external primers did not provide a good sequence, we used these internal primers: ITS3 (White et al 1990Go) and 5.8 S (http://www.biology.duke.edu/fungi/mycolab/primers.htm). We searched for any matching sequences from GenBank with the Basic Local Alignment Search Tool (BLAST).

Sequence and phylogenetic analysis.. To view the placement of sample mycorrhizal fungus phylotypes within a larger phylogeny of Russula and Lactarius species, we aligned the 56 sample root isolate ITS sequences with 44 Russula and Lactarius GenBank ITS sequences. We also aligned 12 M. uniflora root fungal sequences from GenBank, generated by Bidartondo and Bruns (2001)Go and Young et al (2002)Go (TABLE IGo). We added to the alignment 20 ITS sequences of sample nearby Russula sporocarps to assess the range of fungi available to M. uniflora plants. We manually aligned the sequences with Sequence Alignment Editor v. 2.0 (Rambaut 1996Go). A single phylotype (labeled A-KK) represents identical sample sequences. We submitted the sample ITS sequences to GenBank (TABLE IGo) and the alignment to TreeBase (http://www.treebase.org, accession number S1646).

We performed phylogenetic analyses with PAUP*4.0b10 (Swofford 2002Go). We used Gloeocystidiellum aculeatum as outgroup because it is a member of the corticioid clade, which has been shown to form a sister group with members of the Russulaceae (Hibbett and Thorn 2001Go). The parsimony analysis was a heuristic search including all characters with 1000 replicates, starting trees obtained via stepwise addition, random sequence addition, tree-bisection-reconnection (TBR) branch-swapping, and an unlimited number of trees to be saved. We performed bootstrap analysis with 500 replicates with a heuristic search with 100 replicates, random sequence addition, MAXTREES set to 100 not to be increased, starting trees obtained via stepwise addition, and TBR branch-swapping.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
We commonly found Russula, Lactarius, Boletus, Amanita, Cortinarius, and Paxillus sporocarps in the vicinity of the sampled M. uniflora individuals.

Sequencing mycorrhizal fungi obtained from roots of 56 M. uniflora individuals resulted in 20 different phylotypes, and sequencing 25 sporocarps resulted in 20 phylotypes. The sequences of mycorrhizal fungi and sporocarps together represent 37 phylotypes total (labeled A–KK, FIG. 1Go). Most frequent phylotypes among mycorrhizal fungus isolates were phylotypes U (25% of 56 mycorrhizal fungi isolated), J (18%), X (14%), A–C (14%), and O–P (7%). The rest (22%) of the mycorrhizal fungus samples represented unique phylotypes.


Figure 1
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  		FIG. 1. This example maximum parsimony tree (of 5420 trees) shows the 37 sample fungal sequences from root tips and sporocarps (labeled A–KK) and 56 GenBank sequences. Of the 56 sampled root fungi, 20 different phylotypes were found. Boldface emphasizes root fungal sequences. Lightface denotes root fungal sequences from previous studies (i.e. S123, NC2172). Gray print denotes sporocarp sequences generated in this study and nonroot GenBank sequences. Numbers above branches indicate bootstrap values. An asterisk (*) indicates branches that are collapsed in the strict consensus tree. Our samples have been indicated as: number of sequences retrieved, source of the sample, roots "R" or sporocarps "S," and the locations, Concord Field Station (CFS), Estabrook Woods (ES), Flint’s Pond Woods (LN) and Whipple Hill (WH).)

 
All phylotypes clustered with Russula species, except two, which grouped with Lactarius species (FIG. 1Go). GenBank searches of the root fungal sequences gave the same results as closest matches (54 Russula, two Lactarius), although none of the phylotypes matched 100% with any GenBank sequences. Only three of the 25 sequenced sporocarps had sequences that matched those of mycorrhizal fungi (phylotypes C, K and O) (FIG. 1Go). Where sporocarps did match mycorrhizal fungi, they were from different locations. Mycorrhizal fungi isolated from M. uniflora individuals near the sporocarps with phylotypes C, K and O formed mycorrhizae, not with these types but rather with other Russula species. For example for phylotype O mycorrhizal fungus sequences came from Estabrook and the Concord Field Station and the sporocarp with a matching sequence was from the Lincoln site (LN). The fungus from a M. uniflora near the sporocarp of phylotype O at LN was phylotype G.

Furthermore all phylotypes representing more than one root sample consisted of samples from 2–3 sites. For example phylotype U, which represented 14 root samples, included mycorrhizal fungus sequences from Estabrook, Lincoln and Whipple Hill (FIG. 1Go). Also, for all plants with two root tips sampled each (three plants), the fungal sequences from the same plant were identical.

Two fungi from roots grouped with root fungi from previous studies, phylotype U with VT2364 and phylotype W with NC2172, both with 100% bootstrap values (FIG. 1Go). No roots collected in this study had fungi that clustered with the R. pulverulenta-R. parazurea clade (93% boostrap), although two root fungal sequences, S132 and S144/S323, from British Columbia did and from this study six sporocarp phylotypes, I, Q, H, FF, GG, BB (FIG. 1Go), also grouped there.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
In our investigation of M. uniflora mycorrhizal fungus diversity, all root fungi were members of the Russulaceae, despite the availability of other mycorrhizal fungi (FIG. 1Go), and each M. uniflora plant appears to be colonized by a single fungal phylotype, confirming other studies (Cullings et al 1996Go, Bidartondo and Bruns 2001Go, Young et al 2002Go, Bidartondo and Bruns 2005Go). The genus Russula includes more than 750 species (Kirk et al 2001Go), an exceptionally large number. To place our isolates we used the sequences used in a previous phylogenetic study of the genus, which broadly sampled classical taxonomic groups (Miller and Buyck 2002Go). The distribution of sample root fungi and GenBank root fungi in the Russula phylogeny shows that compatible fungi are spread widely across the entire genus, at least so far as it has been sampled currently.

Our results confirm findings that the number of mycorrhizal species from eastern North American M. uniflora populations is greater than that found from western populations (Bidartondo and Bruns 2001Go, Bidartondo 2005Go, Bidartondo and Bruns 2005Go). Similar to the 20 different phylotypes found in this study of eastern plants, Bidartondo and Bruns (2001)Go found four different root fungi from plants of a single population in Vermont. In contrast they found Russula brevipes to be the only mycorrhizal fungus with M. uniflora plants sampled in a 9400 km2 area in Oregon and Young et al (2002)Go found only three different mycorrhizal fungi associating with 15 plants sampled from British Columbia. Furthermore we found the most frequently identified associates in multiple sites (FIG. 1Go), suggesting the genetic diversity of the fungi was not site-specific.

Despite the difference in overall mycorrhizal fungus species-number between eastern and western M. uniflora populations, similar species-abundance patterns are evident between western plants and the eastern plants sampled in this study. First, we found the number of root samples per phylotype was not distributed evenly; 57% of the sampled plants associated with only three of the 20 mycorrhizal fungi. Similarly, in western North America, Young et al (2002)Go found one of three mycorrhizal fungi dominated. Second, we found no species of the R. pulverulenta-R. parazurea clade, even though members of the clade formed mycorrhizae with western plants (S144/S323, S123), and members of this clade were present among sporocarps collected in the vicinity of M. uniflora plants (phylotypes I, Q, H, FF, GG) (FIG. 1Go). Third, no root fungi were identical to nearby sporocarps, even when a nearby fungus had been found to form mycorrhizae with M. uniflora roots in other locations (phylotypes C, K, O) (FIG. 1Go). We conclude that only a few fungal species dominated as M. uniflora mycorrhizae despite the availability of fungal species that are compatible with M. uniflora from other locations. This suggests that specificity might be more complex than random partnering with the available members of the Russulaceae.

Bidartondo and Bruns 2005Go suggest a mechanism for the origin of specificity patterns in mycoheterotrophs. They showed in germination experiments that seedlings of M. uniflora and other plants from the Monotropoideae developed best when associated with the fungus species found with the maternal plant. They suggest that cues for M. uniflora germination are heritable and that fungal performance trade-offs might explain the narrow specificity of individual mycoheterotrophic plants to their mycorrhizal fungi.

Population studies of the genetic diversity of M. uniflora might help to explain if specificity to the Russulaceae arose differently in different lineages (Bidartondo and Bruns 2005Go). M. uniflora plants in North America appear to represent a group genetically distinct from those in Japan (Bidartondo and Bruns 2001Go, Neyland and Hennigan 2004Go), and M. uniflora populations exhibit extensive morphological variation (Wallace 1975Go). Further sampling of the genetic diversity of M. uniflora and its associated fungi across its range might add information to the genetic diversity of the partnership.


   FOOTNOTES
 
Accepted for publication May 19, 2006.

1 Corresponding author. E-mail: sylvia_yang{at}post.harvard.edu


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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———, Bruns T. 2001. Extreme specificity in epiparasitic Monotropoideae (Ericaceae): widespread phylogenetic and geographical structure. Mol Ecol 10:2285–2295.[CrossRef][Medline]

———. 2002. Fine-level mycorrhizal specificity in the Monotropoideae (Ericaceae): specificity for fungal species groups. Mol Ecol 11:557–569.[CrossRef][Medline]

———. 2005. On the origins of extreme mycorrhizal specificity in the Monotropoideae (Ericaceae): performance trade-offs during seed germination and seedling development. Mol Ecol 14(5):1549–1560.[CrossRef][Medline]

———, Redecker D, Hijri I, Wiemken A, Bruns T, Domínguez L, Sérsic A, Leake J, Read D. 2002. Epiparasitic plants specialized on arbuscular mycorrhizal fungi. Nature 419:389–392.[CrossRef][Medline]

Björkman E. 1960. Monotropa hypopitys L.—an epiparasite on tree roots. Physiol Plantar 13:308–327.[CrossRef]

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

Hibbett D, Thorn R. 2001. Homobasidiomycetes. In: McLaughlin D, McLaughlin E, Lemke P, eds. Systematics and Evolution. Berlin: Springer-Verlag: The Mycota VIIB:121–170.

Horton T, Bruns T. 2001. The molecular revolution in ectomycorrhizal ecology: peeking into the black box. Mol Ecol 10:1855–1871.[CrossRef][Medline]

Kirk P, Cannon P, David J, Stalpers J, eds. 2001. Dictionary of the Fungi. 9th ed. Wallingford: CAB International.

Leake J. 1994. The biology of myco-heterotrophic (‘saprophytic’) plants. New Phytol 127:171–216.[CrossRef]

Miller S, Buyck B. 2002. Molecular phylogeny of the genus Russula in Europe with a comparison of modern infrageneric classifications. Mycol Res 106(3):259–276.[CrossRef]

Neyland R, Hennigan M. 2004. A cladistic analysis of Monotropa uniflora (Ericaceae) inferred from large ribosomal subunit (26 S) rRNA gene sequences. Castanea 69(4):265–271.[CrossRef]

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Young B, Massicotte H, Tackaberry L, Baldwin Q, Egger K. 2002. Monotropa uniflora: morphological and molecular assessment of mycorrhizae retrieved from sites in the sub-boreal spruce biogeoclimatic zone in central British Columbia. Mycorrhiza 12:75–82.[Medline]




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