Independent, specialized invasions of ectomycorrhizal mutualism by two nonphotosynthetic orchids

Abstract

We have investigated the mycorrhizal associations of two nonphotosynthetic orchids from distant tribes within the Orchidaceae. The two orchids were found to associate exclusively with two distinct clades of ectomycorrhizal basidiomycetous fungi over wide geographic ranges. Yet both orchids retained the internal mycorrhizal structure typical of photosynthetic orchids that do not associate with ectomycorrhizal fungi. Restriction fragment length polymorphism and sequence analysis of two ribosomal regions along with fungal isolation provided congruent, independent evidence for the identities of the fungal symbionts. All 14 fungal entities that were associated with the orchid Cephalanthera austinae belonged to a clade within the Thelephoraceae, and all 18 fungal entities that were associated with the orchid Corallorhiza maculata fell within the Russulaceae. Restriction fragment length polymorphism and single-strand conformational polymorphism analysis of ectomycorrhizal tree roots collected adjacent to Cephalanthera showed that (i) the fungi associated internally with Cephalanthera also form typical external ectomycorrhizae and that (ii) ectomycorrhizae formed by other Basidiomycetes were abundant where the orchid grows but these fungi did not associate with the orchid. This is the first proof of ectomycorrhizal epiparasitism in nature by an orchid. We argue that these orchids are cheaters because they do not provide fixed carbon to associated fungi. This view suggests that mycorrhizae, like other ancient mutualisms, are susceptible to cheating. The extreme specificity in these orchids relative to other ectomycorrhizal plants agrees with trends seen in more conventional parasites.

Figure 1

Phylogenetic placement of orchid fungi. (a) Each orchid targets a distinct clade of ectomycorrhizal fungi. ML5–6 sequences from symbionts of both orchids were aligned with those from fruit bodies of 112 (mostly ECM) Basidiomycete species. This database has been used previously (19, 34); details will be presented elsewhere. Analysis by the distance method of neighbor-joining under a Jukes–Cantor one-parameter model using paup 4.0 (39) produced a tree in which all symbionts of C. austinae were grouped several nodes interior to the boundaries of the Thelephorales, and all unknown symbionts of C. maculata were similarly grouped well within the Russulales. These placements were strongly supported by a 1000 replicate neighbor-joining boot strap analysis (numbers near branches). Most taxa were then pruned from the tree, leaving a taxon from each major clade in the representation above. Cantharellus was used as the outgroup. (b) ITS sequences provide strong, independent evidence for the placement of the symbionts of C. austinae in a clade within the Thelephoraceae. ITS sequences were generated for each fungal ITS RFLP type associated with C. austinae and from fruit bodies of the Thelephoraceae. ITS sequences were obtained for Sarcodon imbricatum, Hydnum umbilicatum, Hydnellum peckii, and Pseudotomentella trisitis in addition to the taxa shown above. However, none of these could be aligned with the ITS sequences obtained from Cephalanthera associates or Thelephora and Tomentella fruit bodies. Parsimony analysis using 100 random addition replicates of the remaining taxa by paup produced the single tree, shown above. Midpoint rooting was used because of lack of an a priori choice for outgroup. The neighbor-joining tree also agreed with all well supported branches from the parsimony analysis. Parsimony boot strap values from 1000 replicates are shown above branches, and decay indices are shown below branches. Although taxa sampling was limited, this analysis provides evidence that all Cephalanthera associates are more closely related to Thelephora and Tomentella than to other sampled genera of the Thelephoraceae.

Figure 2

The same fungal species forms contrasting mycorrhizal structures when associated with nonphotosynthetic orchid or photosynthetic tree host. All structures were formed simultaneously by the fungus Thelephoraceae 12 on tightly intermingled C. austinae and tree roots recovered from a single soil core. (a) Intact ECM tree root, ×25. (b) Cross section of an ECM tree root showing thick fungal mantle (arrow, m) and Hartig net (arrow, n) but no intracellular hyphae, ×400. (c) Cross section of a C. austinae root demonstrating the lack of a mantle surrounding the epidermis (arrow, e) or a Hartig net and the presence of intracellular coils or pelotons (arrow, p) typical of orchidaceous mycorrhizae, ×100. (d) Cephalanthera root cross section showing a single peloton and typical dark hyphae with clamp connections (arrow, c), ×400.