3Botany Department, University of Tennessee, Knoxville, TN

Flammulina (Basidiomycetes; Agaricales, Tricholomataceae) is a popular edible mushroom that has been cultivated for centuries in Japan and marketed worldwide under the name enoki-take. Until the 1960s Flammulina was thought to consist of a single species with a pan-Northern Hemisphere distribution. As a result the name Flammulina velutipes (Curt.: Fr.) Singer (FIG 1) was more or less uniformly applied to all collections in the genus whether commercially or naturally produced (Buchanan 1993). Beginning in the 1960s a series of papers that described new Flammulina taxa or reported incompatibility between strains cast doubt on the uniform application of the epithet velutipes. Based on the hypothesis that additional taxa might be sheltered within the epithet velutipes, a project was conceived to examine Flammulina from three directions: Morphology, compatibility studies, and molecular data. In the ensuing studies approximately 200 collections of Flammulina were identified to species based on morphology (Redhead and Petersen 1999), mating studies (Petersen et. al. 1999), and RFLP patterns (Table I; Methven et. al. 2000). These species designations were subsequently confirmed by ITS sequences using geographically diverse collections of each morphospecies (Hughes et. al. 1999).

Petersen et. al. (1999) revealed partial compatibility between interspecific crosses of F. ononidis and F. elastica, F. ononidis and F. populicola, F. ononidis and F. velutipes, F. populicola and F. velutipes, and F. rossica and F. velutipes (Table II) and maintained several laboratory generated interspecific hybrids in culture. A number of these hybrids have been fruited in culture (Figure 1) and produced more or less normal basidiomata with viable basidiospores (Petersen and Methven, unpublished data). Hughes and Petersen (2001) recently documented an apparent hybridization event in nature between F. velutipes and F. rossica that resulted in a homogenized ribosomal repeat that contains elements of both parents. To further document the potential patterns of ITS RFLPs in Flammulina hybrids, we surveyed several laboratory generated interspecific hybrids (Petersen et. al. 1999) in this genus. Comparison of the RFLP signatures of hybrids to their parents demonstrated a complicated pattern of ITS evolution; additivity and concerted evolution were observed in the ITS hybrids.

Figure 1. Flammulina hybrid fruiting in culture

Procedures for isolation and maintenance of cultures are given by Hughes et. al. (1999). Interspecific hybrids used in this study (Table II) were generated in the laboratory by Petersen et. al. (1999). DNA extractions and PCR amplifications followed techniques outlined by Hughes et. al. (1999). RFLP digestions adhered to the protocol utilized by Methven et. al. (2000). For restriction digestions ca 200 ng of amplified ITS1-5.8S-ITS2 DNA were digested with 1-10 units of restriction enzyme following manufacturer’s directions. Digestion products were electrophoresed on a 1.5% agarose gel in 1X TBE buffer (Sambrook et. al. 1989), stained for 20 minutes in ethidium bromide, and visualized on a UV transilluminator. Gels were documented using the Kodak EDAS system (Eastman Kodak).

The F. ononidis x F. elastica f. longispora hybrid is olates exhibited concerted evolution in both the Hae III and Bst F51 restriction sites (Figure 3, lane 6) and produced a Hae Hae than F. elastica f. longispora (three Hae III restriction site and four bands; one Bst F51 restriction site and two bands). The F. ononidis x F. populicola hybrid isolates exhibited concerted evolution in the Hae III restriction sites (Figure 2, lane 12) and produced a Hae III pattern characteristic of F. populicola rather than F. ononidis (three Hae III restriction sites and four bands). The F. ononidis x F. velutipes var. velutipes hybrid isolates demonstrated additivity in the Hae III restriction sites (Figure 2, lanes 6-7) with four bands characteristic of the three Hae III restriction sites of F. ononidis Hae

In the F. populicola x F. velutipes var. lupinicola hybrid isolates, both additivity and concerted evolution in the Hae III restriction sites were documented. One F. populicola x F. velutipes var. lupinicola hybrid revealed concerted evolution in the Hae III restriction sites and produced a Hae III pattern characteristic of F. velutipes var. lupinicola (oneHae III restriction site and two bands) instead of F. populicola (two Hae III restriction sites and three bands). The remaining five F. populicola x F. velutipes var. lupinicola hybrids revealed additivity in the Hae III restriction sites with three bands characteristic of the two Hae III restriction sites in F. populicola and two bands characteristic of the single Hae III restriction site in F. velutipes var. lupinicola (Figure 2, lane 10).

In the F. populicola x F. velutipes var. velutipes hybrid isolates both additivity and concerted evolution in theHae III restriction sites were documented. Two of the F. populicola x F. velutipes var. velutipes hybrids revealed concerted evolution in the Hae III restriction sites (Figure 2, lanes 13-14) and produced a Hae III pattern characteristic of F. populicola (two Hae III restriction sites and three bands) rather than F. velutipes var. velutipes (one Hae III restriction site and two bands). A single F. populicola x F. velutipes var.velutipes hybrid also revealed concerted evolution in the Hae III restriction sites (Figure 2, lane 9) but produced a Hae III pattern characteristic of F. velutipes var. velutipes (one Hae III restriction site and two bands) instead of F. populicola (two Hae III restriction sites and three bands). The remaining four F. populicola x F. velutipes var. velutipes hybrids revealed additivity in the Hae III restriction sites (Figure 2, lanes 2-5) with three bands characteristic of the two Hae III restriction sites in F. populicola and two bands characteristic of the single Hae III restriction site in F. velutipes

Figure 2. Hae III digestions of Flammulina hybrids.

Lanes 1, 8, 15 = 8 DNA/Hind III marker Lanes 2-5 = F. populicola x F. velutipes var. velutipes Lanes 6-7 = F. ononidis x F. velutipes var. velutipes Lanes 9 = F. populicola x F. velutipes var. velutipes Lane 10 = F. populicola x F. velutipes var. lupinicola Lane 11 = F. rossica x F. velutipes var. velutipes Lane 12 = F. ononidis x F. populicola Lanes 13-14 = F. populicola x F. velutipes var. velutipes

In the F. rossica x F. velutipes var. velutipes hybrid isolates both additivity and concerted evolution were observed. One F. rossica x F. velutipes var. velutipes hybrid displayed concerted evolution in both the Hae III and Bst F51 restriction sites (Figure 2, lane 11; Figure 3, lane 2) and yielded a Hae III and Bst F51 pattern characteristic of F. velutipes var. velutipes (one Hae III restriction site and two bands; no Bst F51 restriction site) rather than F. rossica (two Hae III restriction sites and three bands; one Bst F51 restriction site and two bands). The second F. rossica x F. velutipes var. velutipes hybrid showed additivity in the Hae III restriction sites with three bands characteristic of F. rossica and two bands characteristic of F. velutipes var. velutipes as well as additivity in the Bst F51 restriction sites (Figure 3, lane 7) with two bands characteristic of F. rossica. The third F. rossica x F. velutipes var. velutipes hybrid exhibited additivity in the Hae III restriction sites with three bands characteristic of F. rossica and two bands characteristic of F. velutipes var. velutipes. However, this hybrid lacked additivity in the Bst F51 restriction site and did not produce the two bands characteristic of F. rossica. We hypothesize that a point mutation has eliminated the Bst F51 site in this hybrid although sequencing is required to demonstrate whether or nor a point mutation has occurred.

 

	Figure 3. Bst F51 digestions of Flammulina hybrids.
	Lanes 1, 5, 9 = 8 DNA/Hind III marker Lane 2 = F. rossica x F. velutipes var. velutipes Lane 3 = F. ononidis x F. velutipes var. velutipes Lane 4 = F. populicola x F. velutipes var. velutipes Lane 6 = F. ononidis x F. elastica f. longispora Lane 7 = F. rossica x F. velutipes var. velutipes Lane 8 = F. fennae

 

Discussion

 

Ribosomal repeats are tandemly repeated and the paralogs are usually identical due to a poorly understood process of concerted evolution (Sanderson and Doyle 1992). Proposed mechanisms of concerted evolution include unequal crossing over (Schlotterer and Tautz 1994) and biased gene conversion (Hillis et. al. 1991). Within-individual diversity for the ribosomal ITS region has been observed and, in some cases, been attributed to hybridization (Sang et. al. 1995). In higher plants, three different outcomes have been reported for the ribosomal repeat following hybridization: 1) Both parental ITS sequences can be retained (Kim and Jansen 1994); 2) The ribosomal repeat may be homogenized to one parental type (Wendel et. al. 1995a); and/or 3) The ribosomal repeat could be homogenized but contained scattered elements of both parents (Wendel et. al. 1995b). This study reports evidence for recombination or gene conversion within the ribosomal repeat followed by homogenization of the repeat in interspecific hybrids of Flammulina. Although rare, interspecific hybrids have been reported for Dutch elm disease (Brasier 2001). While hybrids have been suspected in basidiomycetes based on morphology, few have been confirmed by molecular means. This study suggests that rare hybridization events are possible in mushrooms and that when hybridization occurs, recombination and gene conversion in the ribosomal repeat can result.

This research was supported in part by the Department of Biological Sciences, Eastern Illinois University as well as an Eastern Illinois University Council for Faculty Research Grant and an Eastern Illinois University College of Sciences Seed Grant to ME Mort. We gratefully acknowledge the laboratory assistance of J McGaughey, K Fairfield, and J Archibald.