Apparent Recombination or Gene Conversion in the Ribosomal ITS Region of a Flammulina (Fungi, Agaricales) Hybrid

Karen W. Hughes and Ronald H. Petersen

Department of Botany, University of Tennessee at Knoxville

Internally transcribed spacer (ITS) regions of ribosomal DNA are widely used in reconstructing phylogenies at lower taxonomic levels (see, e.g., Vilgalys and Sun 1994Citation ; Baldwin et al. 1995Citation ). Ribosomal genes are tandemly repeated, and the paralogs are usually identical due to a poorly understood process of concerted evolution. Proposed mechanisms of concerted evolution include unequal crossing over (Schlotterer and Tautz 1994Citation ) and biased gene conversion (Hillis et al. 1991Citation ; Linares, Bowen, and Dover 1994Citation ). Within-individual diversity for the ribosomal ITS region has been observed and has in some cases been attributed to hybridization (Sang, Crawford, and Stuessy 1995Citation ; Hugall, Stanton, and Moritz 1999Citation ).

This report documents an apparent natural hybridization event between two species of Flammulina (F. velutipes and F. rossica) which resulted in a homogenized ribosomal repeat containing elements of both parents. A chromosome carrying the hybrid repeat has apparently introgressed into F. velutipes and persists in nature. Flammulina velutipes is predominantly found in temperate Northern Hemisphere forests but has also been collected in Argentina and New Zealand. Flammulina rossica is a more northern species, so far identified from Alaska, northern Canada, and eastern Russia.

Approximately 200 collections of Flammulina worldwide were identified to species on the basis of morphology (Redhead and Petersen 1999Citation ), mating studies (Petersen et al. 1999Citation ), and restriction fragment length polymorphism (RFLP) patterns (Methven, Hughes, and Petersen 2000Citation ). DNA was extracted from geographically diverse collections of each species. The ribosomal ITS region was amplified and sequenced on both strands using primers ITS5 and ITS3 in the forward direction and primers ITS4 and ITS2 in the reverse direction as described by Hughes et al. (1999)Citation . This combination of primers provided unambiguous sequence data on both forward and reverse strands. Forward and reverse sequences were compared and corrected manually and aligned with other Flammulina sequences using the Genetics Computer Group (1999)Citation family of programs, and all divergent bases were rechecked against the original sequence files. Sequence alignments were deposited with TreeBASE (accession M641; Hughes et al. 1999Citation ).

Two of ca. 150 natural dikaryon (diploid) worldwide collections identified morphologically as F. velutipes exhibited atypical restriction fragment patterns and were examined further. These collections were numbers 8316 (TENN 54820) and 8326 (TENN 54821) from Chubut Province, Parque Nacional de Los Alerces, Argentina. The two collection localities were separated by ca. 50 km. Monokaryon (haploid) cultures from collections 8316 and 8326 were grown in potato dextrose broth (Difco). Monokaryons isolated from both collection 8316 and collection 8326 belonged to one of two different RFLP patterns, one characteristic of European F. velutipes, the other characteristic of North American F. velutipes but with an additional RFLP band. Sequencing of the ITS region from each group of monokaryons confirmed that one monokaryon (haplotype) was F. velutipes, but that the second monokaryon (haplotype) represented a crossover or gene conversion event between F. velutipes and F. rossica in which the ITS1 and 5.8S regions were F. velutipes, but within a 20-bp area starting 45 bp after the 5.8S portion of the sequence, the sequence was that of F. rossica (fig. 1 ).



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Fig. 1.—Collections, geographical origins, and GenBank accession numbers are as follows: c771-vel = Flammulina velutipes, Netherlands AF036928; 7200-vel = F. velutipes, England AF030877; 8326-1vel = F. velutipes, Argentina (monokaryon); Olex-vel = F. velutipes, Michigan AF051700; 8078-vel = F. velutipes, California AF047871; d197-vel = F. velutipes, British Columbia AF141133; dh97080-vel = F. velutipes, China AF159426; 8326-4-hybrid = F. velutipes, Argentina (monokaryon-hybrid); 8171-ros = F. rossica, Alaska AF044194; Bulakh-ros = F. rossica, Eastern Russia AF051699. Bases 1–31 = ribosomal small subunit gene (SSU); bases 32–287 = ITS1; bases 288–446 = 5.8S gene; bases 447–765 = ITS2; and bases 766–799 = large subunit gene (LSU). The probable region in which recombination or gene conversion has occurred is shown by the arrow. Dots indicate identity with collection C771-vel. Dashes indicate gaps

 
Such a circumstance requires formation of an F. velutipes x F. rossica dikaryotic mycelium containing nuclei of both species. Most basidiomycete fungi do not undergo nuclear fusion to form diploid nuclei until just prior to meiosis (see life cycle discussion in Petersen and Hughes 1999Citation ). Recombination or gene conversion of the two ribosomal sequences probably occurred during meiosis, although rare somatic recombination has been reported in dikaryotic Neurospora (Radford 1970Citation ). Concerted evolution of the ribosomal repeat must have occurred at some point to convert the ribosomal repeat to the hybrid sequence, because the ribosomal repeat for the recombined F. velutipesF. rossica ITS sequence showed no detectable heterogeneity based on PCR amplification, sequencing, and restriction digests of the ITS region. Monokaryotic hyphae with the recombined ITS sequence apparently then anastomosed with normal F. velutipes monokaryotic hyphae in nature to produce introgressed hybrid dikaryotic mycelium, which fruited in Argentina. Collections from two different localities showed the same pattern, suggesting that the hybrid ribosomal ITS sequence is persisting and proliferating in nature.

In higher plants, three different outcomes have been reported for the ribosomal repeat following hybridization: (1) both parental ITS sequences were retained, as reported for Krigia (Kim and Jansen 1994Citation ), Arabidopsis suecica (O'Kane, Schaal, and Al-Shehbaz 1996Citation ), and Cardamine (Franzke and Mummenhoff 1999Citation ); (2) the ribosomal repeat was homogenized to one parental type, as reported for Gossypium (Wendel, Schnabel, and Seelanan 1995aCitation ) and Cardamine (Franzke and Mummenhoff 1999Citation ); and/or (3) the ribosomal repeat was homogenized but contained scattered elements of both parents, as reported for Gossypium gossypioides (Wendel, Schnabel, and Seelanan 1995bCitation ), Microseris (van Houten, Scarlett, and Bachmann 1993Citation ), Paenoia (Sang, Crawford, and Stuessy 1995Citation ), and Microthlaspi (Mummenhoff, Franzke, and Koch 1997Citation ). Sang, Crawford, and Stuessy (1995)Citation and Cronn et al. (1996)Citation suggest that concerted evolutionary forces are relatively weak and that homogenization of the ribosomal array may be approximately equal to the rate of speciation.

This study reports evidence for recombination or gene conversion within the ribosomal repeat, followed by homogenization of the repeat in a basidiomycete fungus, Flammulina. The geographical origin of the hybrid ITS sequence identified in collections 8316 and 8326 is uncertain. Flammulina velutipes is a pan–North Temperate species which has also been identified in collections from New Zealand and Argentina. Sequencing, RFLP, and herbarium data suggest that it is probably not native to the Southern Hemisphere (Methven, Hughes, and Petersen 2000Citation ). Flammulina rossica is a North Temperate species and has never been collected in Argentina. Flammulina velutipes and F. rossica distributions overlap in northern Europe, Canada, and Alaska. In in vitro mating studies, these two species showed nearly complete mating barriers (Petersen et al. 1999Citation ), and no natural hybrids have been detected in collections from geographical areas in which these two species are sympatric (northwestern North America and Europe), but a hybrid must have formed at some time and could have been transported to Argentina by human activities (transport of wood products or cultivation of edible mushrooms). Alternatively, both F. velutipes and F. rossica may have been transported independently to Argentina, where adaptation to new substrates may have allowed hybrid survival.

Natural fungal hybrids appear to be rare, in contrast to higher plants. Rare interspecific hybrids have been reported for Dutch elm disease, an ascomycete (Brasier et al. 1998Citation ), and while hybrids have been suspected in basidiomycetes based on morphology, none have been confirmed by molecular means. These studies suggest that rare natural hybridization is possible in mushrooms and that when hybridization does occur, recombination and gene conversion in the ribosomal repeat may occur.

Acknowledgements

This research was supported by NSF grant 95-21526 (PEET Program) to R.H.P.

Footnotes

Thomas Eickbush, Reviewing Editor

1 Keywords: concerted evolution Flammulina fungi gene conversion hybrid introgression ribosomal DNA Back

2 Address for correspondence and reprints: Karen W. Hughes, Department of Botany, University of Tennessee, Knoxville, Tennessee 37996-1100. E-mail: khughes{at}utk.edu Back

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Accepted for publication September 28, 2000.