Variation in the ITS and IGS regions of ribosomal DNA among the biological species of European Armillaria

Variation within the internal transcribed spacer (ITS) and the intergenic spacer (IGS) of the ribosomal RNA gene of isolates representing seven European species of Armillaria was examined by PCR, coupled with RFLP analysis and partial sequencing of the ITS region. The amplified-ITS region was about 840 bp long and uniform in length among the European isolates, while the amplified-IGS region showed four different lengths which corresponded to A. mellea, A. tabescens, A. ectypa and a group including the four other species. A. borealis, A. ostoyae, A. cepistipes and A. gallica. Restriction digestions of the ITS and IGS regions by Alu I and Rsa I, respectively, gave rise to the same four polymorphic groups. However, A. borealis and A. ostoyae were easily separated from A. cepistipes and A. gallica by digestion of the two rDNA spacers with Hinf I and Taq I. Nde II digests of the amplified ITS could distinguish A. borealis from A. ostoyae and each of the seven species were separated by Alu I digestion of the IGS region. A North American A. mellea isolate, partly examined in this work, was found to be different from the European A. mellea isolates, while there was a close similarity between the European A. ostoyae and an isolate of the same species isolated from the U.S.A. Cluster analysis based on the presence or absence of comigrating restriction fragments indicated more than 80% similarity between A. borealis and A. ostoyae and between A. cepistipes and A. gallica, while A. mellea, A. tabescens and A. ectypa were found in separate clusters exhibiting, respectively, about 40, 38 and 32% average similarity with the other species. Although little intraspecific variation was observed in many species, A. gallica and A. cepistipes were found to be heterogeneous. In view of recent results suggesting several groups in A. borealis and A. cepistipes, the Alu I restriction patterns obtained in this work would identify the former species as type B, characterized by an Abi I pattern different from that of A. ostoyae, and the latter species as composed of one isolate (C4) of type B and three isolates (CI, C2 and C3) of type A. Nucleotide sequences of the rDNA internally transcribed spacer I (ITS1) of one isolate of each species showed that A. mellea and A. ectypa differed from the other species by several insertions and point mutations giving rise to a level of similarity ranging from 66 to 79%. This DNA region was highly conserved within the other species which revealed a similarity of 97 to 100%. These results demonstrate that analysis of rDNA spacers provides appropriate data to circumscribe taxonomic entities within the Armillaria complex and that the phylogenic relationships among species deduced from the present study are consistent with previous analyses based on pairing tests as well as morphological and physiological characters.