Phylogenetic Analyses of Armillaria Reveal at Least 15 Phylogenetic Lineages in China, Seven of Which Are Associated with Cultivated Gastrodia elata

Abstract

Fungal species of Armillaria, which can act as plant pathogens and/or symbionts of the Chinese traditional medicinal herb Gastrodia elata ("Tianma"), are ecologically and economically important and have consequently attracted the attention of mycologists. However, their taxonomy has been highly dependent on morphological characterization and mating tests. In this study, we phylogenetically analyzed Chinese Armillaria samples using the sequences of the internal transcribed spacer region, translation elongation factor-1 alpha gene and beta-tubulin gene. Our data revealed at least 15 phylogenetic lineages of Armillaria from China, of which seven were newly discovered and two were recorded from China for the first time. Fourteen Chinese biological species of Armillaria, which were previously defined based on mating tests, could be assigned to the 15 phylogenetic lineages identified herein. Seven of the 15 phylogenetic lineages were found to be disjunctively distributed in different continents of the Northern Hemisphere, while eight were revealed to be endemic to certain continents. In addition, we found that seven phylogenetic lineages of Armillaria were used for the cultivation of Tianma, only two of which had been recorded to be associated with Tianma previously. We also illustrated that G. elata f. glauca ("Brown Tianma") and G. elata f. elata ("Red Tianma"), two cultivars of Tianma grown in different regions of China, form symbiotic relationships with different phylogenetic lineages of Armillaria. These findings should aid the development of Tianma cultivation in China.

Fig 1. Phylogenetic tree generated from the three-locus (ITS, tef1-α and β-tubulin) dataset.

The labels on the right of the phylogram indicate groups identified by phylogenetic analyses. The values of the bootstrap frequencies of ML (BP ≥ 70%), posterior probability (PP ≥ 0.95) and maximum parsimony bootstraps (PB ≥ 70%) are shown above the nodes. Thick black branches indicate high support in the analyses (ML-BP ≥ 70%, BI-PP ≥ 0.95). Green branches were concordantly supported by the majority of the loci (genealogical concordance), or were well supported by at least one locus and not contradicted by any other locus (genealogical non-concordance). Triangles at nodes indicate that all taxa united by it belong to the same phylogenetic lineage (see text for details). Taxon labels indicate strain number and geographic source. If a strain was originally identified as a CBS (Chinese biological species), the CBS taxon is listed before the strain number.

Fig 2. Phylogenetic relationships between samples of Armillaria inferred from tef1-α sequences using ML analysis.

Branch support values are indicated by numbers above branches (ML-BP/BI-PP/MP-PB). Thick black branches received high support in the analyses (ML-BP ≥ 70%, BI-PP≥ 0.95). The accession numbers for the sequences retrieved from the GenBank database are listed in S1 Table.

Fig 3. Phylogeny of Armillaria samples inferred from tef1-α DNA sequences using ML analysis to show the intraspecific level within Lineage 6.

Branch support values are indicated by numbers above branches (ML-BP/BI-PP/MP-PB). The nodes appearing in common between Fig 3 and Fig 2 are indicated by triangles. The species names labeled on our collections correspond to Fig 1. Seven samples with heterozygous sites (HKAS45821, HKAS51692, HKAS86558, HKAS86559, HKAS86564, HKAS85567 and HKAS85572) were analyzed using PHASE 2.0 and every isolate was divided into two haploid genotypes. The four subgroups of Lineage 6 were highlighted in different colors.

Fig 4. The link between 14 CBS (Chinese Biological Species) and phylogenetic lineages identified in this study.

CBS were recognized in previous studies and phylogenetic lineages were delimited in this study. The m/n on the arrows between “CBS” and “phylogenetic lineage” means that m of n isolates of a CBS were assigned to phylogenetic lineages.