High-throughput sequencing-based analysis of endogenetic fungal communities inhabiting the Chinese Cordyceps reveals unexpectedly high fungal diversity

Abstract

Chinese Cordyceps, known in Chinese as "DongChong XiaCao", is a parasitic complex of a fungus (Ophiocordyceps sinensis) and a caterpillar. The current study explored the endogenetic fungal communities inhabiting Chinese Cordyceps. Samples were collected from five different geographical regions of Qinghai and Tibet, and the nuclear ribosomal internal transcribed spacer-1 sequences from each sample were obtained using Illumina high-throughput sequencing. The results showed that Ascomycota was the dominant fungal phylum in Chinese Cordyceps and its soil microhabitat from different sampling regions. Among the Ascomycota, 65 genera were identified, and the abundant operational taxonomic units showed the strongest sequence similarity to Ophiocordyceps, Verticillium, Pseudallescheria, Candida and Ilyonectria Not surprisingly, the genus Ophiocordyceps was the largest among the fungal communities identified in the fruiting bodies and external mycelial cortices of Chinese Cordyceps. In addition, fungal communities in the soil microhabitats were clustered separately from the external mycelial cortices and fruiting bodies of Chinese Cordyceps from different sampling regions. There was no significant structural difference in the fungal communities between the fruiting bodies and external mycelial cortices of Chinese Cordyceps. This study revealed an unexpectedly high diversity of fungal communities inhabiting the Chinese Cordyceps and its microhabitats.

Figure 1. Endogenetic fungal community’s construction in Chinese Cordyceps and its microhabitat samples.

(a) Endogenetic fungal communities at the phylum level; (b) Ascomycota endogenetic fungal communities at the genus level.

Figure 2. Hierarchical-clustering graphics of the weighted UniFrac pairwise distance between Chinese Cordyceps samples and the clustering dendrogram.

(a) All of the samples of Chinese Cordyceps and its soil microhabitat samples; (b) fruiting bodies of Chinese Cordyceps; (c) external mycelial cortices of Chinese Cordyceps; (d) soil microhabitat samples collected from five different areas.

Figure 3. Principal coordinates analysis of endogenetic fungal communities in samples Chinese Cordyceps and soil microhabitat samples.

Percentages on the axes of the graph are the explained variance of total variance. The OUT matrix used in the analyses was clustered at the 97% similarity and the principal coordinate’s analysis was based on Weighted UniFrac distances. The solid points in abbreviations of the sample name indicate the samples distributed in the ordination.

Figure 4. Copy numbers of ITS sequence in Chinese Cordyceps and its microhabitat samples.

The error bars indicate SDs (n = 3). Different letters indicate significant differences (p < 0.05) of the ITS copies between the samples or the groups.

Figure 5. Scanning electron micrograph of mycelia covering the outside of a fruiting bodies of natural Chinese Cordyceps.

(a) Anatomic structure of Chinese Cordyceps clearly showed the worm gut; (b) The spliced image of the Chinese Cordyceps segmentation using scanning electron microscopy (10×); (c) The sclerotia segmentation image shows that the lava was covered with mycelial cortices (10×); (d) The stromata segmentation image shows the fungal filaments on the surface of stromata (500×).

Figure 6. Comparison of the endogenetic fungal communities investigated using different methods.

Different colors revealed the results in the current study and previously published studies using other methods for the analysis of the diversity of the endogenetic fungal communities, i.e., culture-dependent method, PCR-SSCP and clone library. The numbers indicate the shared genera in the studies.

Figure 7. Sampling and sample processing.

“★” collecting site of the Chinese Cordyceps, including three counties of Qinghai, and two counties of Tibet, the maps in the figure were generated using ArcGIS 10.0 software. (A) Plateau meadow; (B,C) growing Chinese Cordyceps collected during early fruiting stage (prior to the stromata production of spores); (D) early harvesting sample; (E) splitting the mycoderma and sclerotium; (F) fruiting bodies of the Chinese Cordyceps (abbreviated as “F”); (G) sample of external mycelial cortices (abbreviated as “M”) and (H) sample of soil (abbreviated as “S”) isolated from of the membrane covering the Chinese Cordyceps. For sample collection sites, the counties of Xinghai, Qumarlêb County and Zadoi of Qinghai province were abbreviated as “X”, “Q” and “Z”, respectively. Biru county of Nagqu and Mainling County of Nyingchi City of the Tibet Autonomous Region were abbreviated as “Na” and “Ny”, respectively.