Understanding the spatial scaling of boreal endophytic fungal communities using environmental cloning and Ion Torrent targeted amplicon sequencing

Background/Question/Methods

Endophytic fungi are cryptic symbionts living innocuously within all lineages of terrestrial plants and have been found in every major biome from the arctic to the tropics. In the last three decades, most studies examining the diversity of fungal endophytes have been conducted at small or large spatial scales using culture-dependent approaches. Although a high phylogenetic diversity was recovered through these studies, culture-based approaches are labor intensive and only capture a small portion of the true diversity. In this context, the objective of our study was to use culture-independent approaches to enhance our understanding of the extent of the phylogenetic diversity and understand the spatial scaling of endophytic fungi at an intermediate level, testing particularly for the ‘distance-decay relationship’ spatial pattern of diversity. During a large scale survey, the endophytic fungal community of two phylogenetically distant plant species (the moss Pleurozium shreberi, and the black spruce Picea mariana) were sampled at 14 sites distributed along a 800-mile East-West transect in the Boreal forest of Québec, Canada. In order to assess the diversity, phylogeny and community structure of endophytic fungi, the commonly used environmental cloning-sequencing of the partial ITS-LSU region approach was compared to a newly developed metagenomic approach using Ion Torrent sequencing of targeted ITS1 amplicons.

Results/Conclusions

The endophytic fungal diversity recovered in this study was higher than previously appreciated using culture-based approaches. Diversity data resulting from both environmental cloning and Ion Torrent approaches were coherent, indicating the same ranking for the most abundant Operational Taxonomic Units. However, while individual-species accumulation curves showed that an asymptote was reached only for low diversity samples using the environmental cloning approach, Ion Torrent sequencing captured the diversity present in all samples analyzed, indicating that this approach was more appropriate for large-scale community studies. However, we found that the intrinsic error rate for Ion Torrent sequencing can easily lead to overestimating the diversity reported, unless stringent read filtering thresholds are applied. Preliminary results revealed that the diversity of endophytic fungal communities varied highly from site to site, but that the ‘distance-decay relationship’ pattern was not observed suggesting that endophytic communities are more likely to be affected by habitat-related factors than geographical distance in the boreal forests.