Diversity measures in environmental sequences are highly dependent on alignment quality--data from ITS and new LSU primers targeting basidiomycetes.

Dirk Krüger,Christiane Fischer,Danuta Kapturska,Tesfaye Wubet,Rolf Daniel

doi:10.1371/journal.pone.0032139

Dirk Krüger, Christiane Fischer + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0032139

Copy DOI

Abstract

The ribosomal DNA comprised of the ITS1-5.8S-ITS2 regions is widely used as a fungal marker in molecular ecology and systematics but cannot be aligned with confidence across genetically distant taxa. In order to study the diversity of Agaricomycotina in forest soils, we designed primers targeting the more alignable 28S (LSU) gene, which should be more useful for phylogenetic analyses of the detected taxa. This paper compares the performance of the established ITS1F/4B primer pair, which targets basidiomycetes, to that of two new pairs. Key factors in the comparison were the diversity covered, off-target amplification, rarefaction at different Operational Taxonomic Unit (OTU) cutoff levels, sensitivity of the method used to process the alignment to missing data and insecure positional homology, and the congruence of monophyletic clades with OTU assignments and BLAST-derived OTU names. The ITS primer pair yielded no off-target amplification but also exhibited the least fidelity to the expected phylogenetic groups. The LSU primers give complementary pictures of diversity, but were more sensitive to modifications of the alignment such as the removal of difficult-to align stretches. The LSU primers also yielded greater numbers of singletons but also had a greater tendency to produce OTUs containing sequences from a wider variety of species as judged by BLAST similarity. We introduced some new parameters to describe alignment heterogeneity based on Shannon entropy and the extent and contents of the OTUs in a phylogenetic tree space. Our results suggest that ITS should not be used when calculating phylogenetic trees from genetically distant sequences obtained from environmental DNA extractions and that it is inadvisable to define OTUs on the basis of very heterogeneous alignments.

Highlights

A large proportion of the world’s biogeochemically important terrestrial microorganisms are Fungi
We have developed simple methods and parameters that can be used to assess the quality of the results obtained with different primer pairs, Operational Taxonomic Unit (OTU) cutoff values, and alignment treatments when conducting large-scale molecular ecology studies on systems whose true evolutionary history is unknown and for which sampling and sequencing errors cannot be ruled out
We focused on tools for exploring data to determine whether bifurcating phylogenetic trees provide realistic representations of diversity for large-scale molecular ecology

Summary

Introduction

A large proportion of the world’s biogeochemically important terrestrial microorganisms are Fungi. Most of the symbiotic and saprobic fungal taxa that degrade plant-derived carbon compounds (e.g. lignin and cellulose) in forest ecosystems belong to the subphylum Agaricomycotina [1], [2]. Recent developments in extremely high-throughput sequencing technologies have made it more feasible to unravel their diversity on a large scale. To most fully survey the diversity of fungi in the environment, the choice of sequencing locus is extremely important, even if more than one locus will result in non-overlapping datasets that have to be evaluated separately. A sequencing target for phylogenetic reconstruction and for identifying different groups should be orthologous, alignable, and not saturated in the mutations that contain the phylogenetic signal. The extent to which some of the most widely used fungal marker genes satisfy above criteria is debatable

Methods

Results

Discussion

Conclusion