Diversity Measures in Environmental Sequences Are Highly Dependent on Alignment Quality—Data from ITS and New LSU Primers Targeting Basidiomycetes Dirk Kru ¨ ger 1 * . , Danuta Kapturska 1,2. , Christiane Fischer 3 , Rolf Daniel 3 , Tesfaye Wubet 1 1 Department of Soil Ecology, UFZ - Helmholtz Centre for Environmental Research, Halle (Saale), Germany, 2 Environmental Biotechnology, International Graduate School of Zittau, Zittau, Germany, 3 Department of Genomic and Applied Microbiology, Georg-August University of Go ¨ ttingen and Institute of Microbiology and Genetics, Go ¨ ttingen Genomics Laboratory, Go ¨ ttingen, Germany 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. Citation: Kru ¨ ger D, Kapturska D, Fischer C, Daniel R, Wubet T (2012) Diversity Measures in Environmental Sequences Are Highly Dependent on Alignment Quality—Data from ITS and New LSU Primers Targeting Basidiomycetes. PLoS ONE 7(2): e32139. doi:10.1371/journal.pone.0032139 Editor: Jason E. Stajich, University of California Riverside, United States of America Received June 30, 2011; Accepted January 23, 2012; Published February 21, 2012 Copyright: ß 2012 Krueger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded in part by DFG Priority Program 1374 on ‘‘Infrastructure-Biodiversity-Exploratories’’ (KA 1241/15-1 and KR 3587/1-1). D. Kapturska also was kindly supported by the Helmholtz Impulse and Networking Fund via the Helmholtz Interdisciplinary Graduate School for Environmental Research (HIGRADE). The funders DFG and HIGRADE had no input into the study design, the data collection and analysis, the decision to publish, or the preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dirk.krueger@ufz.de . These authors contributed equally to this work. 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 com- pounds (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. In general, 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. Phylogenetic analyses are tightly connected with alignment, as can also be seen in the development of tools that simultaneously optimize alignment and phylogenies such as POY and SATe ´-II [3], [4] and attempts to denoise phylogenomic and metagenomic datasets [5]. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA has been the locus of choice for analyzing phylogenetic relationships, especially at lower taxonomic ranks where variability may still allow unambiguous alignment. This is because of its large copy number (and thus high template availability) and the relative ease of designing both broad and selective PCR primers for it [6], [7], [8]. However, it should be noted that different species have different ITS copy numbers and that the copy number may be dependent on fungus’ life stage at the time of sampling. A large amount of ITS sequence data is currently available in rapidly expanding public databases. To illustrate this growth, a keyword search for ‘‘ITS and Fungi and RNA’’ in the NCBI GenBank database conducted on December 1, 2010 yielded ca. 33 160 sequences; a search using the same PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32139