Comparing Sanger sequencing and high-throughput metabarcoding for inferring photobiont diversity in lichens

www.nature.com/scientificreports OPEN Received: 26 March 2018 Accepted: 22 May 2018 Published: xx xx xxxx Comparing Sanger sequencing and high-throughput metabarcoding for inferring photobiont diversity in lichens Fiona Paul 1 , Jürgen Otte1, Imke Schmitt1,2 & Francesco Dal Grande1,3 The implementation of HTS (high-throughput sequencing) approaches is rapidly changing our understanding of the lichen symbiosis, by uncovering high bacterial and fungal diversity, which is often host-specific. Recently, HTS methods revealed the presence of multiple photobionts inside a single thallus in several lichen species. This differs from Sanger technology, which typically yields a single, unambiguous algal sequence per individual. Here we compared HTS and Sanger methods for estimating the diversity of green algal symbionts within lichen thalli using 240 lichen individuals belonging to two species of lichen-forming fungi. According to HTS data, Sanger technology consistently yielded the most abundant photobiont sequence in the sample. However, if the second most abundant photobiont exceeded 30% of the total HTS reads in a sample, Sanger sequencing generally failed. Our results suggest that most lichen individuals in the two analyzed species, Lasallia hispanica and L. pustulata, indeed contain a single, predominant green algal photobiont. We conclude that Sanger sequencing is a valid approach to detect the dominant photobionts in lichen individuals and populations. We discuss which research areas in lichen ecology and evolution will continue to benefit from Sanger sequencing, and which areas will profit from HTS approaches to assessing symbiont diversity. High-throughput sequencing (HTS) technologies have revolutionized the way we study microbial diversity and other complex ecological communities1,2. In particular, DNA metabarcoding - the identification of organisms from environmental samples using HTS of standardized DNA barcodes - has allowed rapid and cost-effective taxonomic assessments of a wide range of microbial groups from a variety of habitats in marine and terrestrial ecosystems3. Metabarcoding has also advanced our understanding of the distribution, abundance, and community structure of both symbiotic and free-living microorganisms4–6. Because HTS of DNA barcodes, e.g. on the Illumina MiSeq, Ion Torrent, or PacBio platforms, allows taxonomic assessment at a far greater depth and resolution than conventional Sanger sequencing2,7, it also facilitates discovery of rare taxa and detection of previously unrecognized eukaryotic and prokaryotic microbiomes8–12. Studying this vastly unexplored microbial biosphere is one of the new frontiers of microbial ecology, currently changing our understanding of species interactions and their ecological and evolutionary dynamics13. One of the most striking examples of the HTS-driven paradigm shifts comes from lichen symbiosis research14–16. For more than a century, lichens have been viewed as a symbiotic association between two, maximally three partners, i.e. a fungus (the mycobiont) and a green alga and/or cyanobacterium (the photobiont). DNA sequences supported this notion: in most cases researchers detected a single species of mycobiont and photobiont in a thallus based on unambiguous Sanger sequence electropherograms. In recent years, HTS-based studies have uncovered a tremendous species diversity associated with lichen symbioses (e.g.17–19). Lichen thalli harbor hyper-diverse bacterial as well as fungal communities (e.g.16,20). This previously unrecognized lichen-associated biosphere may act as a significant functional component of the symbiosis, supporting the growth and regeneration of the thallus, and modulating the holobiont’s response to environmental triggers21–23. 1 Senckenberg Biodiversity and Climate Research Centre (SBiK-F), Senckenberganlage 25, 60325, Frankfurt am Main, Germany. 2Institute of Ecology, Evolution and Diversity, Goethe University Frankfurt am Main, Max-von-Laue-Str. 9, 60438, Frankfurt am Main, Germany. 3Departamento de Farmacología, Farmacognosia y Botánica, Universidad Complutense de Madrid, 28040, Madrid, Spain. Correspondence and requests for materials should be addressed to I.S. (email: ) or F.D.G. (email: ) ScIentIfIc REPOrTS | (2018) 8:8624 | DOI:10.1038/s41598-018-26947-8 1 www.nature.com/scientificreports/ Figure 1. Sanger sequencing success depending on the abundance of the second most abundant photobiont for the full ITS (left) and ITS2 (right) data sets. Abundance categories based on Illumina ITS2 metabarcording24 are defined as follows: cat. 1: only a single photobiont present; cat. 2: secondary photobiont accounts for up to 10% of sequence reads; cat. 3: secondary photobiont accounts for 10–20% of reads; cat. 4: secondary photobiont accounts for 20–30% of reads; cat. 5: secondary photobiont represented by more than 30% of sequence reads. Recent HTS studies on lichen photobiont diversity suggest that the presence of multiple, genetically differentiated algae within a single thallus is a common phenomenon in lichens24–26. This raises the question to what extent Sanger-based studies underestimate within-thallus green algal diversity, and whether the single Sanger sequence obtained from an individual corresponds to the most abundant photobiont in that individual27. These assumptions, however, have never been formally tested. In this study, we compare HTS and Sanger sequencing methods for estimating the diversity of lichen-associated green algal photobionts. In particular, we address the following questions: (i) Are the photobionts identified via Sanger sequencing always the most abundant taxa in the thallus as inferred by HTS? and (ii) If more than one photobiont is present in a lichen individual: Is there an abundance threshold that precludes the generation of an unambiguous Sanger sequence? Results Overall sequencing success. From the Sanger sequencing of the 240 samples, we obtained 183 (full length ITS) and 198 (ITS2) high quality sequences. This corresponds to a sequencing success rate of 76.25% (full length ITS) and 82.5% (ITS2) (Supplementary Table S1). The excluded, low quality sequences were partially sequences containing many ambiguous bases, which reflected variable positions distinguishing the different OTU representative sequences obtained from the Illumina metabarcoding study. We got blast hits of >99% identity to one single OTU representative sequence for all of the Sanger sequences of the full ITS region. In the set of Sanger sequences amplified with the metabarcoding primers covering only ITS2, 99% of sequences were also clearly assignable to a single Illumina metabarcoding OTU. However, one Sanger sequence matched two OTUs at 100% sequence similarity and one Sanger sequence had a highest hit of 98.27% sequence similarity. Both sequences could not be assigned to an OTU, since they contained ambiguous bases at several variable sites that are used to determine OTU membersh (...truncated)