ITS, OTUs and beyond—fungal hyperdiversity calls for supplementary solutions



    1. Section of Mycology, Norwegian Veterinary Institute, N-0106 Oslo, Norway
    2. Microbial Evolution Research Group (MERG), Department of Biology, University of Oslo, N-0316 Oslo, Norway
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Molecular species recognition of fungi emerged years before DNA barcoding (Seifert 2009). While the ideal fungal DNA barcode seems Utopian, two research decades nevertheless highlight the internal transcribed spacer (ITS) as the best available choice (Seifert 2009). Databases providing reliable ITS sequences of known fungi require enormous efforts, but are urgently needed (Abarenkov et al. 2010a,b; Begerow et al. 2010). Any criticism of such a commitment seems unjustified. However, exclusive focus on the development of ITS reference libraries will delay the progress towards a deeper ecological insight. It is widely acknowledged that ITS fails to recognize species, particularly in some ascomycete lineages (Balajee et al. 2009; Seifert 2009). It also appears paradoxical to solely rely on ITS for ecological recognition of fungal species when modern fungal systematics rely on phylogenetic species recognition with concordance of multiple gene genealogies (see Blackwell 2011). Considering that at least 98% of the predicted ∼5 million fungal species remain undescribed (Blackwell 2011), how will reliance on ITS alone influence the biodiversity estimates and ecological understanding? In this issue, Gazis et al. (2011) elegantly demonstrate through multi-locus sequence phylogeny analyses that ITS largely underestimates the species diversity of tropical fungal endophytes and even more importantly obscures fundamental ecological and biogeographical patterns. This thorough reflection on species delimitation criteria and their implications for ecological and biogeographical inferences underline that ITS, particularly in hyperdiverse habitats, provides no shortcut to deeper knowledge of fungal ecology.

Fungal ecology studies largely governed by mycorrhizal research have for years utilized internal transcribed spacer (ITS) sequences for successful detection and identification of fungi in their mycelial or mycorrhizal stages in soil and plant roots (Seifert 2009; Abarenkov et al. 2010a). The approach is increasingly adopted, among others in fungal endophyte surveys (see Gazis et al. 2011) and environmental sequencing surveys (e.g. Jumpponen & Jones 2009). Numerous fungal endophytes dwelling inside above- and below-ground tissues of virtually all plants contribute to the hyperdiversity of Fungi, where particularly tropical trees are considered as hot spots for unknown species (Blackwell 2011; Gazis et al. 2011 and references therein). Gazis et al. have performed a multi-locus approach (MLA) including ITS, gpd and tef1 using three ascomycete endophyte species complexes isolated from rubber trees (Fig. 1) as models. They found that MLA-derived operational taxonomic units (OTUs) either correlated with ecological niche, fungus–host relationships or biogeography (Fig. 2). Not surprisingly, ITS alone lumped isolates of different ecology or origin into composite OTUs, thus underestimating the diversity and obscuring the ecological and biogeographical patterns. Evidence accumulated over the past 20 years has convincingly documented the limited value of ITS as species delimiter in many important ascomycete genera (see references in Balajee et al. 2009; Seifert 2009; Begerow et al. 2010; Gazis et al. 2011). Despite kicking in open doors, Gazis et al. (2011) nevertheless excellently exemplify that the commonly adopted ITS approach weakens our ability to recognize and respond to key questions of ecological and sometimes socio-economic importance. In the latter context, reliance on ITS may camouflage invasive fungal species, often corresponding to disease agents and pests. This cries for group-specific supplementary DNA barcoding loci (Seifert 2009). Leading arguments for the aims and rationale of DNA barcoding stress the link between basic (ecological and taxonomical) and applied (medical, agricultural, social and industrial) interests. For example, biodiversity and ecosystem conservation and management, including precautionary measures and protection against emerging animal and plant diseases, will increasingly rely on alert biosecurity systems. A toolbox of supplementary genetic markers will better equip us to meet and respond to these challenges.

Figure 1.

 The rubber tree Hevea brasiliensis and the ascomycete Pestalotiopsis microspora, one of the rubber tree fungal endophyte species complexes studied by Gazis et al. (2011).

Figure 2.

 Hypothetical phylogenetic trees of multi-locus (MLA; left) and single-locus (internal transcribed spacer (ITS), right) analyses illustrating that reliance on ITS lumps isolates of different origins and ecologies, while MLA recognize operational taxonomic units correlating with ecology and/or origin. Note, the figure is overstated; Gazis et al. (2011) recognized biogeographical and ecological patterns separately and differently for each of the studied species complexes. inline image = pathogenic isolate, inline image = endophytic isolate. Symbol colours reflect different host plants. Black dashed and grey solid lines of the terminal clades refer to origins.

Arguments are accumulating against an exclusive focus on ITS as a DNA barcode sensu stricto (i.e. a single standardized DNA fragment for species identification; Valentini et al. 2009). In addition to the discussion so far, Bellemain et al. (2010) recently demonstrated that commonly used ITS-primers may induce taxonomic biases during PCR, thus questioning the assumed robustness and universality of the ITS-primers, for example in environmental fungal barcoding studies aiming to reflect fungal biodiversity and community composition. A new generation fungal diversity surveys based on next generation sequencing (NGS) of ITS fragments is approaching. Here, arbitrary divergence thresholds (95–97%) commonly compensate for putative sequence errors and intraspecific sequence variation (Jumpponen & Jones 2009). The resulting sequence numbers are breathtaking, and the future perspectives thrilling. However, the tsunami of sequences representing 95–97% ITS–OTUs soon flooding the already packed sequence databases may leave disappointingly limited ecological insight. Deeper knowledge than just a number of composite OTUs with approximate taxonomic affiliation will require supplementary genetic markers serving multiple fungal groups and purposes. Multi-locus sequencing presently seems impractical in environmental NGS surveys, but can be feasible in the near future considering the continuing advances in NGS technology. A fungal ecology toolbox of supplementary genetic markers would nicely fit the flexible ‘DNA barcoding sensu lato’ concept (i.e. identification of any taxonomic level using any DNA fragment; Valentini et al. 2009). This, combined with the recent probabilistic BLAST search approach (Ovaskainen et al. 2010) or other emerging tools, is intriguing prospects. The Ovaskainen et al. (2010) approach abandon the arbitrary sequence similarity thresholds criticized by among other Gazis et al. (2011) and instead utilize the self-consistency of a reference database to transfer sequence similarity to the probability of correct identification to a given taxonomic level.

Comparative and probabilistic identification strategies can only be meaningful combined with well-curated and reliable databases hosting sequence data from ex-type or reference strains (Abarenkov et al. 2010a; Balajee et al. 2009; Ovaskainen et al. 2010). Such databases, for example, united under the expanding umbrella of PlutoF (Abarenkov et al. 2010b), should ideally include DNA sequences from an appropriate and relatively standardized selection of loci fit for purpose. This will heavily depend on a prioritized effort to identify, select and deliver reliable reference sequences. Considering the effort behind any DNA extract of a voucher, ex-type or reference strain, a multi-locus sequence investment running in parallel with planned and future ITS initiatives is a justifiable option. Clearly, more studies following the concept of Gazis and colleagues are needed. Cultivation of the cultivable mycota and multi-locus characterization of specious groups will contribute substantially to a fundament of reference cultures and genetic markers of presently undescribed species. This fundament will further increase the sense and outcome of environmental NGS and next-NGS studies in mycology, better link taxonomic, ecological and socio-economical interests, and deepen our molecular ecological insight into the fungal kingdom.

T.V. works as researcher (NVI) and associate professor (UoO) in mycology with focus on molecular diagnostics, ecology and evolution of fungi and oomycetes. Her special interests include animal parasites and pathogens, fungal diseases, food and feed safety, invasive species, toxigenic fungi, and endophytic- and mycorrhizal fungus-plant relationships.