**2.2.1 The evolution of fruit body morphology**

As mentioned above, traditional fungal classifications were based on morphology, anatomy, and biochemistry. For basidiomycete mushroom-forming fungi, the fruit body shape was traditionally one of the most important characters and as such was used for a long time as the central principle of classification. This approach gave rise to groups such as the *Hymenomycetes* (fungi with exposed hymenium, such as agaricoid species with a cap and stem, fig. 3) and the *Gasteromycetes* (fungi with gasteroid fruit bodies, that is, with internal spores and a truffle-like shape, fig. 3) that we know now are not monophyletic. Detailed anatomical investigations lead to some skepticism about these non-natural groupings, but only with the advent of early molecular studies were these suspicions confirmed: morphological dissimilar taxa could actually be very closely related. The gasteroid and agaricoid habit where shown to occur in very closely related genera, such as *Rhizopogon* and *Suillus* (Bruns et al. 1989) and *Hydnangium* and *Laccaria* (Mueller & Pine, 1994), implying that

and some filamentous fungi), *Saccharomycotina* (the true yeasts), and *Pezizomycotina* (with most of the filamentous and fruit-body producing ascomycetes). There has also been extensive work to understand the arrangement taxa within these higher-level clades, a task complicated by the large numbers of fungal taxa described. As evidenced by fig. 2, the base of the tree is a large polytomy, indicating uncertatinty on the resolution of the earliest

The results of these iniciatives were a big step forward for mycological research. They provided not only a rigorous overview of the main fungal monophyletic groups, but also a framework for understanding and appreciating the evolution of fungi. Although much has been achieved, accurately reconstructing the fungal tree of life is not an easy task and much research effort must be still gathered in order to resolve the earliy branching history of this group in order to have a clear view on how different groups of fungi relate to each other. AFTOL2, an NSF funded sequel to AFTOL1, is ongoing and targetting the unresolved issues and hypotheses raised during the first phase of the project. These include resolving the basal fungal lineages, including the placement of *Microsporidia* and *Glomeromycota*, as well as resolving key lineages within the *Ascomycota* and *Basidiomycota* needed for understanding

The availability of an accurate fungal tree of life allows for not only an appreciation of fungal diversity and evaluation of the fundamental differences across groups, but also an understanding of the evolutionary histories of different lineages that gave rise to the diversity of fungi we see today. For example, estimates point to the split between the *Ascomycota* and *Basidiomycota* having occured ~400 million years ago (Taylor and Berbee, 2006), revealing the ancient nature of the fungal phyla. Reconstructing the timing of such evolutionary events occur can be particularly interesting, allowing for comparisons with diversification patterns in other biological groups and ultimately a more thorough

The use of phylogenetic approaches to reconstruct the fungal tree of life enabled a much better understanding of the evolution of fungi and made testing hypotheses on trait evolution and diversification across the kingdom possible. Two examples of such approaches are discussed below: exploring the evolution of fruit body morphology and the

As mentioned above, traditional fungal classifications were based on morphology, anatomy, and biochemistry. For basidiomycete mushroom-forming fungi, the fruit body shape was traditionally one of the most important characters and as such was used for a long time as the central principle of classification. This approach gave rise to groups such as the *Hymenomycetes* (fungi with exposed hymenium, such as agaricoid species with a cap and stem, fig. 3) and the *Gasteromycetes* (fungi with gasteroid fruit bodies, that is, with internal spores and a truffle-like shape, fig. 3) that we know now are not monophyletic. Detailed anatomical investigations lead to some skepticism about these non-natural groupings, but only with the advent of early molecular studies were these suspicions confirmed: morphological dissimilar taxa could actually be very closely related. The gasteroid and agaricoid habit where shown to occur in very closely related genera, such as *Rhizopogon* and *Suillus* (Bruns et al. 1989) and *Hydnangium* and *Laccaria* (Mueller & Pine, 1994), implying that

the evolution of fungal morphology and ecology (McLaughlin et al., 2009).

branching events.

understanding of how life evolves.

evolution of fungal symbioses.

**2.2 Lessons learned from fungal phylogenetics** 

**2.2.1 The evolution of fruit body morphology** 

Fig. 3 Examples of agaricoid and gasteroid fruit body morphologies. *Amanita muscaria* (upper left corner), *Armillaria* sp. (upper right corner) and *Macrolepiota* sp. (lower right corner) all agaricoid, showing exposed hymenium and *Astraeus hygrometricus* (lower left corner with internal spores. Courtesy of J. Vicente.

overall fruit body morpohology has not been a stable character across fungal evolution. Soon after this dicovery came the realization that monophyletic groups contain multiple morphologies and that these morphologies appear scattered across clades (Hibbett and Thorn, 2001), indicating that certain fruit body forms evolved multiple times independently (see Hibbett, 2007 for a review on the topic).

This phenomenon of labile fruit body morphology is not exclusive to the basidiomycetes. Another interesting example comes from a well-preserved fossil ascomycete fruit body. This flask-shaped specimen was named *Paloepyrenomycites devonicus* and classified as a pyrenomycete (*Sordariomycetes*, within the subphylum *Pezizomycotina*; Taylor et al. 1995). However, this fruit body morphology is found in several other groups within the subphylum, making it difficult to rule out the possibility that this fossil belongs to a more basal *Ascomycota* lineage, such as *Taphrinomycotina* (typically members of this clade do not fruit, however some species have open aphotecial fruit bodies), or even an earlier extinct

Fungal Diversity – An Overview 217

accepted, a Latin diagnosis is still required, as recommended by the International Code of Botanical Nomenclature (McNeil et al., 2006), the code followed by mycologists to name fungi. Describing new species also requires the deposition of voucher specimens in official

The last decade witnessed a substantial increase in studies focused on fungal community ecology. Conducting fungal surveys can be a tedious long-term undertaking and for a long time mycologists relied on fruit body occurrence or culturing of fungal isolates to document species occurrence and site-specific fungal diversity. Although such methods can provide important information, they tend to supply incomplete community descriptions for the

The development of molecular tools to describe diversity allowed a much more straightforward, practical and rapid approach to the study of cryptic organisms such as fungi. These tools permit unveiling the communities colonizing soil (or other rich and dynamic substrates). Not only do they provide DNA-based information for identifying taxa, they also facilitate testing of ecological hypotheses, contributing for a better understanding of the structure and functioning of ecosystems. The vast majority of recent studies targeting

In general, these molecular microbial studies target one specific short DNA region and rely on the identification of operational taxonomic units (OTUs): sequence similarity based surrogates for taxa (Sharpton et al., 2011). Although OTUs are difficult to define, they are the foundation for estimates of richness, frequency, abundance, and distributions. Most fungal environmental DNA-based diversity studies make use of the internal transcribed spacer (ITS), a nuclear ribosomal repeat unit composed of three parts, the rapidly evolving ITS1, the very conserved 5.8S, and the moderately rapid ITS2 (Horton & Bruns, 2001, Bridge et al,

Fig. 4. Structure of the internal transcribed spacer (ITS), the nuclear ribosomal repetitive unit used to describe fungi to the species level. It is composed by the ITS1, 5.8S, and ITS2 regions,

ITS is used for identifying fungi at the species level. While it is far from being perfect, it offers several advantages that make it a popular that will likely be used for a long time. Genomes include numerous ribosomal DNA encoding genes distributed in tandem arrays along the same or different chromosomes (Rooney & Ward, 2005) and these copies are assumed to be extremely similar (Li, 1997). These coupled with the fact that ITS is easily amplified from low-quality samples (as opposed to single- or low-copy regions) makes it a fast and easy way to describe fungal diversity (Nilsson et al., 2008). However, there are several problems associated with using ITS to define fungal species. On the one hand, there are inherent biases associated with the use of DNA to document diversity, in particular problems with DNA extraction and amplification steps that might lead to distorted

and flanked by SSU (ribosomal small subunit) and LSU (ribosomal large subunit).

the description of fungal communities are based on sequence data (Taylor, 2008).

**3.2 The rise of fungal molecular ecological studies** 

reasons described in preceding sections.

collections.

2005; fig. 4).

lineage (Taylor & Berbee, 2006). These doubts make the placement of this fossil into the *Ascomycota* phylogeny difficult and impair its use for calibrating phylogenetic trees.
