MOLECULAR PHYLOGENY OF THE AGARICALES BASED ON 25S rDNA SEQUENCES
J. M. Moncalvo, F. Lutzoni, S. Rehner, J. Johnson, and R. Vilgalys
Department of Botany, Duke University
This material is based upon work supported by the National Science Foundation (NSF Award DEB-9708035,"The Agaricales: Molecular Systematics and Evolution of Mushrooms", R. Vilgalys, PI). Any opinions, findings and conclusions or recomendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation (NSF).
These results were first presented as a poster at the Asilomar Fungal Genetics Conference on March 20-22 1996 (see http://www.kumc.edu/research/fgsc/asilomar/evopop.html),
and also at the annual meetings of the Mycological Society of America on August 3-7, 1997, in Montreal, Canada.
MATERIALS AND METHODS
FIGURE 1. Phylogenetic relationships of the Agaricales inferred from 25S rDNA sequences. A phylogram of one of the 60 trees obtained using unweighted parsimony (length = 3100, CI = 0.207, RI = 0.555). The tree was rooted using Ganoderma (a polypore). Bootstrap support > 70% = . Clades supported with less than 70% bootstrap value but also present in weighted parsimony and various neighbor-joining trees are bracketed in bold. The arrow and question mark indicate that the node for monophyly of the Agaricales is not supported above the 50% of bootstrap frequencies and that there is no single morphological synapomorphy to define the clade.
CLICK ON FIGURE TO SEE AN EXPANDED VERSION OF THE TREE
FIGURE 2. Phylogenetic relationships of the Agaricales and related taxa inferred from 18S rDNA sequences. Sequences from GenBank were used to confirm rooting of the Agaricales. Strict consensus of 4 equally parsimonious trees obtained using unweighted parsimony (length = 1112, CI = 0.603, RI = 0.484). The tree was rooted with Auricularia. Bootstrap values > 50% are noted above respective branches. Names in bold show taxa of the Agaricales as defined in Fig. 1. The base of the Agaricales clade is noted with an arrow
CLICK ON FIGURE TO SEE AN EXPANDED VERSION OF THE TREE.
The identities of natural groups within the order Agaricales were addressed using ribosomal DNA sequences. Approximately 900 bases of the 5' end of the nuclear-encoded large subunit RNA gene (25S rDNA) were sequenced for over 300 broadly selected taxa representing most families within the Agaricales. A phylogenetic tree was estimated from 154 of the most diverse taxa using maximum parsimony. Many groups were supported by moderate to high bootstrap levels, or else were consistent with morphologically-based classification schemes. Some well recognized groups were the families Amanitaceae, Coprinaceae (excluding C. comatus and subfamily Panaeolideae), and Agaricaceae (excluding the Cystodermateae), and the genera Tricholoma, Termitomyces and its ally Podabrella, Pleurotus and Hohenbuehelia, etc. Nonmonophyletic groups revealed were the families Tricholomataceae and Hygrophoraceae, and the genera Clitocybe, Omphalina, and Marasmius. This first-order estimate of phylogenetic relationships among major lineages will serve as a starting point for further comparative studies on mushroom biology and evolution.
Mushrooms and their allies (Agaricales, Basidiomycota) are simultaneously the most familiar and the most mysterious of fungi: these conspicuous and diverse organisms are characterized by a wide variety of fruit body forms and ecological habits. Examples include the gilled mushrooms, boletes, puffballs, and polypores. Although mushrooms are an important component of most terrestrial ecosystems, mycologists still disagree about taxonomic limits of the Agaricales and the identity of natural groups within the order.
The terms "agaric" and "agaricoid fungi" are still most often used in reference to the Agaricales of Singer (1986), which includes both poroid and gilled fungi in suborders Agaricineae (which contains some polypores as well as agarics), Russulineae, and Boletineae. While monophyly of Singer's Agaricales has never been demonstrated and is not supported by molecular evidence (Hibbett and Donoghue, 1995; Hibbett, 1996; also Figs.1 & 2 below), recent and ongoing studies suggest that several lineages can be identified using both molecular and morphological evidence.
In recent years, the emergence of phylogenetic mycology as a paradigm for all fungal biology studies has been greatly accelerated by numerous advancements in phylogenetic methods, especially in the area of molecular systematics. Molecular phylogenetic studies have helped to define many higher level clades of true and pseudo fungi, as well as the major lineages within the two "crown fungi" clades Ascomycota and Basidiomycota (summarized in Blackwell et al., 1996). Evidence from recent phylogenetic studies of basidiomycetes supports the recognition of several major clades within the homobasidiomycetes (Hibbett, 1996; Hibbett and Donoghue, 1995; Swann and Taylor, 1993, 1995) . One realization emerging from these studies is that although many groups such as "mushrooms" and "polypores" may be polyphyletic (as first suspected), molecular systematics can help to better define these taxonomic groups based on phylogenetic principles.
We have addressed the higher-level relationships among the major groups of agarics by sequencing the nuclear large subunit (25S) RNA gene for more than 300 taxa. Preliminary phylogenetic analysis based on 154 diverse taxa reveals many well-defined groups of agarics that are consistent with modern taxonomic systems.
MATERIALS AND METHODS
Taxonomic sampling -- Taxa used for phylogenetic studies were defined as in Singer's (1986) "Agaricales in Modern Taxonomy". At least one representative was included from each of Singer's (1986) families (except Crepidotaceae and Gomphidiaceae).
Molecular techniques -- We used standard protocols for DNA isolation and PCR amplification of fungi with the use of amplification and sequencing primers for ribosomal genes developed in our laboratory. Most sequencing reactions, electrophoresis, and data collection were performed using dye termitator sequencing chemistries (Perkin Elmer/ABI). Sequence chromatograms were compiled using Sequencher software (vers. 2.0, GeneCodes Corp.). Alignments were completed using PILEUP (GCG, 1991) followed by manual optimization of hypervariable regions. Regions with ambiguous alignment were removed for the analyses.
Phylogenetic analysis -- Maximum parsimony searches were employed using PAUP* (Swofford, in prep.) on a UNIX Spark Station. Topological robustness of the trees was assessed by using "fast bootstrap" methods with PAUP*.
Higher level relationships
Basal relationships remain poorly resolved for both the 25S (fig.1) and 18S (fig.2) rDNA data sets. However, phylogenetic analysis of both data sets suggest that the Boletaceae and Russulaceae are basal to a large group of agarics which may represent the Agaricales sensu stricto.
These results are consistent with earlier findings that show the Boletaceae belonging to a distinct evolutionary lineage containing mostly poroid species along with gilled taxa and reduced forms (Baura et al., 1992; Bruns and Szaro, 1992; Bruns et al., 1992). The Russulaceae, defined by reticulate spores and heteromerous trama with spherocysts, also represents a distinct clade that includes both agaricoid and non-agaricoid genera (e.g., Bondarzewia , a polypore with striking micro-anatomical similarities to Russula (Singer, in Clémençon, 1977).
Phylogenetic relationships within the Agaricales suggest that there are two major clades of agaric fungi (Fig. 1): Groups A to Q include only light spored agarics, whereas groups R to U include mostly dark-spored species. This pattern is in agreement with the earliest Friesian classifications that emphasized spore color as a character at higher taxonomic levels.
Our results support the monophyly of several traditional groups, for instance:
1. The Amanitaceae (Group E: Amanita and Limacella).
2. The Agaricaceae (Group S) including Agaricus, Lepiota, Leucocoprinus, etc... but not Ripartitella and Cystoderma (Group R); the Coprinus comatus group is also nested within the Agaricaceae (Hopple, 1994; Vilgalys et al., 1994; Johnson, 1997).
3. The Coprinaceae pro parte (Group T: Coprinus, Psathyrella and Lacrymaria), excluding C. comatus and subfamily Panaeoloideae (Group U2).
4. The Strophariaceae, Bolbitiaceae, Cortinariaceae (with exclusion of Cortinarius and Dermocybe, Group R) and subfamily Panaeoloideae (Group U); relationships between taxa within group U, however, are poorly resolved.
5. The mycorrhizal species of Tricholoma (Group N) including Leucopaxillus (also mycorrhizal) but excluding the only non-mycorrhizal Tricholoma sampled (T. giganteum in Group G).
6. Termitomyces and its ally Podabrella (Group J).
7. Pleurotus and Hohenbuehelia pro parte (Group H); etc...
Several nonmonophyletic groups were also revealed by these analyses, for instance:
1. The Tricholomataceae, the largest family of Agaricales, is paraphyletic (spread across Groups A, C, D, G, I pro parte, and J to N).
2. The Hygrophoraceae is polyphyletic (split among Groups O and Q, and between E and F).
3. Omphalina ss. lato (across Groups I, O, Q and W) contains several distinct groups and is clearly in need of revision (Lutzoni, 1996; Redhead and Kuyper, 1987).
4. Clitocybe (Groups I, L, N and O, and also G (Ossicaulis).
5. Marasmius (Groups A and C) is polyphyletic.
1) rDNA phylogeny supports recognition of the Agaricales, a monophyletic clade containing most conventional families of mushrooms. Two other major groups of agaricoid fungi - the Boletaceae and the Russulaceae - are excluded from the order Agaricales.
2) There are no major conflicts between our results and the classification of Singer (1986), although problematic taxa are noted.
3) More taxa (including reduced and secotioid forms, sterile and lichenized members, and other poorly known taxa) and more sequences (complete 18S and 25S rDNA) will be sampled in the future in order to more fully address the evolution of the Agaricales.
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