Persoonia 23, 2009: 147–176 www.persoonia.org RESEARCH ARTICLE doi:10.3767/003158509X480944 Molecular phylogeny and spore evolution of Entolomataceae D. Co-David1, D. Langeveld1, M.E. Noordeloos1 Key words Abstract The phylogeny of the Entolomataceae was reconstructed using three loci (RPB2, LSU and mtSSU) and, in conjunction with spore morphology (using SEM and TEM), was used to address four main systematic issues: 1) the Clitopilus monophyly of the Entolomataceae; 2) inter-generic relationships within the Entolomataceae; 3) genus delimitation Entoloma of Entolomataceae; and 4) spore evolution in the Entolomataceae. Results confirm that the Entolomataceae (Ento­ Entolomataceae loma, Rhodocybe, Clitopilus, Richoniella and Rhodogaster) is monophyletic and that the combination of pinkish Rhodocybe spore prints and spores having bumps and/or ridges formed by an epicorium is a synapomorphy for the family. Rhodogaster The Entolomataceae is made up of two sister clades: one with Clitopilus nested within Rhodocybe and another Richoniella with Richoniella and Rhodogaster nested within Entoloma. Entoloma is best retained as one genus. The smaller spore evolution genera within Entoloma s.l. are either polyphyletic or make other genera paraphyletic. Spores of the clitopiloid type are derived from rhodocyboid spores. The ancestral spore type of the Entolomataceae was either rhodocyboid or entolomatoid. Taxonomic and nomenclatural changes are made including merging Rhodocybe into Clitopilus and transferring relevant species into Clitopilus and Entoloma. Article info Received: 21 April 2009; Accepted: 14 October 2009; Published: 19 November 2009. INTRODUCTION Monophyly of the Entolomataceae and intergeneric relationships The euagaric family Entolomataceae Kotl. & Pouzar is very The members of Entolomataceae have been classified together species-rich. It is composed of more than 1 500 species and because they all share the property of spore prints that are occurs worldwide, from arctic to tropical habitats (Horak 1980, pink to brownish or greyish pink in combination with spores 2008, Baroni 1981, Largent 1994, Noordeloos 2004, Gates & that are bumpy, ridged, or angular in polar or in all views. The Noordeloos 2007, Noordeloos & Hausknecht 2007). The fam- spore wall ornamentations are unique, being formed by local ily is highly variable in terms of sporocarp morphology (tiny to thickenings in the spore wall, the epicorium (Clémençon et al. large; pleurotoid, omphalioid, collybioid, mycenoid, and tricholo- 2004). The presence of pink, angular spores has been consid- matoid, as well as sequestrate), and micromorphology (spore ered so unique that Entolomataceae, in contrast to many other shape, pileipellis structures, pigmentation types, cystidia pres- Agaricales families, has been widely regarded a natural group ence and shape, etc.; Noordeloos 2004; Fig. 1). Lifestyles are (Kühner 1980, Singer 1986). equally varied: Most species are saprotrophic on soil, wood or moss, but some are parasitic on other mushrooms (Noordeloos Species from other genera had, in the past, been placed within 2004), parasitic on plants or ectomycorrhizal (Antibus et al. Entolomataceae. However, recent studies have excluded them. 1981, Agerer & Waller 1993, Agerer 1997, Kobayashi & Hatano Macrocystidia Joss. and Rhodotus Maire had been classified in 2001, Montecchio et al. 2006). The family traditionally contains the family on account of their pink spores, but molecular phylo- three main agaricoid genera: Rhodocybe Maire, Clitopilus (Fr. genetic studies have placed them outside the family (Moncalvo ex Rabenh.) P. Kumm. and Entoloma (Fr.) P. Kumm. s.l. The et al. 2002). Comparison of the spore wall of Rhodotus palmatus latter genus is sometimes split into more genera (e.g. 13 gen- (Bull.) Maire and members of Entolomataceae showed that era; Largent 1994). Additionally, three smaller non-agaricoid their bumps are not homologous (Clémençon 1997). Also, the genera have been distinguished on the basis of habit, namely, phylogenetic study by Moncalvo et al. (2002) suggested that the monotypic Rhodocybella T.J. Baroni & R.H. Petersen (with Catathelasma Lovejoy and a strongly supported clade contain- a cyphelloid habit), Rhodogaster E. Horak (secotioid) and Ri­ ing Callistosporium Singer, Macrocybe Pegler & Lodge and choniella Costantin & L.M. Dufour (gasteroid). Pleurocollybia Singer were best included in Entolomataceae. The more recent phylogenetic study by Matheny et al. (2006) It is no surprise that Entolomataceae, being such a large and highly variable family, raises questions that analysis of morpho- has excluded Catathelasma and Callistosporium from Entolo­ logical characters alone cannot answer, either due to scarcity mataceae with strong support. of characters and/or difficulty in interpreting the significance of Both phylogenetic studies (Moncalvo et al. 2002, Matheny et the characters. Molecular phylogenetic methods are therefore al. 2006) were based on relatively small samples of Entoloma, used in our study to address four main systematic issues: Rhodocybe and Clitopilus and none of Rhodogaster, Richoniella 1. the monophyly of the Entolomataceae; and Rhodocybella. Thus, phylogenetic relationships among 2. inter-generic relationships within the Entolomataceae; these six genera had remained unresolved. 3. genus delimitation of Entolomataceae and, with the addition of spore morphology; Spore evolution 4. spore evolution in the Entolomataceae. Spore characters have been important both to characterize 1 National Herbarium of the Netherlands, Leiden University branch, P.O. Box the family (having pink, angular spores) but also to separate 9514, 2300 RA Leiden, The Netherlands; corresponding authors e-mail: its three main agaricoid genera, Rhodocybe, Clitopilus and [email protected], [email protected]. Entoloma, from each other. Rhodocybe has spores with orna- © 2009 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 148 Persoonia – Volume 23, 2009 a b c d e f g h i j k Fig. 1 Entolomataceae variation in basidiocarp morphology. a. Entoloma prunuloides; b. E. sinuatum; c. E. catalaense; d. E. conferendum; e. E. camarophyllus; f. E. roseum; g. E. rodwayi; h. Clitopilus prunulus; i. Rhodocybe gemina; j. Richoniella pumila; k. E. uranochroum. — Photos by: a, b. Y. Deneyer; c. G. Con- siglio; d. J. Vesterholt; e, g–i. M.E. Noordeloos; f. H. Huijser; j. M. Pilkington; k. M. Meusers. mentations in the form of bumps and undulate ridges having 1980), rhodocyboid spores represent the plesiomorphic con- various arrangements resulting in spores that are undulate to dition since they are the more similar to what he considered weakly angular in profile and face views, and angular in polar the closest relative of Entolomataceae, Lepista (Fr.) W.G. Sm. view (Baroni 1981). Clitopilus is characterized by spores with (Tricholomataceae). Species of that genus have pinkish, rough- an ornamentation of longitudinal ridges. Entoloma has spores ened spores. The spores of Clitopilus are the evolutionary that are angular in all views due to its network of intercon- intermediate between Rhodocybe and Entoloma. Entoloma nected ridges that form facets and are highly varied in shape spores are the most complex and represent the most evolved (Romagnesi 1974, Pegler & Young 1978, 1979). spore form. The second theory (Baroni 1981) is similar in that There are two main theories on how spore shapes within Ento­ rhodocyboid spores are ancestral. However, Baroni based his lomataceae evolved. According to the first theory (Kühner argument that the rhodocyboid spore is the most primitive on D. Co-David et al.: Molecular phylogeny and spore evolution of Entolomataceae 149 the assumption that since pink angular spores do not exist 1. Is Entolomataceae monophyletic?; elsewhere in the Agaricales, the first Entolomataceae evolved 2. Are the main genera Rhodocybe, Clitopilus and Entoloma from an unknown member of Tricholomataceae with slightly monophyletic?; rounded-angular, pinkish spores. More pronounced angularity 3. What is the relationship of these three genera to each then derived from this. Furthermore, in contrast to Kühner’s other?; theory, clitopiloid and entolomatoid spores evolved independ- 4. What theories on spore evolution in Entolomataceae are ently from rhodocyboid spores. Note, however, that modern inconsistent with the phylogeny and should therefore be phylogenies support neither the sister relationship of Lepista rejected, and what theories are consistent with it?; nor any member of Tricholomataceae with a pink spore print. 5. How does the phylogeny inform the debate on the various Rather, it is suggested that the Lyophyllaceae are the sister taxonomic proposals for a possible generic delimitation clade of Entolomataceae and that it