Phylogeny of Lepiota (Agaricaceae) Evidence from nrits and nrlsu sequences

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1 Mycological Progress 2(4): , November Phylogeny of Lepiota (Agaricaceae) Evidence from nrits and nrlsu sequences Else C. VELLINGA1 The phylogeny of Lepiota, Cystolepiota, Melanophyllum, Coprinus comatus, and Chamaemyces fracidus was examined with molecular techniques, and the consequences for morphological classification were considered. This group (excluding Chamaemyces fracidus and Coprinus comatus and allies) can be split up into several clades, which do not correspond to currently recognized genera, subgenera and sections. Structure of the pileus covering appears to be a key morphological character; colour and shape of the spores do not reflect the new phylogeny. Lepiota sect. Ovisporae is not monophyletic, and a separate genus for Cystolepiota, Lepiota sect. Echinatae, Melanophyllum and Pulverolepiota combined seems warranted. More research, especially on developmental morphology and on more tropical taxa, will be necessary to solidly erect a new classification. The genus Lepiota (Pers.: Fr.) S.F. Gray, with its satellite genera, Cystolepiota Singer, Pulverolepiota Bon, and Melanophyllum Velen., forms a monophyletic group within the family Agaricaceae (VELLINGA 2003). This group also includes Coprinus comatus and its allies. However, Chamaemyces fracidus, often assigned to the same tribus as Lepiota, viz. tribus Lepioteae, is excluded from it and appears to be basal to the family Agaricaceae. The family as a whole is a widely distributed monophyletic group of saprotrophic fungi, which display a huge diversity in spore colour, and structure of the pileus covering2. Recently it has been shown that secotioid (e.g. Endoptychum agaricoides, Podaxis pistillaris, and Longula texensis) and gasteroid taxa (Lycoperdaceae and Tulostomataceae) also belong to this family (VELLINGA, DE KOK & BRUNS 2003; KRÜGER et al. 2001; MONCALVO et al. 2002). Morphologically, the clade to which Lepiota s.l. belongs, comprises species with a regular lamella trama, and in most cases with clamp-connections. Some authors, e.g. BON (1993), included Melanophyllum with coloured spores in the same tribus as Lepiota, whereas other authors placed Melanophyllum close to Agaricus L.: Fr., e.g. KERRIGAN (1986), SINGER (1986) and WASSER (2002). Generally speaking, the other lineages in the family either have pale spores and a trabecular lamella 1 Department of Plant and Microbial Biology, University of California at Berkeley, 111 Koshland Hall, Berkeley CA , U.S.A., vellinga@uclink.berkeley.edu 2 The neutral term pileus covering is used as it is not known whether the covering layers are developmentally of velar origin or a real pileipellis. trama, or brown spores and a regular lamella trama, or are secotioid or gasteroid. Clamp-connections are rare in those groups. Genus delimitations and the number of genera within tribus Lepioteae have been hotly disputed over the last decades. A very wide concept of the genus Lepiota, close to the original Friesian concept (FRIES 1821) is still used by many American authors of popular guide books (e.g. ARORA 1986; LINCOFF 1981); Lepiota in their sense includes the genera Cystolepiota, Lepiota, Leucoagaricus, Leucocoprinus, and Macrolepiota of other classifications. A much more restricted concept of the genus Lepiota has been applied by recent European authors (e.g. BON 1993; CANDUSSO & LANZONI 1990; VELLINGA 2001a), and by SINGER (1986). BON (1993) applied the narrowest genus concept of all, excluding members of sect. Echinatae and transferring them to a separate genus, Echinoderma (Locq. ex Bon) Bon. The genus Lepiota, in the rest of this paper, is taken in the sense of VELLINGA (2001a), i.e. including sect. Echinatae, but excluding Chamaemyces, Cystolepiota, Melanophyllum, and Pulverolepiota. JOHNSON (1999) and JOHNSON & VILGALYS (1998) elucidated the phylogenetic relationships of Lepiota s.l. but used only seven species to represent Lepiota s. str. and Cystolepiota. On this limited basis they concluded that Lepiota consists of an assemblage of paraphyletic lineages, with the two Cystolepiota species and L. aspera (as L. acutesquamosa) forming a monophyletic clade. Coprinus comatus (O.F. Müll.: Fr.) Pers. and C. sterquilinus (Fr.: Fr.) Fr., and Montagnea arenarius (DC.) Zeller were included in their studies, and were placed either in the middle of Lepiota (based on ITS-sequences), or paraphyletically between Lepiota and the Leucoagaricus/Leucocoprinus

2 306 Phylogeny of Lepiota assemblage (JOHNSON 1999; JOHNSON & VILGALYS 1998) (see also REDHEAD et al. (2001) on the position of Coprinus comatus and its allied species). These taxa are morphologically quite different from the rest of the group; the spores are black, with a germ pore, the basidia are of different length, and separated from each other by pavement cells, and the lamellae are autolytic. The hymenium characters and the presence of a germ pore in the spores hint at a relationship with Leucocoprinus Pat., but that is not supported by the molecular data. The genus Lepiota embraces species with quite varied types of pileus covering, ranging from a hymeniderm, a cutis, a trichoderm to a special type of epithelium (rounded cells in chains which are agglutinated to form acute pyramidal warts). A second group of very variable characters involves the spores, viz. their shape and size, chemical reactions and the number of nuclei per spore. Most striking is the diversity in spore shape, from broadly ellipsoid and ellipsoid to spurred, to fusiform or penguin-shaped, resembling typical bolete spores. Combinations of spore shape and the structure of the pileus covering, have been used in the past to distinguish at least five groups within the genus, on subgenus and genus levels (e.g. BON 1993) or on section level (e.g. SINGER 1986; VELLINGA 2001a). Older infrageneric classifications, like KÜHNER s (1936) are more difficult to compare with present day ones, as tribus Leucocoprineae and the genus Cystolepiota were still included. BON s classification is slightly deviant, as he recognized the groups at different ranks than other systematists, and he used less known names for most of them. It should be kept in mind that the classifications especially below genus level, were all based on European and other temperate material. The systematic position of the taxa used in the present study according to BON s classification (1993) is displayed in Table 1, as his ideas are widely followed by European authors. VELLINGA & HUIJSER (1999) used ITS-sequences to show that Lepiota cristata (Bolt.: Fr.) Kumm., a species characterized by spurred spores and a hymenidermal pileus covering, is more closely related to species with ellipsoid spores and a hymenidermal pileus covering, than to spurred-spored taxa which have a trichodermal pileus covering. This is an indication that the spurred spore might have evolved several times, but the hymenidermal pileus covering only once. ITS sequences have also proven to be a powerful tool to reveal specific and infraspecific variation that was hitherto unrecognized (VELLINGA 2001b on L. magnispora and L. spheniscispora; VELLINGA 2001c on L. cristata and L. castaneidisca). The present study sets out to explore the circumscription of the genus Lepiota itself and its satellite genera, and to evaluate and test the existing morphological infrageneric classifications, in which so much emphasis is placed on spore morphology and colour. For these purposes an ITS data set, and a combined ITS-LSU data set were used, encompassing a wide sample of species. The ingroup comprised 81 species, with four other Agaricaceae for comparison, viz. Agaricus subrutilescens (Kauffman) Hotson & Stuntz, Macrolepiota procera (Scop.: Fr.) Singer, Chlorophyllum rachodes (Vittad.) Vellinga, and Leucoagaricus leucothites (Vittad.) Wasser. The outgroup comprised two species from outside the Agaricaceae: Limacella glioderma (Fr.: Fr.) Maire and Pseudobaeospora pyrifera Bas & L.G. Krieglst. The new data may help us understand the evolution of several characters, suggest hypotheses concerning speciation and distribution of the taxa, and indicate some fruitful directions for further morphological and molecular studies. Background information genera involved Lepiota (Pers.: Fr.) Gray, Natural Arrangement of British Plants 1: FRIES (1821) gave a wide definition of the genus, which included squamose and annulate white-spored species: Cystoderma, Limacella, and all the more recent split-offs from the genus, like Leucocoprinus, Leucoagaricus, Macrolepiota, and Cystolepiota. The present definition (VELLINGA 2001a) is much narrower, while still allowing for variation, and is mainly based on microscopical characters: pileus and stipe context not confluent, universal and partial veils present, lamellae free, spore print white to cream, microscopically with (rarely without) clamp connections, dextrinoid (rarely non-dextrinoid), non-metachromatic (rarely metachromatic), bi-nucleate (rarely uni-nucleate) generally non-ornamented spores, without a germ pore, with pileus covering made up of either long elements (trichodermally arranged or in chains and radially adnate or ascending), or narrowly clavate elements (hymeniderm-like), regular lamella trama. The following infrageneric groups are generally recognized: i. species with fusiform to penguin-shaped spores, and a trichoderm as pileus covering Section Lepiota. ii. species with spurred spores, and a trichoderm Section Stenosporae. iii. species with ellipsoid spores and a trichoderm Section Ovisporae (with two subsections: subsect. Helveolinae with a trichoderm made up only of long, cylindrical elements, and subsect. Felinae with a mixed trichoderm made up of both long, cylindrical and short, clavate elements). iv. species with a hymeniderm and ellipsoid or spurred spores Several names are available for this group, on section level sect. Lilaceae is the oldest. BON (1993) recognized two sections, sect. Integrellae with a pileus covering which does not break open into squames, and mainly uninucleate spores, and sect. Lilaceae with a pileus covering which does break open into squames, and mainly binucleate spores. Species with spurred spores are sometimes accommodated into a section of their own. v. species with acute warts on the pileus, made up of round elements Section Echinatae / genus Echinoderma (see below).

3 Mycological Progress 2(4) / Lepiota fuscovinacea is placed in its own section Fuscovinaceae by all authors, as it has a unique combination of characters: no clamp-connections, uni-nucleate spores, and a cutis-like pileus covering. Lepiota parvannulata and L. nigrescentipes are assigned to sect. Helveolae subsect. Parvannulatae in BON s classification (1993), and were tentatively placed in sect. Ovisporae subsect. Helveolinae by VELLINGA (2001a). SINGER s classification (1986) is different in that he recognized sect. Amyloideae for L. lignicola P. Karst., which is now accommodated in Leucopholiota (Romagn.) O.K. Mill., T.J. Volk & Bessette. Furthermore, SINGER (1986) recognized sect. Anomalae, for species without clamp-connections a very artificial group, with species belonging to Leucoagaricus as well as to other sections within Lepiota and sect. Amylosporae for species with amyloid (non-dextrinoid) spores. Melanophyllum Velen., Ceské Houby: This small genus of currently three species was erected to accommodate species with a pileus covering made up of globose cells, and spores which change from green when fresh to black when dry. Nowadays, species with persistent green spores are included, and the spores of all species are small and rough (warted when observed with Scanning Electron Microscope). Lepiotula (Maire) Locq. ex Horak, Synopsis Generum Agaricalium: Lepiota species with spurred spores, disregarding the type of pileus covering, were combined in this genus. Cystolepiota Singer in Singer & Digilio in Lilloa 25: 281. ('1951') Cystolepiota is a genus for species with generally small basidiocarps which have a veil composed of loosely arranged globose cells. The spores are uni-nucleate, and in most species non-dextrinoid. Clamp-connections are present in most species. This concept was amended by KNUDSEN (1978) to accommodate Lepiota sect. Echinatae, and amended again to remove that section (KNUDSEN 1980). VELLINGA (1992) included L. pulverulenta, with inflated, non-globose, loosely arranged velar cells, bi-nucleate spores, and without clampconnections, in Cystolepiota. Echinoderma (Locq. ex Bon) Bon in Documents Mycologiques 21 (82): This genus was split off from Lepiota for the species with rounded to ellipsoid elements in chains, arranged to form acute to pyramidal warts on the pileus. The main differences from Cystolepiota are the bi-nucleate, dextrinoid spores, and the presence of so-called intermediate cells in the pileus covering (intermediate between globose and cylindrical) (KNUD- SEN 1980). Developmentally, a distinct metablematogenic layer stands out between the veil and the pileus trama (GREIS 1937; terminology according to CLÉMENÇON (1997)), a layer which is absent in Cystolepiota. Pulverolepiota Bon in Documents Mycologiques 22 (88): This genus was erected for L. pulverulenta, a species resembling Cystolepiota, but differing in the elongate inflated elements of the pileus covering, the absence of clamp connections, and the bi-nucleate, rough spores (versus uni-nucleate and in most cases smooth spores in Cystolepiota). The American species L. petasiformis Murrill belongs here as well, though BON (1993) put it together with L. pseudogranulosa (Berk. & Br.) Sacc. in section Fuscovinaceae. Leucopholiota (Romagn.) O.K. Mill., T.J. Volk & Bessette in Mycologia 88: This monotypic genus harbours L. decorosa (Peck) O.K. Mill., T.J. Volk & Bessette, a species treated by KNUDSEN (1980) and SINGER (1986) (as Lepiota lignicola) in Lepiota sect. Amyloideae. HARMAJA (2002) erected the genus Amylolepiota Harmaja for L. lignicola, disputing the synonymy of L. decorosa and L. lignicola. He considered it closely related to Lepiota. It differs from Lepiota species in the gelatinized hymenophoral trama, and the amyloid thin-walled spores (VEL- LINGA 2003). MILLER, VOLK & BESSETTE (1996) placed the genus in the Tricholomataceae, and a position outside the family Agaricaceae was confirmed by molecular evidence (VELLINGA 2003). Chamaemyces Battarra ex Earle in Bulletin of the New York Botanical Garden 5: Chamaemyces was originally distinguished from Lepiota because of the non-dextrinoid spores. It differs from all other members of tribus Lepioteae in a number of characters: the confluent stipe and pileus context, presence of pleurocystidia, the (ixo)hymenidermal pileus covering, and the presence of clusters of caulocystidia, which are different from the pileus covering. The development is monovelangiocarpic and stipitocarpic, whereas all other Agaricaceae are bivelangiocarpic and not stipitocarpic. Coprinus (Pers.: Fr.) Gray, Natural Arrangement of British Plants 1: It has been shown repeatedly that Coprinus is a polyphyletic group (see REDHEAD et al for an overview), and that the type of the genus, C. comatus, together with a few other species, belongs to the Agaricaceae, close to Lepiota. Morphologically this genus (in its restricted sense) is quite different from Lepiota, because of the structure of the lamellae with the pavement cells between the basidia, the heteromorphic basidia themselves, the autolytic lamellae, and the black spores with a germ pore. Currently it is debated whether the genus Coprinus should not be typified by C. atramentarius, in which case the genus name Annularius would be used for the present group (JØRGENSEN et al. 2001).

4 308 Phylogeny of Lepiota Montagnea Fr., Flora Scanica: Montagnea is a sister taxon of Coprinus (in the restricted sense), and harbours coprinoid taxa with a secotioid habit. Three tropical genera are not included in the present study: Smithiomyces Singer (see VELLINGA (1999) for a description of S. mexicanus (Murrill) Singer, the type species); Janauaria Singer from the Amazon region (SINGER 1986), and Hiatulopsis Singer & Grinling, described from tropical Africa (SIN- GER & GRINLING 1967). Morobia Horak is regarded by some authors as a synonym of Lepiota (e.g. SINGER 1986), but the adnate (not free) lamellae, the structure of the pileipellis and the presence of rhizomorphs, connecting the basidiocarps (HORAK 1979), suggest a closer relationship with Marasmius and allies. Material and methods In total 81 species and varieties of the ingroup, two outgroup species not belonging to the family Agaricaceae, and four species from other clades within the family Agaricaceae have been sampled. Most collections originated from Europe and North America; Asia, tropical regions and the Southern Hemisphere are strongly under-represented. An overview of all taxa studied is given in Tab. 1, which also shows to which infrageneric taxon the Lepiota species were assigned. GenBank accession numbers are given there as well. Molecular analysis DNA was extracted from fresh and herbarium material; the methods described by GARDES & BRUNS (1993 and 1996) were either followed completely, or instead of the alcohol precipitation the GeneClean kit with Glassmilk (Bio101 Systems, Carlsbad, California, U.S.A.) was used. The internal transcribed spacer (ITS), and part of the Large SubUnit (LSU) of the nuclear ribosomal repeat were amplified by the polymerase chain reaction (PCR) with the fungal specific primers ITS1F and ITS4 (GARDES & BRUNS 1993) for the ITSregion, and primers LR0R, LR3R, LR7 and LR16 for LSU (see Large subunit RNA (25-28S) primer sequences at Sequencing of both strands was performed with an ABI model 377 sequencer or an ABI model 3100 automated sequencer (Applied Biosystems, Foster City, California, U.S.A.) using a Thermo Sequenase Dye terminator Cycle Sequencing Pre-Mix Kit (Amersham Pharmacia Biotech, Piscataway, New Jersey, U.S.A.) or a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems); the primers ITS1 or ITS5, ITS2, ITS3, and ITS4 were used for the ITS-region, and primers LR0R, LR3R, LR7, and LR16 for the LSU-region. The raw data were processed with the use of DNA Sequencing Analysis v and Sequence Navigator v (also of Applied Biosystems). Alignment The ITS sequences were aligned with the program Partial Order Alignment (POA) (LEE, GRASSOW & SHARLOW 2002) with minimal visual alignment. CLUSTAL X 1.81 (THOMPSON et al. 1997), with default settings, needed such heavy visual alignment that objectivity of the data could not be guaranteed. The LSU sequences were visually aligned only. The alignments have been deposited in TreeBase under number SN1461. Phylogenetic analysis Maximum parsimony analyses were performed using PAUP* version 4.0b10 (SWOFFORD 2002). One hundred heuristic searches were conducted with random sequence addition and tree bisection-reconnection (TBR) branch-swapping algorithms, collapsing zero-length branches and saving all minimal length trees (MulTrees), with MaxTrees set at Limacella glioderma was chosen as outgroup. Non-informative characters and a part of c. 150 base pairs (the number depends on the actual alignment) of the ITS1-region that was not unambiguously alignable, were excluded from all the analyses. Gaps were treated as missing data. To measure relative support for the resulting clades, 3000 bootstrap replications (FELSENSTEIN 1985) were performed using the fast stepwise addition mode; groups with a support of 50% were retained. To test alternative phylogenetic relationships, the Kishino-Hasegawa maximum likelihood ratio test (KISHINO & HA- SEGAWA 1989) with default parameters as implemented in PAUP* was performed. Several hypotheses, mainly based on the morphologically based existing classifications, were formulated and tested. Results Alignment The total length of the ITS fragment studied (including the 5.8S gene) ranged from 610 base pairs in L. clypeolaria to 711 in L. luteophylla. The ITS data set comprises 81 taxa and 87 collections, and is made up of 855 characters in total, of which 316 are parsimony informative. The aligned combined ITS-LSU data set, comprising 42 taxa, consists of a total of 1776 characters, of which 415 are parsimony informative. The LSU data set consists of 967 characters, of which 154 are parsimony informative. Part of the ITS1 segment was excluded from all analyses, as it was not unambiguously alignable. Phylogenetic analyses Three distinct clades were recovered from all data sets, Clade 1, comprising sect. Lepiota and sect. Ovisporae pro parte; Clades 2 and 3 (sometimes combined, sometimes separate), comprising the species with trichodermal and hymenidermal pileus coverings, and ellipsoid to spurred spores (sect. Ovisporae pro parte, sect. Stenosporae, and sect. Lilaceae) (Figs. 1-4).

5 Mycological Progress 2(4) / Fig. 1. One of 448 most parsimonious trees, based on all 314 parsimony informative characters of the ITS data set. Three morphological characters are indicated for each species; sp sh indicates the spore shape, and p cov stands for pileus covering. L = 2768, CI = , RC =

6 310 Phylogeny of Lepiota Fig. 2. Strict consensus tree, based on 448 most parsimonious trees, yielded from the ITS data set. Bootstrap values > 70 % are indicated below the branches.

7 Mycological Progress 2(4) / Fig. 3. One of seven most parsimonious trees, with best lnl, based on ITS and LSU data together. Bootstrap values 50 % are indicated below the branches. 414 Characters are parsimony informative. L = 1987, CI = , RC =

8 312 Phylogeny of Lepiota Fig. 4. One of 290 most parsimonious trees, based on the LSU data set. Bootstrap values > 60 % are indicated below the branches. 154 Characters are parsimony informative. L = 490, CI = , RC =

9 Mycological Progress 2(4) / Clade 4, the combination of Cystolepiota, Melanophyllum, and sect. Echinatae, can be recovered from a CLUSTAL X aligned ITS data set (after visual alignment; data not shown), but it is paraphyletic in the phylogeny based on the POAaligned ITS sequences. Several taxa do not fit into one of those four clades, and cluster together into morphologically diverse groups. Only Clade 1, or even only part of Clade 1 is bootstrap supported, though the whole clade is present in all consensus trees. The other major clades are not bootstrap supported, but again, the clades are all recovered in the consensus trees. Fourteen different hypotheses, mostly concerning the validity of the sections, were thoroughly tested for the ITS data set. Inclusion of both Chamaemyces fracidus and Coprinus comatus (and C. sterquilinus) cannot be rejected (Tab. 2) (the combined ITS-LSU data set gives the same results; Tab. 3); but Coprinus within Lepiota is very unlikely (in both analyses) (Tab. 2 & 3 note that Lepiota has been taken in a restricted sense in the analyses for Tab. 2, and in a wide sense in the analyses for Tab. 3). The hypothesis that Chamaemyces fracidus belongs to Lepiota (in restricted or in a wide sense) is not unambiguously supported, as the two different data sets give different outcomes (Tab. 2 & 3). The ITS data set does not support sect. Ovisporae, whose members are divided between Clade 1 and Clade 2 in the phylogenies. Monophyly of sect. Lilaceae, of sect. Echinatae, and of Cystolepiota, Pulverolepiota, Melanophyllum and sect. Echinatae (Clade 4) together cannot be rejected (Tab. 2). But monophyly of Lepiota including Cystolepiota, Pulverolepiota, Melanophyllum, and sect. Echinatae, nor Lepiota excluding those taxa can be accepted; in all those cases Coprinus comatus and allies, and Chamaemyces fracidus were not included in Lepiota. Discussion Overview In the analysis of the entire family, the tribus Lepioteae without Chamaemyces, but including Coprinus comatus and allies, was shown to be monophyletic; Melanophyllum was shown to belong to this very clade (VELLINGA 2003). These same results emerge from the present analyses of a much more restricted data set. The resulting clade again also contains Coprinus comatus and C. sterquilinus. Chamaemyces The position of Chamaemyces fracidus is still not unambiguous. In the analyses of the ITS, LSU or combined ITS-LSU data sets of the family Agaricaceae as a whole (VELLINGA 2003), Chamaemyces fracidus is always basal to the family, and inclusion in Lepiota has to be rejected. In the present analyses, based on a smaller sample from fewer lineages, its placement varies with the data; according to the ITS data a place within Lepiota is acceptable (Fig. 1; Tab. 2), but it is excluded based on the results of the Kishino-Hasegawa test on the combined data set, where Lepiota is taken in a wider sense (including Cystolepiota, sect. Echinatae, Melanophyllum and Pulverolepiota) (Table 3). The morphology of Chamaemyces fracidus, and especially its development (see under Developmental characters ), is quite different from Lepiota itself, and a position at the base of the family seems much more plausible than one within Lepiota. Coprinus comatus Despite ending up within the Lepiota clade, the position of Coprinus comatus and C. sterquilinus is ambiguous. Inclusion of Coprinus comatus and C. sterquilinus within Lepiota is not supported by the ITS data, nor by the ITS-LSU data; though a position within Lepiota for this group and for Chamaemyces cannot be rejected (Tab. 3). However, MONCALVO et al. (2002) showed, based on LSU analyses, that these taxa together with three Montagnea species form a distinct clade, separate from Lepiota and the other monophyletic groups. These authors examined the same seven Lepiota and Cystolepiota taxa as JOHNSON & VILGALYS (1998), plus two Melanophyllum sequences. Morphologically, the coprinoid taxa are totally different from all taxa in the Lepiota clade, and a separate position, which cannot be ruled out by our analyses, does seem an appropriate solution. Lineages Contrary to expectations, essentially none of the morphologically recognized genera, subgenera and sections form at first glance monophyletic groups; Pulverolepiota and Melanophyllum form the exception. Though when tested with the Kishino-Hasegawa test, monophyly of sections Lilaceae and Echinatae cannot be rejected (ITS data; Tab. 2). Based on morphology and outcome of the present analyses the following four clades are accepted, and briefly described and discussed. Clade 1 Clade 1 is characterized by trichodermal pileus covering made up of long and short, cylindrical to narrowly clavate, elements. The spore shape varies: fusiform (e.g. in L. erminea and L. clypeolaria), penguin-shaped (in L. spheniscispora, L. magnispora, and L. cortinarius), and broadly fusiform to ellipsoid (e.g. in L. subgracilis, L. xanthophylla). Within the Lepioteae, Clade 1 always gets relatively high bootstrap support. This clade harbours all species from sect. Lepiota and a part of sect. Ovisporae. There has been disagreement about the position of L. cortinarius; BON (1981; 1993) placed the species in sect. Stenosporae, but VELLINGA (1992) placed it in sect. Lepiota close to L. ventriosospora. Both authors used spore shape as the overruling character for their decision. A position in sect.

10 314 Phylogeny of Lepiota Lepiota is supported by the present, molecular data, and by the structure of the pileus covering. The collection from which the GenBank sequence of L. subincarnata has been derived must have been misidentified, as the present data do not place it with our collections of this species, characterized by the lack of short elements in the pileus covering. Several collections, morphologically identified as L. clypeolaria, turned out to be polymorphic with regard to their ITS sequences; these collections were not included in the present data set. Clade 2 This clade harbours species with similarly varied spore shape, and with two types of pileus covering, either a trichoderm of long elements only or a loosely arranged cutis with articulate, clamped hyphae. Species with ellipsoid and with spurred spores both occur in this clade, representing sect. Stenosporae (part of genus Lepiotula) and sect. Ovisporae subsect. Helveolinae. Three subclades can be recognized: 2a with ellipsoid spores and a trichoderm, 2c with spurred spores and a trichoderm, and 2b with spurred spores and a cutis-like pileus covering. The most infamous amanitin containing species, L. subincarnata (and its synonym L. josserandii), L. elaiophylla, and L. brunneoincarnata constitute Clade 2a. Species limitations in the complex of Lepiota castanea have been examined by MIGLIOZZI & ZECCHIN (1997), who recognized three species, mainly on spore size. However, VEL- LINGA (2001a) found the boundaries between their taxa were not clear-cut and still considered L. castanea a single very variable species. Two samples of this group were sequenced, and it appears that indeed, a small-spored taxon, tentatively called L. ignicolor, with relatively short elements in the pileus covering can be separated from a long-spored taxon, viz. L. castanea, with long elements. Lepiota ignicolor is a sister taxon of L. subalba, with equally short pileus covering elements. A thorough examination of this group is needed, using a wide sample from all over the Northern Hemisphere; the morphological variability and the molecular data deserve equal attention. The morphologically relevant characters are general basidiocarp shape and colour, spore size, length and number of septa of the pileus covering elements and the position of these on the pileus. Clade 3 Clade 3 is characterized by a hymenidermal pileus covering, whereas the spore shape is variable, and so are the chemical reactions and the number of nuclei per spore (see VELLINGA & HUIJSER (1999) for an extensive morphological overview of the relevant species). Hymenidermal species assigned to Lepiotula, and to sect. Lilaceae belong here. Three species, L. pyrochroa, L. lilacea and L. ochraceofulva, do not fall into this clade on molecular grounds, though they fit morphologically; their inclusion cannot be rejected (Tab. 2). Surprisingly, L. rufipes and L. pyrochroa are not sister taxa, as suggested by VELLINGA & HUIJSER (1999), nor does L. rufipes stand out from the other taxa in this clade, despite its distinctive morphology. Spores of several species in this clade have metachromatic, non-dextrinoid walls. Pink colouration of the spore wall in Cresyl Blue, a metachromatic reaction, is characteristic for, but not restricted to, the genera Leucoagaricus, Leucocoprinus, Macrolepiota and Chlorophyllum. In these genera, the spore wall is also dextrinoid. Lepiota scaberula, L. cystophoroides, and L. luteophylla do not have a simple hymenidermal pileus covering, but one giving rise to loose cells. On account of this architecture of the pileus covering, L. luteophylla was accommodated in Cystolepiota (KNUDSEN 1978). That position does not seem acceptable, in light of the present data. As in many other species of this clade, the spores are not dextrinoid. Chamaemyces fracidus possesses the characteristic hymeniform pileus covering of Clade 3, and is accommodated there in trees based on the combined data set. However, the development of the basidiocarp in Ch. fracidus is quite different from that in L. cristata (REIJNDERS 1975; ATKINSON 1916 for L. cristata). Clade 4 The fourth clade is not monophyletic in phylogenies based on the ITS-data, though its monophyly cannot be rejected (Tab. 2). This group is made up of species assigned to sect. Echinatae, and the genera Cystolepiota, Melanophyllum and Pulverolepiota. In the ITS-phylogenies, Coprinus comatus and C. sterquilinus also belong to this clade. The position of these two taxa is far from resolved, as ITS and LSU data give conflicting signals. Section Echinatae is split into two subclades, a large-spored group (L. aspera, L. hystrix, L. perplexa), and a small-spored group (L. jacobi, L. echinacea and some undescribed species from California). Amazingly, in this case spore shape and size do seem to distinguish the two subclades. Variation in ITS among the species in this clade as a whole is quite substantial. Additional visual alignment of the POA-aligned ITS data set renders clade 4 paraphyletic, and split up into small subclades (results not shown). It is not difficult to distinguish Cystolepiota species from species in Lepiota sect. Echinatae; spore characters and pileus covering are decisive. However, misidentifications have been made, e.g. ENDERLE & KRIEGLSTEINER (1989) misapplied the name L. pseudoasperula Knudsen to C. moelleri Knudsen. A close relationship of Melanophyllum and Cystolepiota is not surprising, as both taxa are morphologically and developmentally very similar, they differ only in colouration and ornamentation of the spores. It is also clear that Melanophyllum haematospermum is more likely to be a species complex, than one phylogenetic species. The three collections sampled here differ in size of the basidiocarps, from small to medium-sized, with short or long stipe. World-wide sampling and morphological and molecular comparisons of the collections are necessary.

11 Mycological Progress 2(4) / Taxa outside the four main clades The position of some species which are morphologically distinct from all the others is just as problematic on molecular grounds. For example, L. fuscovinacea and L. nigrescentipes are not closely related to any other species in the present data set. Lepiota nigrescentipes is closely related to L. parvannulata (Lasch: Fr.) Gillet (the two may be synonymous), and is characterized by small, non-dextrinoid, uni-nucleate spores, absence of cheilocystidia, and a cutis-like pileus covering, with clamp-connections. Lepiota fuscovinacea differs from all other examined species in the absence of clamp-connections, the uni-nucleate spores, and the cutis-like copious pileus and stipe covering. The position of L. lilacea, L. ochraceofulva, and C. icterina in the cladograms based on ITS data (e.g. Fig. 1) is hard to explain. The first two species have a hymenidermal pileus covering, and the latter is a unique Cystolepiota species because of its yellow tinges and the dextrinoid spores. Character combinations Clades 1, 2 and 3 each harbour species with different spore shapes, an indication that features such as spurred spores have evolved independently several times. Spore shape in Clade 4 also varies, though less dramatically. Since spore shape fails to characterize the clades, is there any other morphological character that does? An answer may be found in the structure of the pileus covering, and perhaps in the developmental stages of the basidiocarps that give rise to it. The pileus covering of the species in Clade 1 is a trichoderm, made up of both long cylindrical elements and short, clavate to cylindrical elements. Clade 2 is characterized by having a trichoderm or a cutis-like pileus covering, but both types of covering are made up of long cylindrical elements only. Taxa in Clade 3 have a hymenidermal pileus covering. The elements of the pileus covering in Clade 4 are globose to inflated, either loosely arranged or in agglutinated chains. Despite the variation in spore shape within each clade, some character combinations have not evolved; for example, there are no taxa with a hymenidermal pileus covering and fusiform uni-nucleate spores. Developmental characters A key character in the Agaricaceae is the presence of protective layers (veils or vela); their structure and developmental pathways are both important. Ontogenesis has been morphologically studied in several species: L. clypeolaria (ATKINSON 1914), L. cristata (ATKINSON 1916), L. aspera (as L. acutesquamosa; GREIS 1937), L. castanea (as L. ignicolor; HUGUE- NEY 1966), C. seminuda (ATKINSON 1916), C. hetieri and Melanophyllum haematospermum (REIJNDERS 1963). REIJNDERS (1975) treated the family as a whole. CLÉMENÇON (1997) introduced a new terminology for the carpogenesis, and reinterpreted some of the existing data. The development of L. clypeolaria differs substantially from that of L. castanea (CLÉMENÇON 1997); in L. castanea a separate hyphal cutislike layer covers the whole pileus, and the underlying trichoderm is interpreted as the pileipellis by CLÉMENÇON (1997). This outer layer is absent in L. clypeolaria, and the trichoderm is interpreted as of velar origin. This may supply a clue to the differences between Clades 1 and 2, though without further work we should not assume that the development of all the species in those clades is the same as for the two representatives studied. Lepiota cristata deviates in the absence of velar material on the stipe of the mature basidiocarps, though in other members of Clade 3, e.g. L. hymenoderma, there are remnants of the universal veil present at the stipe base. It also seems that C. seminuda follows a different developmental pathway than the other species, so it is surprising that it is so close to L. aspera in the molecular analyses, which place both taxa in Clade 4. The absence of the metablematogenic layer in the primordial stages of C. seminuda, C. hetieri, and Melanophyllum haematospermum is striking, as this layer is very prominent in the early stages of the development of L. aspera (compare GREIS (1937) and ATKINSON (1916)). Unfortunately nothing is known about the genetic basis of these different developmental pathways. Chamaemyces fracidus differs considerably in development from Lepiota (REIJNDERS 1975). It is monovelangiocarpic, whereas all other taxa are bivelangiocarpic, and furthermore, the stipe is the first to develop (stipitocarpy); Lepiota species are pileostipitocarpic. Character evolution As the deeper branches are poorly resolved, and the various analyses yield slightly different results, it is not easy to say anything with certainty about the characters of the ancestral Lepiota. BON (1993), speculating on the evolutionary lines within the group, considered Pseudobaeospora ancestral, and regarded L. parvannulata as the most primitive species. He envisaged lineages originating with L. parvannulata and leading to Cystolepiota; to sect. Lepiota, and, via sect. Ovisporae, to Echinoderma, while sect. Stenosporae was a side branch. Lepiota parvannulata was not included in our sample but L. nigrescentipes, which is morphologically very close to it, does not seem to be a good candidate for the proto-lepiota. Other aspects of BON s phylogeny are contradicted by the present analyses, e.g. the close relationship of Cystolepiota and Echinoderma. But it is premature to speculate further. Spurred and penguin-shaped spores do seem to be a derived character, having originated several times in different lineages. Vicariance and speciation An intriguing aspect of the present study is that it draws attention to several pairs of sister taxa with the same disjunct distribution, with one species in Europe and the other in western North America. Examples are L. cystophoroides paired with L. scaberula, and C. pulverulenta with L. petasiformis. This phenomenon also occurs in other members of the family. It might indicate that speciation took place after the two areas became isolated from each other. Unfortunately, for many of

12 316 Phylogeny of Lepiota these taxa data on their occurrence in eastern North America and in Asia are not available. Data on sexual compatibility of these taxa are also needed to be more explicit on the mechanisms of speciation. Secotioid forms Recently a secotioid species was described from Sardinia, viz. Notholepiota sardoa Padovan & Contu, which resembles the grassland species L.oreadiformis Velen. and L. erminea (PADOVAN & CONTU 2001). Judging from the description by PADOVAN & CONTU (2001) this species belongs to Lepiota Clade 1. Notholepiota areolata (Stevenson) Horak from New Zealand, which was suggested to be closely related to Lepiota on account of the pale fusiform spores and the dextrinoid reaction of the spore wall (HORAK 1971), has turned out to be closely related to Boletus (VELLINGA 2003). Other secotioid forms within this group of the family Agaricaceae are Montagnea species in the Coprinus clade. The phenomenon of adapting to dry conditions by becoming secotioid has evolved several times within the genus Agaricus; examples are Gyrophragmium dunalii (Fr.) Zeller, Longula texensis (Berk. & M.A. Curtis) Zeller, and Agaricus inapertus Vellinga (syn. Endoptychum depressum Singer & A.H. Sm.). In conclusion It is premature to base a new classification on the phylogenetic hypotheses presented here; bootstrap supports for the clades are low, the different data sets do not always present the same topologies, and the deeper branching is unresolved. However, morphological characters strongly support the clades, and the main clades are always present in the consensus trees. In other basidiomycete genera ITS sequences have successfully been used to recognize infrageneric taxa, e.g. in Rhizopogon (GRUBISHA et al. 2002). Chamaemyces fracidus should be excluded from Lepiota; a place at the base of the family Agaricaceae seems appropriate on account of both morphological and molecular characters. Inclusion of Coprinus comatus and allies within Lepiota does not seem acceptable either. Results indicate that sections Lepiota and section Stenosporae have to include species with different spore shapes than accepted hitherto, and a re-evaluation of section Ovisporae is needed. Also the recognition of separate genera for Cystolepiota, sect. Echinatae, Melanophyllum and Pulverolepiota has to be reconsidered. One genus for this group seems a good alternative as already advocated by KNUDSEN (1978). The structure of the pileus covering, and the development of the basidiocarps are key characters in the evolution and classification of this group. The present study suggests several areas where further research may elucidate the phylogeny of Lepiota and its allies: i. molecular research focusing on other genes, preferably markers which give a good phylogenetic signal above species level to elucidate the basal branching; ii. molecular and morphological research on tropical taxa, and on taxa with unique character combinations, such as the neotropical species L. rubella Bres. with a cutis-like pileus covering of thick-walled elements and small ellipsoid spores, and the neotropical genus Smithiomyces, with its unique structure of the pileus covering, a genus which might be closely related to Lepiota; iii. developmental studies, especially focusing on the ontogeny of the basidiocarps and the origin and formation of the velar layers, but also paying attention to the developmental differences between agaricoid and secotioid basidiocarps. But the foremost requirement is for descriptive taxonomic and morphological work in all areas of the world. Even in Europe, where the situation is best, new Lepiota species are described almost every year. Some recent examples: FILLION (1997) described L. glareophila from the French Alps, a species which probably belongs to Clade 1 in the present presentation, and VELLINGA & HUIJSER (1999) published two new species in sect. Lilaceae (Clade 3). In North America, our knowledge of the Lepiota flora is still very limited, and many undescribed species occur. Acknowledgments The basis for this analysis was formed by collections made by many people. Curators of the herbaria at C, L, and MICH, and the following individuals are acknowledged for their contributions: Henk A. Huijser, Rob Chrispijn, Manfred Enderle, Thomas Læssøe, Pierre Roux, Nico J. Dam, Fred Stevens, Nancy Ironside, Rod E. Tulloss, Zhu-Liang Yang, P. Brandon Matheny, and Lothar J. Krieglsteiner. 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