New and rare taxa inAgaricussectionBivelares(Duploannulati)

Richard W. Kerrigan; Philippe Callac; Luis A. Parra

Agaricus is a genus of saprobic basidiomycetes including species of nutritional and medicinal interest. Historically the temperate species have been grouped into eight classical sections. Recent phylogenetic analyses however, revealed that two-thirds of the tropical taxa do not cluster in these sections, but form exclusively tropical clades. Seven (TR I to TR VII) strongly supported tropical clades have been revealed and it was hypothesized that clade TR I might represent Agaricus section Brunneopicti. This section was initially characterized by the presence of punctiform squamules, the remains of the veil, on the pileus and stipe. The present morphological study and phylogenetic ML, MP and Bayesian analyses based on ITS1+2 sequences show that clade TR I corresponds to Agaricus section Brunneopicti and includes 16 taxa grouped in four strongly supported subclades and two isolated branches. The six species with punctiform squamules which initially characterized the section constitute...

The genus Agaricus was recently rearranged to accommodate numerous tropical taxa. Accordingly, the genus was split into six subgenera and 22 sections of which 12 are included in A. subg. Pseudochitonia. Preliminary data indicated that three putative new species belong to this subgenus. Our objectives were to describe these species, to determine to which sections they belong, and to experience the interest of some traditional traits in this new context. We morphologically described Agaricus coniferarum from France and Portugal, Agaricus iranicus from Iran, and Agaricus lusitanicus from Portugal. Multi-gene phylogenetic analyses based on ITS, LSU, and tef1 sequence data of representatives of the 12 sections clearly indicated that A. coniferarum and A. lusitanicus are placed in Agaricus sect. Bohusia, while A. iranicus is in A. sect. Sanguinolenti. Incidentally, we replaced the illegitimate name Agaricus magnivelaris by Agaricus fiardianus. In a phylogenetic tree, based on all availabl...

Mycologia, 100(6), 2008, pp. 876–892. DOI: 10.3852/08-019 # 2008 by The Mycological Society of America, Lawrence, KS 66044-8897 New and rare taxa in Agaricus section Bivelares (Duploannulati) Richard W. Kerrigan1 and subsectional names have been applied to this group, most often with A. bitorquis (or its heterotypic synonym Psalliota rodmanii) or A. hortenses (a heterotypic synonym of A. bisporus) as the type species (see Challen et al 2003). Agaricus section Duploannulati Wasser ex Wasser (1980), typified by A. bitorquis, has been the subject of two recent phylogenetic reassessments based on sequence analysis of the ITS1+2 region of the nuclear rDNA. Challen et al (2003) evaluated six species-level taxa and several infraspecific entities and restricted the section to a clade containing the type species, A. bitorquis, while excluding species that we believe should be placed in section Chitonioides Romagn. Didukh et al (2005) extended this analysis with an emphasis on taxa from Israel, according to Wasser’s (1980; see also Wasser et al 1976) original, broader concept of Duploannulati. Given Singer’s (1975) selection of P. rodmani (Peck) Kauffman, a synonym of A. bitorquis, as the type of Kauffman’s (1919) Psalliota section Bivelares, Bivelares is the oldest epithet for the section containing A. bitorquis, with priority from 1919. Consequently a new combination in Agaricus has been created at the sectional level (Parra 2008). We have continued to collect and evaluate both field material and cultures having unresolved affinities or status. As a result three new species are described here: Agaricus cupressophilus and A. tlaxcalensis, both belonging to the new Agaricus subsection Cupressorum, and A. subsubensis, in Agaricus subsection Hortenses Heinem. (1956). In addition we formally treat two entities that were discussed in our earlier publications, segregating the new species A. agrinferus from the accumulated concept of A. subfloccosus ( J.E. Lange) Hlaváček and recognizing A. devoniensis subsp. bridghamii. Yet another new member of Bivelares from France (collection CA103 [INRA]) is given a preliminary report here. The latter three new taxa (and the unnamed entity) are placed in subsection Hortenses. Finally we present comments intended to supplement information provided in Challen et al (2003). Sylvan Research, 198 Nolte Drive, Kittanning, Pennsylvania 16201 Philippe Callac INRA, MYCSA (Mycologie et sécurité des aliments) BP 81, 33883 Villenave d’Ornon cedex, France Luis A. Parra Avenida Padre Claret 7, 5th G, 09400 Aranda de Duero, Burgos, Spain Abstract: Ongoing field and laboratory studies have led to our recognition of new taxa in Agaricus section Bivelares, a recent combination and now the earliest synonym and correct name of section Duploannulati. Agaricus cupressophilus and A. tlaxcalensis, in the new Agaricus subsection Cupressorum, and A. agrinferus, A. devoniensis subsp. bridghamii, and A. subsubensis in Agaricus subsection Hortenses, are described. Agaricus subfloccosus is lectotypified. Phylogeny reconstruction methods with ITS1+2 DNA sequences were used to determine appropriate placements of the new taxa. Collectively these new taxa and phylogenetic associations represent a substantial augmentation and clarification of our knowledge of section Bivelares; described, sequenced species-level taxa in the northern hemisphere are increased from six to 10, a distinct subsectional lineage is revealed and infraspecific resolution within A. devoniensis is improved. An anomalous ITS1+2 sequence is documented in one collection of A. subsubensis. Preliminary data on another novel member of Bivelares from France also are provided. Several of these taxa are rare, highlighting opportunities and challenges for documenting biodiversity in this group. Additional comments on related taxa treated in recent publications are also provided. Key words: button mushroom, cultivation, Cupressus, ITS sequence evolution, phylogeny, taxonomy INTRODUCTION The familiar cultivated button mushroom species Agaricus bisporus ( J.E. Lange) Imbach and the cosmopolitan A. bitorquis (Quél.) Sacc. belong to a relatively small infrageneric group. Several sectional MATERIALS AND METHODS Collection, preservation and study methods, and cultural practices, were routine and have been described elsewhere (Kerrigan et al 1999). Collection data are given below. North American material is deposited at SFSU unless otherwise indicated. CA collections and cultures are housed Accepted for publication 25 August 2008. 1 Corresponding author. E-mail: rwk@sylvaninc.com 876 KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES at the Collection du Germplasm des Agarics à Bordeaux (CGAB), France. Cultures were deposited at the American Type Culture Collection (ATCC) or at CGAB. DNA sequencing and sequence analysis also has been described elsewhere (Challen et al 2003). Briefly, PCR amplification of the ca. 710 nt ITS1+2 nuclear genomic rDNA region, here defined as beginning at ‘‘position 1’’ with 59-ggaaggat in the SSU-rRNA gene and ending with gaacttaa-39 in the LSUrRNA gene, was performed with primers ITS1 or ITS5 and ITS4, or ITS2 and/or ITS3 as needed (White et al 1990). Sequences for both strands were determined with an automated DNA sequencer either at the Nucleic Acid Facility-Pennsylvania State University, University of Pittsburgh, McMaster University or INRA and were deposited in GenBank (accession numbers are given below). Sequences were aligned with the Clustal W routine followed by manual inspection and correction and the final alignment was deposited in TreeBASE (study accession number 5 S2128; matrix accession number 5 M4027). In the special case of A. subsubensis JB 101 (discussed below) the ITS1+2 sequence, independently amplified twice and sequenced twice for verification, was divided into three parts; the first and third segments were aligned independently for comparisons but were not used in tree-building. Both maximum parsimony (MP) trees (FIG. 1; PAUP* v 4.0b8: Swofford 2000) and a neighbor-joining (N-J) tree (FIG. 2; MegAlign, Lasergene v. 6; unweighted nucleotide substitution model) were generated. Evaluation of the nonredundant sequences, including some that were too short for N-J analysis, under MP was performed as per Challen et al (2003) with the following heuristic search settings: random addition sequence, one tree held at each step during stepwise addition using the tree-bisection-reconnection algorithm, branch collapsing if maximum branch length was zero, gaps treated as missing data, stepwise descent option not in effect, topological constraints not enforced, bootstrapping (n 5 1 000 000) and MAXTREES 5 100. Distinctive DNA sequence characters are reported with position numeration (see above) specific to individual sequences. We report such characters when they appear to be useful in the context of available data; as with any taxonomic character, new data might lead to a reassessment. Nomenclature follows Kerrigan et al (2006: 1299) in agreement with ICBN-2006 (Vienna Code). Rank relationships presented herein are hypotheses based on all available data. No single criterion is sufficient to the task; however in general we have observed in earlier studies (Challen et al 2003, Kerrigan et al 2005) and in ongoing work that conventionally accepted species of Agaricus generally differ by two or more ITS DNA characters, whereas infraspecific taxa differ by two or fewer ITS characters. This emergent, rough phenetic standard cannot serve as a sole criterion for defining taxon boundaries and was considered as only a single line of evidence. In selecting the most appropriate rank hypothesis we also considered, as available, DNA sequences from other genes (e.g. from manuscripts in preparation; see A. subsubensis), morphology (see A. tlaxcalensis and A. cupressophilus), physiology, cultural behavior, reproductive mode (for all see A. agrinferus), 877 mating studies, other molecular data, ecological and geographical separation of populations/taxa and any patterns of association of ITS or other characters among the isolated populations, evidence for hybrids among putatively differentiated entities (see A. cupressicola, below, contra A. xanthodermus and A. moelleri in Kerrigan et al 2006), the state of knowledge and complexity of the immediate group (see the A. devoniensis clade, below), nomenclatural practice and precedents, our preference for conservative rank placements, and, to a lesser (or greater) extent, the unexpectedly adamant guidance of reviewers. New names were recorded in MycoBank (http://www. mycobank.org) and the assigned unique identifiers are presented below after the Latin diagnoses in the format MB ######. DESCRIPTIONS OF TAXA Agaricus section Bivelares (Kauffman) L.A. Parra 2008. Fung. Europ. 1(1):157. Type: Psalliota rodmanii (Peck) Lloyd, designated by Singer, The Agaricales in Modern Taxonomy. 3rd ed.: 461. 1975. ; Psalliota section Bivelares Kauffman, Agaric. Michigan: 234. 1919 [‘‘1918’’]. [basion.] 5 Agaricus section Duploannulati Wasser ex Wasser, Fl. Fung. RSS Ucrain.: 125. 1980. [‘‘DuDuploannulatae’’] [nom. illeg., nom. superfl.; art. 52.1 since Psalliota rodmanii (Peck) Lloyd, type of the section Bivelares Kauffman (1919) is cited as a synonym of Agaricus bitorquis (Quél.) Sacc., the type of the section Duploannulati designated by Wasser (1980: 125)] 5 Agaricus section Bitorques (Heinem.) Bon & Cappelli, Doc. Mycol. 13(52): 16. 1983. Type: Agaricus bitorquis (Quél.) Sacc., designated by Heinemann, Sydowia 30: 13. 1978. ; Agaricus subsection Bitorques Heinem., Sydowia 30: 13. 1978. Orig. diag.: Annulus double, with thick flocculose patches on under side. MB 512252. Delimitation of the section: Schäffer’s reaction negative, KOH reaction negative (at least away from the disk), o-tolidine generally blue or negative on peripheral tissues (pelles, veils, exposed stipe base) while violet-purple or negative on exposed interior tissues, a-naphthol generally violet-purple (or rarely negative) on the basal stipe context while orange to pink on exposed interior tissues. Exposed context rufescent: becoming pink or reddish, rarely blood red or reddish-orange or sordid, or sometimes unchanging or almost so. Veils forming various types of annuli or occasionally volva-like basal structures. Stipe cylindrical, clavate or slightly bulbous, usually not tapering at the base, stuffed-hollow (having a pith-filled central cavity) except for Agaricus bitorquis in which it is 878 MYCOLOGIA FIG. 1. One of six most parsimonius trees from a run of 1 000 000 iterations on a set of nonredundant ITS1+2 sequences from taxa of Bivelares and other sections of Agaricus. Three trees had the topology shown for the Cupressorum clade, while the other three trees had the alternative topology shown at the top of the figure. Abbreviations: BERN: A. bernardii; CPCL: A. cupressicola; CPHL: A. cupressophilus; TLAX: A. tlaxcalensis; DEVO: A. devoniensis (and variants); FRGV: A. fragilivolvatus; BRID: A. devoniensis ssp. bridghamii; SBPR: A. subperonatus (misapplied); BISP: A. bisporus; BISP BURN: A. bisporus var. burnettii; BISP EROT: A. bisporus var. eurotetrasporus; SFLC: A. subfloccosus; AGRN: A. agrinferus; BITQ: A. bitorquis; CAPL: A. capellianus; SBSB: A. subsubensis; XANT: A. xanthodermus; SRTL: A. subrutilescens; SBRF: A. subrufescens; CAMP: A. campestris; PATR: A. pattersonae. Bootstrap values are based on 1000 replications. usually about solid. Basidiomata with mild or indistinct odors, most often like that of Agaricus bisporus, or occasionally more complex, as in some species of section Sanguinolenti Jul. Schäff. & F.H. Møller ex L.A. Parra (a fruity/spicy odor following Kerrigan [1986]). Cheilocystidia usually present. Spores lacking a rudimentary apical pore. Culture on routine media such as PDA or compost extract agar produces ample growth KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES 879 FIG. 2. Neighbor-joining tree of all ITS1+2 sequences evaluated in the present study, except (i) the too short sequences for the New Zealand A. devoniensis variant (GenBank AF059225: ITS1 data are missing) and CA 504 / A/9.1.2, the type of A. fragilivolvatus (which matches that of DEVO CA 100 / Dv2), and (ii) the highly modified, nonaligning sequence from SBSB JB-101. The Clustal W alignment was hand corrected. Abbreviations are as in FIG. 1. (relative to cultures of Sanguinolenti). Characteristic ITS polymorphisms (Challen et al 2003): cttt(k/-) tCAggta @ 120–121, at (r) tAag (r) a @ 167, ttat (Y ) atac @ 311, attaTattc @473, c(y)c(y) (R) atac @488, (y)cgtCtgcg @580, and tnag [-8-]gaca @663–670. We are aware that the sectional name Bivelares has not been used for many years. However under the rules of nomenclature Bivelares, as typified by Singer (1975) has priority over Duploannulati and Bitorques and is therefore the correct name of the section to which A. bitorquis belongs. A more complete discussion is provided by Parra (2008). Two closely related sections are sect. Xanthoderma- tei and sect. Chitonioides. As in sect. Bivelares these two sections also have negative Schäffer’s reactions (although in a lapsus in an earlier paper [Challen et al 2003], those authors wrote that Xanthodermatei is characterized by positive Schäffer’s reactions, when of course ‘‘negative’’ was meant [see also Kerrigan et al 2006]). KEY TO SUBSECTIONS Pileus margin typically not strongly inrolled in young basidiomata, with a thin sterile marginal rim not (or barely) exceeding the lamellae; annulus 880 MYCOLOGIA thin, ‘‘simple’’, pendant or remaining appended to the pileus margin, stipe without conspicuous velar remnants below the annulus; habitat typically under Cupressaceae, occasionally under other trees . . . . . . . . . . . . . . . . . . subsection Cupressorum. Pileus margin strongly inrolled in young basidiomata (often even in mature basidiomata) with a thick, sometimes ‘‘swollen’’ rim conspicuously exceeding lamellae, seldom appendiculate; annulus/veils various: usually not thin, usually intermediate, or peronate or occasionally pendant, but if appearing pendant then sometimes with an ornamented undersurface or with some velar remnants towards the base; habitat various including under Cupressaceae. . . . . . . . . . . . . . subsection Hortenses. See also distinguishing molecular characters (below). Another helpful field character is a tendency (only) toward a slender stature in most species of Cupressorum (or not, in A. cupressicola) vs. a nonslender stature (with the exception of some A. devoniensis) in Hortenses. Generally speaking the pendant annulus in Agaricus (see Kerrigan 1982) corresponds to the descending (Nauta 2001) or superous (see Boisselet 1990, Parra 2003, Cappelli 1984) veils of European authors. These terms all apply to the veils of the new subsection Cupressorum, but in their strict senses are not typical of subsection Hortenses. Agaricus [section Bivelares] subsection Cupressorum Callac & Kerrigan, subsect. nov. Stipes cylindricus ad clavatum. Margo pilei non valde involutus. Annulus superus pendulus, tenuis, simplex. Caro fracta aurantiascens vel rubescens. Odor inconspectus vel Agarico bisporo similis. Cheilocystidia praesentia. Sporae apicis poro destitutae. Cultura in PDA bona. Polymorphismi propii regionis ITS: in positionibus: ctttGtcag @ 115, tattgGgAgaag @ 126, 128, ccttCgctg @ 152, tgcaTtgtg @ 173, gaaaGcGgtgc @ 181, 183, tgtaGagga @ 491, atctGcact @ 600, aggaTtacc @ 689 (ex typi Agarici cupressicolae sequentia positiones captae, L 5 705 nt). MB 512253. Typus: Agaricus cupressicola Bon & Grilli in Bon, Doc. Mycol. 17(67):11. 1987. Stipe cylindrical, clavate, neither clearly bulbous nor tapering at the base. Pileus margin not strongly inrolled, sometimes at least partially appendiculate with velar remnants. Annulus simple, pendant-superous. Context becoming orange to red. Odor indistinct or similar to Agaricus bisporus. Cheilocystidia usually present. Spores lacking an apical pore. Growth on PDA good. Schäffer reaction negative; KOH usually negative, or yellow on disk. Distinctive ITS1+2 region characters: ctttGtcag @ 115, tattgGgAgaag @ 126, 128, ccttCgctg @ 152, tgcaTtgtg @ 173, gaaaGcGgtgc @ 181, 183, tgtaGagga @ 491, atctGcact @ 600, aggaTtacc @ 689 (in the LSU) (positions taken from the A. cupressicola type sequence, L 5 705 nt). Type species: A. cupressicola Bon & Grilli in Bon, Doc. Mycol. 17(67):11. 1987. Also included: A. tlaxcalensis and A. cupressophilus, described below. Agaricus has a rich association with Cupressaceae. Several dozen Agaricus species are found under Cupressus, most particularly C. macrocarpa; more than 30 species have been recorded from a single hectare of this habitat (Kerrigan 1982). Many Agaricus spp. from California are known only from groves or plantations of Monterey cypress, possibly implying a specific habitat association. On the other hand, where Cupressus has been introduced, Agaricus usually soon arrives, and novel, apparently indigenous species are encountered (Orton 1960, Callac 1994). Considering the extensive cohabitation of Agaricus (and other genera of Agaricaceae including Leucoagaricus, Lepiota, Macrolepiota, Chlorophyllum, Cystoagaricus and Tulostoma) with Cupressaceae, we speculate that the ecological association might be ancient. Thus far three cypress-linked species are known to belong to subsection Cupressorum (FIGS. 1–2). All are of slender to medium stature (none have very short stems), with pendant annuli and rufescent flesh. Agaricus cupressicola, the type of Cupressorum, was originally (Grilli 1988) placed in section Sanguinolenti (type species: A. sylvaticus Schaeff.), and by casual criteria (simple pendant annulus, red discoloring) A. tlaxcalensis and A. cupressophilus could be placed there as well. However we noted that mycelia of these three species grow well and are white on compost extract agar or PDA, while A. sylvaticus and other species of Sanguinolenti grow very slowly and become brown as the medium around them becomes browner. We have successfully cultivated A. cupressicola and A. cupressophilus (A. tlaxcalensis has not been tried). Phylogenetic analyses indicate that these latter three species are not close to Sanguinolenti but instead belong to a single basally joined clade within section Bivelares. In MP bootstrap analysis, a clade containing Cupressorum within Bivelares had 96% support; Cupressorum itself had 99% bootstrap support (FIG. 1). A neighbor-joining tree also indicated a sister relationship between subsections Cupressorum and Hortenses (FIG. 2). On balance we prefer to recognize these two subsections within Bivelares, while noting that three of six MP trees obtained did not include a unified clade containing both subsections. Comments on A. cupressicola.—Agaricus cupressicola was first described under Cupressus sempervirens, in the Abruzzi, in central Italy (Grilli 1988). It is also relatively frequent on the French Atlantic coast under C. macrocarpa (Guinberteau et al 1998) and has been found by Callac under Juniperus phoenicea along the coast in Portugal. The latter small tree produces a KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES litter of small branches very similar to those of Cupressus spp.; such trees might have represented an ecological niche for cypress-loving species of Agaricus during the absence of cypresses in continental Europe after their local extinction during the glacial period, before their (re)introduction by humans. Contu (1992) also has encountered Agaricus cupressicola in coastal sand dunes under Juniperus phoenicea, and Contu indicated that Heinemann (pers comm) had found it in Belgium under broadleaf trees with Taxus baccata. Guinberteau et al (1998: 26, pl. 3, photo 5) reported it beneath Robinia. Lanconelli (2003) cited it under Cedrus atlantica and indicated that Grilli (pers comm) had found it under Populus. Agaricus cupressicola has never been found in North America. (Similarly A. cupressophilus, collected ca. 100 km from the 19th century Californian refuge area of C. macrocarpa around Carmel Bay, is not known from Europe and apparently has not followed the introduction of this tree.) We have observed a moderate degree (3/705 characters) of ITS sequence differentiation between (i) the Italian type specimen (coll. 84.112 deposited in Grilli’s private herbarium) (GenBank EU363031) of A. cupressicola and (ii) two collections of French (CA554: France, Budos, Laulan, 30 Sep 2006, under Cupressocyparis leylandii, leg. & det. J. Guinberteau: [GenBank EU363030]; CA72 [5Cp1; GenBank AJ418769: Challen et al 2003]) and (iii) Greek (CA99 [5Cp2; GenBank AJ418770: Challen et al 2003]) origin (vouchers and/or strains at CGAB). A collection from Portugal (CA548: Portugal, Odeceixe, Monte Novo, 29 Oct 2006, under Juniperus phoenicea in paleodunes at the top of a coastal cliff (leg. & det. P. Callac and A. Tancrède [CGAB]: GenBank EU363029) was heteromorphic for all three of the sequence characters differing between the French/ Greek and Italian material, suggesting that interbreeding might be occurring within a single, somewhat subdivided species. Using these (nonredundant) sequences, an A. cupressicola clade had 58% bootstrap support. In N-J trees and in half of the nonredundant MP trees our A. cupressicola sequences group together. A speciation process may be in flux among Mediterranean A. cupressicola. Two new species in subsection Cupressorum are now described from North America: Agaricus tlaxcalensis Callac et G. Mata, sp. nov. FIGS. 3–7 Figura saepe gracilis. Pileus 2–8 cm pallide brunneogriseus; primum convexo-cuboideus, conico-campanulatus fit, deinde plano-convexus cum panditur, modice umbonatus, radiose fibrillosus, nonnumquam fibrillo-squamulosus, tactu rufescens. Stipes saepe curvus vel flexuosus (4)5–8(12) 881 FIG. 3. Cheilocystidia of A. tlaxcalensis (Holotype, MATA 681). cm 3 5–15 mm. Simplex annulus membranaceus ac descendens. Caro a roseo ad rutilum admodum transit, deinde siccati sanguinis colorem ducit. Agaricum bisporum olet. Sporae ellipsoideae, forte ovato-ellipsoideae, (4.5–) 4.8–6.3(–6.9) 3 (3.4–)3.6–4.8(–5.5) mm. Basidia tetrasporigera (21–27 3 6–8 mm). Cheilocystidia articulosa cylindroutriformiaque 18–30 mm 3 6–12 mm. Sub cupressis lectus. In septentrionalis Americae superioribus locis situs. HOLOTYPUS: ‘‘Mexico, 11 October 2003, MATA 681’’ in herbario XAL depositur. MB 512234. Pileus at first cuboidal-convex, becoming conicocampanulate, later expanding to plano-convex with a low umbo, 2–8 cm diam; pileipellis beige-gray, exceeding whitish margin, radially fibrillose, sometimes fibrillose-squamulose, the background layer concolorous, rufescent on handling. In drier weather the surface can become smooth to polished, with concentric cracks. Lamellae free, ca. 10 per cm at 1 cm from stipe, at first vinaceous pink, rufescent on handling, later chocolate brown; margin not distinctive. Stipe subequal, progressively thicker toward the base, sometimes curved or flexuous, spindly, with a length up to 2.5 times the width of the pileus, (4)5– 8(12) cm long 3 5–15 mm thick; surface longitudinally fibrillose, silky, partly rugulose, white, quickly becoming sordid brown; basal bulb absent but rhizomorph often present at the base; context white, becoming strongly red-orange when sectioned, then vinaceous beige-gray, and finally dried-blood colored. Odor faint, typical of Bivelares. Veils distinctive, thin, membranous, sometimes remaining attached to the pileus margin, forming an apical descending (pendant) annulus, white then vinaceous brown, persistent. Spores dark brown at maturity, ellipsoid to ovoideoellipsoid, (4.5–)4.8–6.3(–6.9) 3 (3.4–)3.6–4.8(–5.5) mm, mean 5 5.3 3 4.2 mm (N 5 240), L/W 5 1.28; hilar appendix prominent. Basidia tetrasporic, cylindro-clavate, 21–27 3 6.0–8.0 mm; sterigmata 2–3 mm long. Cheilocystidia present, often in chains of 882 MYCOLOGIA FIGS. 4–12. Basidiomata. 4–7. Four collections of Agaricus tlaxcalensis under Cupressus benthamii in Tlaxcala State, Mexico; bars 5 10 cm, to demonstrate variation in proportions. 4–6. In Rancho el Tejocote. 4. 19/07/2001 (not accessioned); 5. 02/ 10/2002, GANDARA 377; 6. 10/10/2003, MATA 675. 7. In the National Park of the Malinche, MATA 6817, 11/10/2003: Holotype. 8. A. cupressophilus. FS-22*: Holotype. Specimens cultivated from culture FS-22, prepared from material from San KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES broadly cylindrical elements, the terminal element being cylindrical-utriform; 18–30 mm 3 6–12 mm. Chemistry: not determined. ITS1 + 2 sequence (GenBank EU363032– 33): L 5 705 nt (estimated). Distinctive characters: agagTttggaCtggt @ 486 & 492. The first might be unique in Agaricus, the second is unique within Bivelares. Habit, habitat, known distribution, occurrence: Isolated, gregarious or cespitose in litter under planted Cupressus benthamii, at more than 2400 m altitude in the state of Tlaxcala (Mexico); possibly frequent. Specimens examined: MEXICO. Tlaxcala state: National Park of the Malinche (near the restaurant of the park), under Cupressus benthamii, 11 Oct 2003, MATA 681, P. Callac and G. Mata, HOLOTYPE (XAL); Rancho El Tejocote at 2 km from Cuapiaxtla, under Cupressus benthamii, 19 Jul 2001, lost specimen, P. Callac and G. Mata; 02 Oct 2002, GANDARA 377 (XAL), G. Mata; 10 Oct 2003, MATA 675 (XAL), P. Callac and G. Mata; Mycelium cultures of MATA 675 and MATA 681 both are available at MYCSA-INRA Bordeaux (CGAB: strains CA 219 and CA 221 respectively). Etymology: from Tlaxcala state, Mexico. Agaricus tlaxcalensis is most closely related to A. cupressicola and A. cupressophilus. It resembles A. cupressicola in the small size of the sporocarp, the dull color of the pileus, the simple membranous descending annulus, the red discoloring when sectioned, the spore size, and the cypress habitat. It differs by its more slender, elongated silhouette with a stipe often curved, the stronger discoloring when sectioned or touched, the larger basidia (20–25 3 5–6 mm for A. cupressicola, per Bon [1987]), the cheilocystidia in chains, which are rather cylindrical-utriform than broadly clavate (see FIG. 4), and the highland Mexican geographic distribution. A. tlaxcalensis differs from A. cupressophilus by its substantially longer spores and larger cystidia, by its cap that tends to remains umbonate at maturity, while the cap of A. cupressophilus tends to become depressed, and possibly by its geographic distribution because A. cupressophilus thus far is known only from under Monterey cypress in coastal California. ITS1+2 sequences of two A. tlaxcalensis collections consistently differ from A. cupressicola by seven polymorphisms and from A. cupressophilus by two polymorphisms. In 883 view of all of the data A. tlaxcalensis and A. cupressophilus are best treated as separate species. Edibility of A. tlaxcalensis is unknown. Agaricus cupressophilus Kerrigan, sp. nov. FIG. 8 Figura gracilis. Pileus 1–7 cm latus, pallide griseus, mutabilis, brunnescens. Stipes plerumque aequalis, e farcto cavus, cavitum amplo, rufescens. Sporae ellipsoideae, (4.2–) 4.6(–4.9) 3 (3.4–)3.7–3.9(–4.2) mm, basidia tetrasporigera, 19–23 3 6–7 mm, cheilocystidia fasciculata, 16–22 3 6–7.5 (–8.0) mm, plerumque cylindro-clavata. HOLOTYPUS: FS22*, in herbario SFSU depositur. MB 512229. Pileus (1–) 4–7 cm diam., convex to broadly convex, center slightly depressed, margin slightly incurved; pileipellis glabrous to minutely appressedsquamulose, the fibrils grayish to gray-brown to buff or light tan, potentially developing dark brown stains hours after handling; context (3–)5–8 mm thick, white becoming sordid vinaceous-flesh color, odor indistinct to spicy. Lamellae free, close, ca. 13 per cm at 1 cm from stipe, 2–4 mm broad, margin concolorous or faintly paler, dull flesh-colored becoming dark brown. Stipe subequal to subclavate, (5–)10–12 mm above, (7–)11–20 mm below 3 (1.5–)4–6 cm long; stipitipellis buff to tan, lustrous-fibrous, coloring as context or more reddish when injured, potentially darkening toward the base in age or long after handling, lacking velar remnants; context lustrousfibrous, rapidly becoming sordid vinaceous-flesh color near cuticle and base when exposed, stuffedhollow, the cavity relatively broad (to 10 mm), filled with white, unchanging pith. Veils forming a thin, whitish, subapical to supramedian pendant annulus, often tending to remain partially attached to the pileus margin, and/or tearing into radial strands and fibers, readily collapsing. Spores dark brown at maturity, ellipsoid, (4.2–) 4.6(–4.9) 3 (3.4–)3.7–3.9(–4.2) mm, mean 5 4.6 3 3.8 mm (N 5 30, C 5 1); hilar appendix moderately prominent; apical pore not evident. Basidia tetrasporic (some bisporic), cylindro-clavate, 19–23 3 6–7 mm; sterigmata 3–4 mm long. Cheilocystidia present in occasional fascicles, mostly about cylindrical to clavate, sometimes broader, often irregular to somewhat contorted, 16–22 3 6–7.5 mm, apices sometimes mucronate, occasional cells to 12–16 mm broad present; elongated cells arranged parallel to the margin generally predominate. r Mateo County, California. Scale ca. 0.45 3. 9–10. A. subsubensis. 9. Field collection JB-101, Riverside County, California, which was cultured and from which holotype JB-101* was cultivated. Scale ca. 0.4 3. 10. A. subsubensis RWK 1789*, cultivated from a field spore sample from Monterey County, California. Scale ca. 0.7 3. 11. A. agrinferus RWK 1397, Monterey County, California: Holotype. Scale ca. 0.3 3. 12. A. devoniensis ssp. bridghamii RWK 1899, Clear Creek County Colorado: Holotype. Credit: 4, 5, 7 P. Callac; 6 G. Mata; 8–12: R.W. Kerrigan. U.S. penny is 18.5 mm diam. 884 MYCOLOGIA Chemistry: KOH yellow on disk (only); aniline 3 GAA (glacial acetic acid) negative. ITS1+2 sequence (GenBank EU258676): L 5 708 nt. Distinctive characters: gagg[TG]ggat @ 612– 613. Habitat, known distribution, occurrence; material studied: Known only from collection FS-22 (no longer extant), under Cupressus macrocarpa, USA, CA, San Mateo County, Pacifica, Skyline Blvd., Fred Stevens, 26 Aug 1990. HOLOTYPE: FS-22*, basidiomata cultivated from tissue culture ARP 042 of FS-22 (5MYA-4431 at ATCC) at Sylvan Research, Nov 2005 [SFSU]; paratype (second gathering from crop that produced holotype) [NY]; paratype 5 preserved culture MYA-4431, ATCC. Etymology: A cypress-loving species. The ITS sequence of A. cupressophilus is most similar to that of the Mexican A. tlaxcalensis, followed by the European A. cupressicola. The collector of the only known field specimens, Dr F. Stevens, provisionally identified the fresh FS-22 material as A. bisporus. He cultured this material but unfortunately no voucher was preserved. RFLP analysis done on the culture by RWK while in the J.B. Anderson lab, University of Toronto at Erindale, demonstrated that FS-22 was not A. bisporus or any other RFLPcharacterized species. An opportunity to obtain the ITS1+2 DNA sequence later arose, thanks to J.-P. Xu of McMaster University. Availability of the unique sequence allowed placement of this entity within the emergent Cupressorum clade and inspired the successful attempt to cultivate specimens, from which the description of the basidiomata was prepared. The yellow color appearing with KOH on the disk is unusual, not normally reported for section Bivelares. This is likely to have been associated with velar hyphae, which can remain as a substantial but obscure presence on the disk. We would not be surprised if this reaction were not observed on other material that had developed under different (e.g. less humid) conditions. No other tissue exhibited a yellowing reaction with KOH. For a comparison with A. tlaxcalensis see that species. Edibility is unknown. Agaricus [section Bivelares] subsection Hortenses Heinem., Bull. Jard. Bot. Etat 26:22. 1956. ; Agaricus section Hortenses (Heinem.) Bon, Doc. Mycol. 15(60):9. 1985. Orig. diag.: Vela complexa. Acies lamellarum semper cheilocystidiis praedita. Sporae breviter ellipticae, crasse tunicatae, poro non praeditae. Species ruderales, interdum halophilae. Type: Agaricus hortensis ( J.E. Lange) S. Imai [‘‘Agaricus hortensis (Cooke) Pilát’’], designated by Heinemann, Bull. Jard. Bot. Etat 26:22. 1956. We regard A. hortensis ( J.E. Lange) S. Imai 1938 (nom. illeg., art. 53.1 non A. [Clitocybe] hortensis Pers. : Fr., 1801), also known in literature as A. hortensis (Cooke) Pilát, 1951, to be a heterotypic synonym of A. bisporus ( J.E. Lange) Imbach 1946. The correct name for the subsection including A. bisporus is therefore Agaricus subsect. Hortenses Heinem. (1956). This subsection is distinguished from subsect. Cupressorum as noted above: the common tendency toward complex and/or thickened, even cottony or somewhat rubbery, often intermediate to occasionally peronate velar structures, and the pileus edge strongly involute at least in young basidiomata in Hortenses are likely to be the most useful macroscopic characters in this regard. Stature and habitat also can be useful. DNA ITS1+2 characters have been noted above. Three new taxa are described: Agaricus subsubensis Kerrigan, sp. nov. FIGS. 9–10 Pileus 1–9 cm latus, convexus vel undulato-planus, laxe intertextus, albus, mutabilis, aetate rufobrunneis; contextus ex albo obscure rufescens vel vinaceus; stipes 5–15(–25) mm crassus, 0.5–4.5 cm longus, plerumque aequalis, plerumque solidus; vela alba, friabilis; sporae (5.7–)6.2–6.7(–7.4) 3 (4.3–4.9–)5.3–5.6(–6.2) mm; basidia 24–30 3 7.4–9.2 mm, bi-, tri-, et tetraspora; cheilocystidia nulla vel cylindrica vel clavata. HOLOTYPUS: JB-101*, in herbario SFSU depositur. MB 512226. Description of JB-101: Pileus broadly convex, uneven, with inrolled margin, becoming approximately plane, wavy, 1–9 cm broad; cuticle somewhat loosely interwoven, more scurfy than glabrous, with concolorous velar patches toward and on disk, obscurely rimose toward margin, whitish, becoming light brown (7C4 or lighter) when dried, or if bruised when young, quickly turning reddish brown (8D-E8, 9F5), then drying a mahogany brown (9F6); context semifirm, whitish, unchanging in a V-shaped zone beneath disk, becoming sordid above lamellae and pinkish above stipe and at boundary of sordid and unchanging zones, odor mild, indistinct, like that of A. bisporus. Lamellae free, not close, ca. 10–11 per cm at 1 cm from stipe. Stipe about equal, 5–15 mm 3 0.5–4.5 cm long; cuticle semismooth beneath velar remnants; context whitish, almost unchanging or becoming sordid, except becoming pinkish at cuticles above and below, and in basal context, with an indistinctly defined central region of white pith, especially evident and tending to become hollow near apex, base potentially subtended by copious stringy mycelia (at least in culture). Veils forming a narrow, indistinct, band-like, supramedian, subperonate annulus, more obviously forming a series of thin broken ring-like remnants on the lower twothird of stipe, and sometimes velar patches on pileus (disk). Spores dark brown at maturity, broadly ellipsoid, (5.7–)6.2–6.6(–7.2) 3 (4.9–)5.3–5.6(–6.2) mm, mean KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES 5 6.4 3 5.5 mm (N 5 30, C 5 1); hilar appendix unremarkable; no apical pore evident. Basidia tetrasporic, cylindro-clavate, 24–30 3 7.4–9.2 mm; sterigmata 4–5 mm long. Cheilocystidia absent or basidiolelike, the lamellar edge bearing basidia and basidiolelike cells in regions one to several mm long, otherwise naked, with exposed hyphae oriented parallel to the edge. Chemistry: KOH negative; aniline 3 GAA negative; o-tolidine blue on cuticles and veils, stipe and disk context near cuticle, stipe pith and basal zone, purple on lamellae and somewhat obscurely so above basal context, otherwise unchanging; alpha-naphthol brownish to scarlet on pileus cuticle, unchanging or slightly orangish on pileus and stipe context except becoming purple in portions of lower stipe. ITS1+2 sequence of the type (GenBank EU131641): L 5 661 nt, anomalous, with major deletions, substitutions and/or modified sequence from pos 124 (gggtatA.) to pos 171 (.Gcctgtc), with up to 28 other possible recently modified characters throughout the ITS. See discussion below, including ITS1+2 sequence of RWK 1789 (GenBank EU257802). Habit, habitat, known distribution, occurrence; material studied: USA. California. Riverside County circa Thermal: JB-101, R.W. Kerrigan 7 Mar 1992 (paratype, SFSU); gregarious, fascicled to cespitose, in sandy soil in sparsely landscaped area near dwelling, Sonoran Desert, elevation 26 m [5type locality]; specimen designated JB-101* (HOLOTYPE, SFSU; isotypes: NY, MA), cultivated at Sylvan Research, Kittanning, Pennsylvania, from MYA-2981 (paratype [preserved culture], ATCC), a culture derived from JB-101; Monterey County, Pacific Grove: RWK 1789* (SFSU), cultivated at Sylvan Research, Kittanning, Pennsylvania, from MYA-4432 (preserved culture, ATCC), derived from RWK 1789 (no longer extant; collected from soil and Cupressus litter at Esplanade Park by R.W. Kerrigan). Etymology: The epithet ‘‘subsubensis’’ honors the tradition in Agaricus taxonomy of prefixing epithets of the less-distinctive species with ‘‘sub’’. It is tempting to say that this species is noteworthy for its lack of distinguishing macroscopic features. Specimens from the desert were easily cultivated; they fruit prolifically and are therefore better known to us. These basidiomata tend to be small and irregularly shaped, with an indistinct pileipellis. They are unlikely to be deemed attractive. The indelible rubro-brunnescence of the pileus surface observed in sound young specimens is an unusual feature sometimes seen in a few species of sect. Agaricus and sect. Sanguinolenti (Kerrigan 1986). RWK 1789 was not recognized initially as conspecific with JB-101; in addition to its very different 885 habitat, it is not easy to cultivate and only a few basidiomata were obtained from the original culture. The two collections differ most remarkably in the divergence of their ITS sequences. The ITS1+2 sequence of JB-101 was obviously anomalous within its group, and reproducibly so: two independent amplifications of genomic DNA obtained from mycelia grown in broth yielded a single shared sequence with multiple deletions and other alterations. One segment of the ITS1 is unalignable with other Agaricus ITS1 sequences. At 661 nt the JB-101 ITS1+2 is much shorter, by about 40–50 nt, than in any other known Agaricus; whereas in RWK 1789 this DNA segment is unremarkable at 707 nt long. Even the alignable portions of JB-101 and RWK 1789 have a great number of differences between them but also some unusual shared unique characters. Both sequences have characteristics of section Bivelares, for example an 8 nt deletion about 55 nt 59 of the 39 end of the ITS1+2 region (as we define it). The phylogenetic situation became clear when uniquely matching sequences from the LSU, EF1-a, and RPB2 genes were obtained from the two cultures (Kerrigan and Xu unpubl; GenBank EU284015–20). Thus the ITS1+2 region of JB101 evidently has been altered in a major way by an event or process that violates the assumptions of nucleotide substitution based phylogenetic analysis. While interesting, such a sequence cannot be used in a meaningful tree-building exercise. The phylogenetic and geographical scope of the altered sequence is not known. However, based on comparisons of other gene sequences from the two cultures, we regard it to be an intraspecies event, hypothetically marking disjunct populations. This rare species has been collected once (among an abundance of A. bisporus var. burnettii) near Indio, California, in desert sand below sea level, and once (among an abundance of A. bisporus var. bisporus) in Pacific Grove, California, within meters of the coast (see Kerrigan 1995, FIG. 3, for site map and position of RWK 1789). Because the JB-101 material is much better documented, in part because of its cultivability, it therefore was selected as the type of the species. The significance of its anomalous ITS sequence is unknown; the species concept includes both of the known ITS sequences. Given the cultural and habitat differences between the two collections, while keeping in mind the ITS sequence differences, it remains possible that two divergent populations or even rare sister taxa are involved. For that reason a separate description of cultivated RWK 1789 (FIG. 10) is provided: Pileus 4–6.5 cm diam, oval in top view, convex to broadly convex, becoming subplane or with two uplifted sides, center slightly depressed, the margin 886 MYCOLOGIA incurved until maturity, ultimately with semishallow notched radial fissures; pileipellis glabrous to broadly, indistinctly appressed-squamose, the squamae large, obscurely imbricate, apparently due to velar separations, the surface white when young, becoming unevenly vinaceous-brown from button stage through maturity; context (3–)5–8 mm thick, white or whitish becoming salmon pink-orange to darker vinaceous in places when cut, odor indistinct, mushroom–like (as in A. bisporus), of sweet chicory at pileus after velar rupture. Lamellae free, close (distally) or not (proximally), with a pronounced bifurcating zone about 1 cm from stipe, ca. 15 per cm at 1 cm from stipe, 1– 2 mm broad, margin concolorous, dull grayish fleshcolored becoming medium brown. Stipe subequal, tapering toward base, oval in cross-section, 8–13 3 15–25 mm above, 7–8 mm below 3 3.5–4.5 cm long; stipitipellis whitish, semilustrous, adorned above the annulus with broad, appressed, dentate velar patchscales, these very light buff color, coloring as context or more orangish when injured; context dull, whitish, rapidly becoming salmon-orangish to vinaceous color in places and more sordid overall near base when exposed, stuffed-hollow, the cavity relatively broad (to 11 mm), wedge-shaped, filled with white, rufescent pith. Veils forming a short, narrow, submedian bandlike intermediate annulus, whitish or becoming brownish on the edge in age, the edge a two-limbed groove of 1–2 mm width when young, this indistinct in age, upper and lower annular surfaces smooth, remaining white. Spores dark brown at maturity, ellipsoid, sometimes subglobose, occasionally narrowed toward one end, (5.7–)6.4–6.7(–7.4) 3 (4.3–4.9–)5.3–5.4(–6.0) mm, mean 5 6.5 3 5.4 mm (N 5 40, C 5 1); hilar appendix prominent; no apical pore evident. Basidia predominantly tri- and bisporic, cylindro-clavate, 24.5–27.5 3 (5.5–)7.5–8.5 mm; sterigmata 3–4 mm long. Cheilocystidia absent; lamellar margin fertile. Chemistry: KOH gray-green on pileus surface, elsewhere indistinct; aniline 3 GAA negative. ITS1+2 sequence (GenBank EU257802): L 5 707 nt, unique characters: ctgtTcttg @ pos. 191. Description is of basidiomata cultivated from a spore culture of RWK 1789 at Sylvan Research, Nov 2005. This unique collection was taken along with many others during an unexpected collecting opportunity under challenging travel conditions. It was only possible at the time to collect and process air-dried lamellae bearing viable spores, which later were germinated, and to record spatial locations within the site. Growth and basidiomatal development are slow in this culture. Superficially RWK 1789 bears some resemblance to A. pequinii, a member of section Chitonioides; the latter species has a peronate veil, solid stipe, deeply reddening stipe context, and abundant cheilocystidia. Agaricus agrinferus Kerrigan et Callac, sp. nov. FIG. 11 Pileus 5–8(–10) cm latus, late convexus, mediocriter fuscus vel raro subalbidus; pileipellis implexa, levis vel paulum squamosa, continuo integra in margine, cetera autem parte sordide aurantiaca evadente; stipes subaequalis, 3–8 cm longus 3 1–2 cm latus, externa parte levi, cetera autem parte paulum fibrata atque medullosa in medio, colorem sordide aurantiacum ducit; sporae circa 5.7–6.5 3 4.4–4.7 mm, late ellipsoideae vel tantum ellipsoideae, basidia tetrasporigera, cheilocystidia cylindracea forte etiam claviformia, 15–33 3 3– 15 mm. Dispersus sub coniferis crescit in litoris regionibus parva altitudine. HOLOTYPUS: Point Lobos State Reserve, Monterey County, California, USA: RWK 1397, 10 Jan 1986, in herbario SFSU depositur. MB 512235. Pileus 5–8(–10) cm broad, at first broadly convex, with moderately inrolled margin, becoming plane, disk sometimes slightly depressed; surface dry, glabrous on disk, elsewhere innately fibrillose, color pallid to sordid (when young) or medium brown, if brown then becoming appressed fibrillose-squamose, squamae ca. 10 mm long 3 5–15 mm broad, background color whitish to pale buff; context white, becoming reddish near lamellae and stipe apex when sectioned, firm, up to 10–16 mm thick, odor of A. bisporus plus fruity/spicy as in some species of Sanguinolenti after exposure. Lamellae free, close, up to 10 mm broad, at first pallid to dingy pinkish, finally dark blackish-brown, margin somewhat pallid. Stipe 3–8 cm long 3 1–2 cm broad, equal to subclavate, occasionally ventricose (in culture), interior white, quickly becoming orange-red, ultimately red after longitudinal sectioning, in median transverse section becoming orangish, stuffed-hollow; surface glabrous, or minutely floccose or with fibrillose zones below, white, coloring as above when incised; base firmly rooted in litter and/or soil. Veils forming a supramedian, white intermediate-type annulus with a pendant aspect, sheathing upward and with a broadly flaring limb, unrolling with the pileus margin, upper surface smooth to striate, lower surface interwoven to scurfy, sometimes subtended by one or two small limbs, margin thin, uneven; universal veil also sometimes leaving a scale ring above the base of the stipe. Spores (4.5–5.3–)5.7– 6.5(–7.1–7.5) 3 (4.1–)4.4–4.7(–5.3–6.0) mm, dark brown, broadly ellipsoid to ellipsoid, hilar appendix semiprominent, no apical pore evident. Basidia 17– 28.5 3 (4–)7–9(–12) mm, clavate to cylindro-clavate, tetrasporic; sterigmata 1–4 mm long. Cheilocystidia 15–33(–45) 3 3–15 mm, clustered, not abundant; lamellar margin primarily composed of hyphae oriented parallel to the margin, sterile. KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES Chemistry: KOH negative; aniline 3 HNO3 negative; o -tolidine blue on basal context of stipe, elsewhere violet; alpha-naphthol purple on basal context of stipe, elsewhere pinkish-orange to orange, weakly so on pileus context. Habit, habitat, known distribution, occurrence: Solitary to subgregarious under Cupressus macrocarpa in California, and under that tree and others in low elevation coastal areas of the U.K. and France. Fall– winter. ITS1+2 sequence (GenBank EU257801 and AF432888 for RWK 1397 and FS-5 respectively; see also ITS1 sequences AJ418738-42, for RWK 1397, Sf5, FS-10, FS-13, and WAT1 respectively: Challen et al 2003): L 5 703 nt. Distinctive characters: atgtTattg @ pos 259, vs C for A. subfloccosus, is unique within Bivelares. Material examined: USA. California. San Francisco County: RWK 738, 770b, 770c, 770*, 775, 884, 1117, 1140, 1040*, 1153, 1175, FS-2 (5ARP 003, 5ATCC 66250), FS-5 (5ARP 006, 5ATCC 66253), FS-13 (5ARP 030); San Mateo County: RWK 969, 1192, FS-10 (5ARP 020, 5ATCC 76561), FS-29 (5ARP 073, 5ATCC 200200); Monterey County: RWK 1030, 1397 (TYPE: SFSU; Paratype: ATCC MYA-3441 [preserved culture]), 1819, [1910 or 1911], NW-1 (5ARP 205); UK. Scotland: ATCC 34842; England: W4 II* (5ATCC 200593), W4 IV*, A 119* (5ATCC 200592), A 11*; France: Sf2 (5CA47), Sf5 (5CA70). (See also Kerrigan et al 1999, Challen et al 2003.) All California collections were made under Cupressus macrocarpa. Etymology: ager/agri: territory, land; inferus: lower. The epithet refers to the distribution of the species at elevations of about 5–200 m at middle northern latitudes. This mushroom was discussed in detail and figured by Kerrigan et al (1999). At that time we showed that, based on morphological, cultural, and molecular (RFLP, isozyme) characters, this lowland entity was phylogenetically distinct from the highland, spruce/ fir/pine associated form of A. subfloccosus (see Cappelli 1984, Parra and Suárez 2002, Kerrigan et al 1999; this association is frequent but not absolute). Both entities have been referred to A. subfloccosus by various authors. We also indicated that the two entities might be sister species, however without sequence data it was difficult to be confident of the best rank-relationship for them. We further noted the problem presented by the lack of a type specimen for Lange’s taxon. Lange (1926) mentioned only this syntype: ‘‘Hollufgård forming large fairy-rings in naked ground in plantation of Picea, Aug. 1915’’. No original specimens of Psalliota subfloccosa exist in the Copenhagen Natural History Museum (H. Knudsen pers comm; Kerrigan et al 1999), therefore a lectotype designation is in order: 887 Agaricus subfloccosus (J.E. Lange) Hlaváček, Mykol. Sborn. 28(4–6):67. 1951 [‘‘subflocosus’’] Lectotype (hic designatus): [icon] ‘‘Psall. hortensis v. subfloccosa JEL.’’, in Lange, Danmarks Agaricaceer: plate 647 (unpubl), deposited in the Copenhagen Natural History Museum. Remarks: Plate 647 was reproduced by Lange (1939) as plate 139 D as Psalliota subfloccosa and the original plate is currently shown on the Copenhagen Botanical Museum web site at http://130.225.211. 158/agaricina/FMPro?-db5agaricina.fp5&-format5 flagar-storbillede.htm&-sortfield5fadtavle&-op5cn& FADogCurrent5Agaricus%20subfloccosus&-max5 15&-recid533495&-find5 Briefly the key field characters useful in differentiating A. agrinferus from A. subfloccosus are: the former has an interwoven pileipellis that is usually not pronouncedly scaly nor rimose toward the margin (as in the latter) and is most often a somewhat darker medium-brown (except in rare specimens with a nearly white pileus when young) than the pallid to lighter brown of the latter; A. agrinferus has a relatively uniform stipe context, vs. the more fibrous stipe context of A. subfloccosus; the two species are separated by habitat and by elevation differences reaching hundreds or thousands of meters at middle latitudes in the northern hemisphere. Although these two lineages differ at only one position in their ITS sequences, the rank of species was chosen for A. agrinferus because both species are homothallic and their genomes and lineages therefore are phylogenetically isolated, as indicated by the nonreticulating pattern of RFLP markers within and between the two species (Kerrigan et al 1999); they are homomictic analogues of asexual species. The single ITS character difference is consistent among studied strains: compare with GenBank AJ418743, AJ418745–47, AF432887, and EU131638–40 for A. subfloccosus collections SUB1, RWK 1542, 21552, 21553, 21441, LAPAG 113, RWK 1568, 21993 and 21994 respectively. The two phylogenetic lineages however are much more substantially diverged: the observed extent of RFLP molecular marker dissimilarity between the two sister species is 92% 6 2% SE (Kerrigan et al 1999). Abundant diverging cultural and molecular features, as well as the agreement of characters within each species found on both sides of the Atlantic, also were evaluated (Kerrigan et al 1999). Morphologically the two species are quasicryptic under some conditions. Because both species are homothallic, and each comprises multiple nonrecombining sublineages (genets) (Kerrigan et al 1999), the process of homogenization that occurs within an outcrossing species via independent association of most 888 MYCOLOGIA traits in each generation is absent. Instead, homothallic (homomictic) species can diverge into genets that increasingly vary among themselves with respect to the presence of various traits that might useful for recognition of the species (e.g. absent in one vs. present or absent in the other); hence we describe ‘‘tendencies’’ when presenting characteristics of these species (see Stebbins 1950). We believe that Lange (1926) described the Pinaceae-associated entity as P. hortensis subfloccosa, the basionym of A. subfloccosus. His material was collected from ‘‘large fairy-rings’’ in a ‘‘plantation of Picea’’ in Denmark. Danish material that we examined had the nuclear RFLP genotype of the Pinaceaeassociated entity, confirming its existence in the region of the type locality (Kerrigan et al 1999). In Denmark spruce will grow near sea level, while to the south it occurs, along with this highland mushroom, in montane settings. Finally, although macroscopic differences between the two entities are subtle and inconsistent, Lange’s (1939) figures of Psalliota subfloccosa more closely resemble material of the Pinaceae-associated entity, particularly with respect to the more fibrous appearance of the stipe context. We accept this interpretation. In contrast Bohus (1994) published the illegitimate name A. cappellianus Bohus (;A. bohusianus L.A. Parra 2005) (non A. cappellianus Hlaváček 1987), based on an Hungarian type believed to correspond to A. subfloccosus ss. Cappelli (1984, Pl. 26). Both Cappelli and Bohus placed their mushroom(s) in section Sanguinolenti, and our study (Parra unpubl) of the relevant material illustrated by Cappelli (1984, Pl. 26) indicates that it is a pale collection of A. sylvaticus. Our attempt to study the type material of Bohus (BP) was unsuccessful; however his description, particularly the spore size of 5.2–7 3 3.5–4.5 mm, is a better fit to A. sylvaticus than to Lange’s A. subfloccosus. Therefore it appears that no new name actually has been typified by specimens of the montane, Pinaceae-associated A. subfloccosus, simplifying the correct application of the original name to such material. In much of Europe south of Scandinavia and in North America A. subfloccosus occurs from 500 m to more than 1700 m above sea level, typically with Picea (Italy, Switzerland, Canada, USA) or other Pinaceae (Spain). It can be common in the bottoms of glaciated valleys in Alberta, Canada (e.g. Banff ca. 1400 m and, farther north, Jasper ca. 1100 m), where it may form arcs (partial fairy-rings). In Colorado it occurs with spruce or with broadleaf trees and shrubs in urban parks at 1600 m (Denver: M. Shaw, G. Pickett and R. Kerrigan, pers obs). Parra and Suárez (2002) report it with Pinus and Populus at 1500 m in Spain. In contrast the lowland entity that is known from Scotland into France and from California usually occurs near the coast and is most commonly associated with Cupressus. We describe this entity here as A. agrinferus. Like A. subfloccosus it consists of homothallic genets; however each genet may be more widely distributed in A. agrinferus (Kerrigan et al 1999). Agaricus agrinferus has sometimes been (incorrectly) called A. subperonatus ( J.E. Lange) Sing. (see Kerrigan et al 1999 for literature review). The latter species, recently proposed to be synonymous with A. cappellianus Hlaváček (;A. vaporarius [Pers.] Imbach 1949, nom. illeg., non A. vaporarius Schrank 1789) (e.g. Nauta 2001; Parra 2003, 2005, 2008), has been consistently illustrated with radially oriented brown fibrils in appressed scales on the pileus surface (Lange 1939, Essette 1964, Bon 1988, Cortecuisse and Duhem 1994). Close inspection of fresh A. agrinferus reveals that the fibrils of the pileipellis are highly interwoven, not radially arranged (Kerrigan 1986); it is a distinct entity. Based on our review of its ITS1 sequence, strain WC721 from the PSUMCC (provided by RWK as FS-2/ARP003, from a collection by F. Stevens in San Francisco, California), deposited by Geml et al (2004) in GenBank (AY484698), belongs to A. agrinferus, consistent with our own evaluation of this culture (Kerrigan unpubl). Agaricus devoniensis subsp. bridghamii Kerrigan, subsp. nov. FIG. 12. Pileus 3–7.5 cm latus, convexus demum late convexus, fibrillis adpressis, albus demum sordidus; contextus albus, immutabilis; stipes 5–8 cm longus, 1–1.5(–2.0) cm crassus, subaequalis, e farcto cavus; contextus albus; velum peronatum, inferus; sporae (4.8–5.3–)6.0–6.6(–7.3–7.4) 3 (4.3– 4.8–)5.1–5.5(–5.9) mm; basidia 19–23(–29) 3 9–10 mm, tetrasporigera; cheilocystidia (12.5–)20–31 3 11–12.5(–15– 19) mm, equalis vel clavata vel subglobosa. HOLOTYPUS: RWK 1899: Colorado, Clear Creek Picnic Area, Clear Creek County, 16 Jul 1995: in herbario SFSU depositur. MB 512227. Pileus broadly convex, 3–7.5 cm broad, margin initially inrolled, disk slightly depressed in age; surface glabrous or obscurely appressed-fibrillose, color whitish or with irregular sordid brownish areas; context white, virtually unchanging when cut, later (or in age) becoming brownish near lamellae and stipe, to 1.5 cm thick, odor mushroomy, unremarkable. Lamellae free, close, drab when young, obscurely if at all marginate (paler), later dark blackishbrown. Stipe approximately equal, slightly broader at base and sometimes at pileus, 5–8 3 1–1.5(–2.0) cm, surface finely erect-fibrillose above veils, glabrous or innately fibrillose below, basal 5 mm covered with substrate; Veils submedian, peronate, sheathing, with a flaring upper margin and a flaring, or appressed, or KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES obscure lower margin, all weak and soon collapsing; context white, or obscurely rufescent in places, becoming brownish near pileus, with a 3–5 mm broad pith-filled central cavity. Spores dark brown, broadly ellipsoid (Q 5 1.13– 1.16 to 1.24–1.26), (4.8–5.3–)5.9–6.6(–7.3–7.4) 3 (4.3–4.8–)5.0–5.5(–5.9) m m, the mean 6.21 3 5.20 mm, (N 5 120, C 5 3); hilar appendix not prominent; apical pore not evident. Basidia clavate or cylindro-clavate, 19–23(–29) 3 9–10 mm, mostly tetrasporic, the sterigmata acute, to 4 mm long. Cheilocystidia in scattered to semicontinuous fascicles, mostly broadly clavate to subglobose or almost pyriform, otherwise quasi-cylindrical, 20–27 3 11–12.5(–15) mm. Chemistry: unavailable. ITS1+2 sequence (GenBank AF432890– 91, EU131637): L 5 707 nt. Distinctive characters: gtgaTaaca @ 180 and actcActtg @ 530 distinguish the subspecies. Habit, habitat, known distribution, occurrence: Solitary or in small groups, under Picea, in fairly level areas in valley bottoms, at middle to high elevations in the eastern Rocky Mountains of Colorado and Alberta. Material studied: USA. Colorado. Clear Creek County Near Empire: Clear Creek Picnic Area, elev. 2900 m. Under Picea, 16 Jul 1995, RWK 1899 (HOLOTYPE [SFSU]; paratypes: preserved culture MYA-2671 [ATCC]); Mizpah Campground, elev. 3000 m. Under Picea, 16 Jul 1995, RWK 1900 (NY), preserved culture 5 MYA-2672 [ATCC]); CANADA. Alberta. Canmore. Bow River Recreation Area campground, elev. 1300 m. Under Picea, 5 Aug 1990, RWK 1664 (SFSU). Etymology: The name honors Joseph Bridgham, one of 19th century North America’s foremost illustrators of fungi and other natural subjects, who illustrated several Agaricus spp. for the Icones Farlowianae (Farlow 1929). Two ITS characters unify subsp. bridghamii contra devoniensis; one of these characters is unique within the section and possibly the genus. This level of ITS1+2 sequence divergence of subsp. bridghamii from A. devoniensis P.D. Orton subsp. devoniensis, which itself is somewhat heterogeneous as presently constituted (Challen et al 2003, Callac et al 2006; see FIG 1), is in the rank-range of both established species and varieties in the section. Because the morphological differention of the two entities is slight and because subsp. devoniensis at least comprises one or more heterothallic, out-crossing populations (Callac et al 2006), and furthermore because the ‘‘devoniensis clade’’ is a complex group with additional undescribed entities of uncertain rank, we believe that it is more conservative and appropriate at present to recognize bridghamii at subspecific (vs. species) rank. Considering the other extreme, the physiological differentiation of the two subspecies is greater than 889 what is seen among isolated populations or even varieties in related Agaricus species (cf. Callac et al 1993). To underscore this point we also considered partial RPB2 gene sequence from RWK 1899 and from RWK 1800 (A. devoniensis subsp. devoniensis from California) obtained for a separate study (Kerrigan and Xu unpubl; GenBank EU285571–72). We found 1.5% sequence divergence between the two sequences, whereas for this gene segment A. subrufescens Peck from California and Spain diverged by 0.6%, two A. placomyces Peck sequences differed by 0.3%, and two A. depauperatus (F. H. Møller) Pilát sequences differed by 0.4% (Kerrigan and Xu unpubl). Morphologically subsp. bridghamii tends to have a more compact shape, with a stipe length/width ratio of 1.2–3.0 (vs. (2.4–)3.4–8.3 in subsp. devoniensis, per auct.), and shorter cheilocystidia (for subsp. devoniensis, 36–50 mm, per Møller (1952), although LAP notes that the terminal elements in the type are often only 15–26 3 9–14 mm (we also note that subsp. devoniensis collection RWK 1800 from California lacked cheilocystidia). The habitat and range differences (sand dunes and Cupressus macrocarpa groves near sea level for subsp. devoniensis, vs. montane Picea forests above 1300 m for subsp. bridghamii) are of uncertain value in delimiting the taxa; however, the physiological divergence implied by the differing abilities of cultures of the two subspecies to use mushroom compost (RWK 1899 and RWK 1900 grow poorly and ultimately unsuccessfully on commercial Agaricus mushroom compost, while Californian, French and Greek subsp. devoniensis grow and fruit well on it) is distinctive. In a contrasting example individuals from all populations of A. bisporus, including those from coastal cypress and montane spruce forests, use compost approximately equally well (Kerrigan and Callac unpubl). Considering the degree of sequence divergence, and the morphological, cultural, and ecological attributes of the Rocky Mountain organism, subspecific rank within A. devoniensis appears to be most appropriate for this entity. Only certain other isolates of subsp. devoniensis show clear mating reactions, so the negative results of mating confrontations attempted by Callac between the subspecies are ambiguous at this time (Callac et al 2006). Sordid discolorations on pilei of A. devoniensis subsp. bridghamii also were reported for A. litoralis (Wakef. & A. Pearson) Pilát, a species sometimes linked with A. devoniensis (;Psalliota arenicola Wakef. & A. Pearson; Cappelli (1984) provides a good summary and discussion of variation reported for European A. devoniensis and its purported relative A. litoralis) but now considered to be a synonym of A. 890 MYCOLOGIA spissicaulis F.H. Møller, which is the type of section Spissicaules (Heinem.) Kerrigan (Nauta 2001; Parra 2005, 2008). Stains have not been noted otherwise on A. devoniensis proper. The variation RWK has observed in spore length/ width ratios for the two Colorado collections strengthens our sense that members of the ‘‘devoniensis clade’’ may be morphologically plastic and correspondingly difficult to delimit and/or identify. The entire ‘‘A. devoniensis clade’’ remains challenging, as discussed below. Since 2003 we also have also studied additional material of A. devoniensis not noted above. Collection data on this material is: CA 504 (5A. fragilivolvatus holotype): Italy, Sardegna, prov. Cagliari, Serramanna, under Eucalyptus camalduensis, 23 Oct 1992, leg. M. Contu and P. Dessi, coll. M. Contu 92/60 (CAG) (GenBank EU363035); CA 445 (originally determined as A. fragilivolvatus): Italy, Sardegna, prov. Sassari, Golfo Araui, La Marinella, under Eucalyptus sp., 24 Nov 2004, leg. & det. M. Contu (without collection number, private herbarium) (EU363036); CA457: Spain, La Rioja, Calahorra, under Pinus halepensis, 28 Nov 1997, leg. & det. Agustı́n Caballero, coll. AC2160 (private herbarium) (EU363034); CA 568 (5Dv9; GenBank EU258677): Greece, Papakosta (close to Agiokampos), Thessalie, under C. sempervirens on the coast, 17 Oct 2000, leg. & det. I. Theochari, P. Callac, & R. Kerrigan (CGAB); CA 117 (5Dv7; GenBank AF432895): Greece, Feres, Velestino, under C. sempervirens, 13 Jan 1999, leg. I. Theochari, det. P. Callac (CGAB). DISCUSSION It is fair to say that section Bivelares encompasses a number of rarely seen species, some of which remain undescribed. Collecting and describing ultrarare species of agarics present special challenges. We realize that we presently can provide only partial information on the ranges and variation of some of the taxa described here. Some conjecture remains, for example about the relationship of the two known collections of A. subsubensis. Particularly with the advent of molecular data, especially DNA sequences, it is possible to provide robust markers for biological novelty even when material for study is limited. We think it is important to place such data in the public record. Our intent is to document such species as well as possible at present, in aid of encouraging and improving the recognition, preservation and reporting of future collections. Cultures of JB-101, FS-22, and RWK 1789 date from 1990–1992. As far as we know these species have not been encountered otherwise in nature. Their novelty has long been evident from RFLP and other molecular data. Unfortunately the latter two collections were made under conditions that precluded voucher preservation; only a culture or viable spores were obtained. Both A. subsubensis and A. cupressophilus have now been cultivated successfully from original cultures, which have been deposited at ATCC. The French CA103 (INRA), sequenced by Callac from a culture (available at MYCSA) from mushrooms obtained by J. Guinberteau under cypress on Oléron Island, France, is also a unique entity that, lacking an extant voucher, is in need of recollection. A. agrinferus is uncommon in the Monterey cypress habitat of coastal California, the only place it is known to occur in North America, where it has perhaps one-tenth the abundance of A. bisporus (Kerrigan 1982). Agaricus tlaxcalensis is presently known only from scattered sites planted with C. benthamii in Tlaxcala, Mexico. The A. devoniensis clade is challenging not only due to rarity but also due to apparently recent speciation processes, which in the case of subsp. devoniensis in Europe might include re-integration of diverged populations (cf. Callac et al 2006 and unpubl). On one hand, because we have observed no differences in the types of both species, Agaricus fragilivolvatus Contu is considered here a synonym of A. devoniensis. The type of A. devoniensis has spores 6–7 3 5–5.5 mm and clavate cheilocystidia 16–26 3 9– 14 mm, while from the type of A. fragilivolvatus we measured spores and cheilocystidia respectively as 6–7 3 (4.5–)4.8–5.5 mm and 17–34 3 8–13 mm. The cheilocystidia in the A. fragilivolvatus type were clavate to pyriform and not vesiculose as described by Contu. Compare also GenBank sequences AF432892, –93, –94, –96, –97 and EU363034 with EU363035–36) (FIGS. 1, 2). On the other hand, some real heterogeneity exists. A. devoniensis subsp. bridghamii has been found twice in Colorado and once in Alberta. Its range is therefore considerable; however in our experience it is rare or at least uncommon. Another member of the clade, known only from two collections (see CA 117, CA 568) from Greece has distinctly divergent ITS1+2 sequences and might deserve taxonomic recognition after further study (see GenBank AF432895 and EU258677). Two additional members of this clade (see GenBank AF432889, AF059225) in need of further study have been reported from New Zealand by Mitchell and Bresinsky (1999), indicating a broad geographical scope for this phylogenetic radiation. We discussed in 2003 our sequence data from material determined by Mitchell and Bresinsky as A. subperonatus but which instead is closely related to A. devoniensis (Challen et al 2003). Based on our review and in comparison with European material, the ITS2 sequence deposited by Mitchell and Bresinski (1999) as A. devoniensis from New Zealand also appears to KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES belong to a related but distinct entity in the ‘‘A. devoniensis complex’’ (the unresolved phylogenetic clade encompassing A. devoniensis and any variants and closely related putative species) (FIG. 2). During this study we have evaluated some sequences present in GenBank. We note that ‘‘A. padanus HAI 0312’’ (GenBank AJ884647) recorded by Didukh (2004) and sequenced by Didukh et al (2005) is a very close match for A. bitorquis. We also have sequenced the type of A. padanus, and it has a distinct sequence similar to those of section Sanguinolenti (unpubl). We therefore regard GenBank AJ884647 to be of A. bitorquis. Some comments on our earlier report with M. Challen on Duploannulati (5Bivelares) (Challen et al 2003) are in order. Nomenclature of A. subfloccosus s.l. and A. vaporarius are discussed above, as is the challenge presented by Agaricus devoniensis and its associates. The possibility of the existence of a distinct, rare A. subperonatus elicits a range of opinions but cannot be fully resolved at present. M.M. Nauta (pers comm) has noted that our statement concerning the lack of rufescence in A. bitorquis (Quél.) Sacc. is at least an overgeneralization. We are inclined to agree, in view of our emerging belief that A. bitorquis var. validus (F.H. Møller) Bon & Cappelli is most likely to be a phylogenetically irrelevant name applied to occasional individuals of A. bitorquis exhibiting discernible rufescence, a trait hypothetically (for example) due to a single gene change resulting in over-expression of tyrosinase enzyme activity (see van Leeuwen and Wichers 1999). We sequenced the ITS1+2 region of one such individual (LAPAG 517: duplicate from P. Arrillaga, garden, Zarauz, Guipúzcoa, Spain, 8 Apr 1996 [coll. 501-8/4/96 deposited in Arrillaga’s private herbarium] Arrillaga 2004) (GenBank EU257804) and found no differences from var. bitorquis sequences other than one heteromorphism. We also have added new A. bitorquis sequences to GenBank from CLO 4465 (Clarke Ovrebo, Oklahoma [OKL]; EU258675) and JWS-1 ( John Sparks, New Mexico [SFSU]; EU258674) for comparison. According to Nauta the rufescent phenotype is not uncommon in Europe, but in our own experience it is not common and less so in North America. At best the rufescence of typical A. bitorquis is limited to an indistinct sordid discoloration of the stipe and of pileus context near the lamellae, primarily in mature specimens. There are now 10 named species and several infraspecific taxa formally placed in section Bivelares. Two subsections are recognized. Entities needing names, after adequate further study, are known from France, Greece and New Zealand. Additional discoveries would not be surprising. 891 ACKNOWLEDGMENTS We thank D. Arora, P. Arrillaga, P. Boisselet, A. Caballero, K. Chadwick, M. Contu, J. Guinberteau, G. Maddalena, G. Mata, A.D. Mitchell, M. Moinard, D. Murietta, A. Mua, C. Ovrebo, K. Peterson, G. Pickett, S. Rextoueix, M. Shaw, J. Sparks, F. Stevens and A. Tancrède, and the Colorado Mycological Society, for specimens, cultures and/or collecting assistance. J. Guinberteau provided FIG. 3 and other assistance. Advice on Latin and translations were provided by G. Heuclin, R. Halling and J. Santos. Some sequences were kindly provided by M. Challen and J.-P. Xu. P. Callac acknowledges ECOS-Nord and ANUIES (M06A01 FrancoMexican program) and the BRG (Bureau des Ressources Génétiques, program No. 51, 2007/2008) for financial support. LITERATURE CITED Arrillaga P. 2004. Guı́a de los champiñones del Paı́s Vasco y su entorno, estudio del género Agaricus L. : Fr. Munibe, (Suppl. 17):1–197. Bohus G. 1994 [‘‘1993’’]. Agaricus-Studien XII. Beitr Kenn Pilze Mitteleurop 9:51–56. Boisselet P. 1990. Clé de détermination provisoire de quelques espèces et principales sections du genre Agaricus d’après la nature, le développement, la structure et la combinaison des différents voiles. Bull Féd Mycol Dauphiné-Savoie 117:18–21. Bon M. 1987. Novitates. Doc Mycol 17(67):11–13. ———. 1988. Champignons d’Europe occidentale. Paris: Arthaud. 368 p. Callac P. 1994. Prospections pour la recherche d’Agaricus bisporus en France: contexte historique et scientifique, premiers résultats. 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New and rare taxa in Agaricus section Bivelares (Duploannulati)

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