Morphological and molecular characterization of three Agaricus

Mycologia, 106(6), 2014, pp. 1220–1232. DOI: 10.3852/14-076 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897

Morphological and molecular characterization of three Agaricus species from tropical Asia (Pakistan, Thailand) reveals a new group in section Xanthodermatei Naritsada Thongklang

introduced based on molecular and morphological studies, whereas A. microvolvatulus is recorded for the first time in Asia. Specimens from Thailand however have a much larger pileus than the type specimens from Congo. In maximum likelihood (ML) and maximum parsimony (MP) phylogenetic analyses these three species cluster with A. pseudopratensis from the Mediterranean area and A. murinocephalus recently described from Thailand. In Agaricus section Xanthodermatei this new group is monophyletic and receives low bootstrap support whereas the two previously known groups receive strong support. Within the new group, the most closely related species share some traits, but we did not find any unifying morphological character; however the five species of the group share a unique short nucleotide sequence. Two putatively toxic species of section Xanthodermatei are now recognized in Pakistan and six in Thailand. Key words: basidiomycota, morphology, new species, phylogeny, tropical biodiversity

Institute of Excellence in Fungal Research and School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand

Rizwana Nawaz Abdul N. Khalid Department of Botany, University of the Punjab, Lahore, Pakistan

Jie Chen Kevin D. Hyde Institute of Excellence in Fungal Research, and School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand

Ruilin Zhao The State Key Lab of Mycology, Institute of Microbiology, Chinese Academic of Science, Beijing 100101, China

Luis A. Parra Avda. Padre Claret 7, 5u G, 09400 Aranda de Duero, Burgos, Spain

Muhammad Hanif Department of Botany, Government College University, Lahore, Pakistan

INTRODUCTION Agaricus L. is a genus of saprobic fungi of the order Agaricales that includes species of nutritional and medicinal interest, such as the button mushroom (A. bisporus [J.E. Lange] Imbach) and the almond mushroom (A. subrufescens Peck, syn. A. blazei sensu Heinemann; Wisitrassameewong et al. 2012). Zhao et al. (2011) recognized 386 species in genus Agaricus; this number now exceeds 400 in that at least 23 novel species were described from Europe (Parra et al. 2011, Parra 2013), Thailand (Chen et al. 2012; Zhao et al. 2012, 2013) and Australia (Lebel and Syme 2012, Lebel 2013). Although almost half of these 400 species are tropical it remains certain that many more undescribed species are present than in temperate areas. For instance we have collected more than 50 new species in Thailand that are waiting further study and formal description (Zhao et al. 2011). This is also probably the case in Pakistan where only 20 species of Agaricus have been recorded so far (Ahmad et al. 1997, Sultana et al. 1997). Because Agaricus is a familiar genus whose species are collected for consumption, it is important to know which species could be toxic. All or most of the toxic species of the genus belong to Agaricus sect. Xanthodermatei Singer. Members of this section are

Magalie Moinard Philippe Callac1 INRA, UR 1264, Mycologie et Se´curite´ des Aliments, CS 20032, 33882 Villenave d’Ornon CEDEX, France

Abstract: The genus Agaricus is known for its medicinal and edible species but also includes toxic species that belong to section Xanthodermatei. Previous phylogenetic reconstruction for temperate species, based on sequence data of nuc rRNA gene (rDNA) internal transcribed spacers (ITS), has revealed two major groups in this section and a possible third lineage for A. pseudopratensis. Recent research in Agaricus has shown that classifications need improving with the addition of tropical taxa. In this study we add new tropical collections to section Xanthodermatei. We describe three species from collections made in Pakistan and Thailand and include them in a larger analysis using all available ITS data for section Xanthodermatei. Agaricus bisporiticus sp. nov. and A. fuscopunctatus sp. nov. are Submitted 26 Mar 2014; accepted for publication 28 Jun 2014. 1 Corresponding author. Email: [email protected]

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THONGKLANG ET AL.: THREE AGARICUS commonly characterized by a negative Scha¨ffer’s reaction, a positive KOH reaction (yellow), an odor of phenol, a yellow discoloration when cut or touched (Kerrigan et al. 2005, Parra 2013). Toxic compounds such as phenol cause gastrointestinal distress, progressing in severe cases, to violent vomiting (Gill and Strauch 1984, Wood et al. 1998). It can be noted that two proposed novel species, A. biannulatus A. Mua, L.A. Parra & Callac from Sardinia (Parra et al. 2011) and A. murinocephalus R.L. Zhao, Desjardin & K.D Hyde from Thailand (Zhao et al. 2013), have neither yellow discoloration when bruised nor a phenol-like odor. A phylogenetic reconstruction of Agaricus sect. Xanthodermatei based on temperate species from Europe and North America by Kerrigan et al. (2005) showed that this section is monophyletic and closely related to Agaricus sect. Chitonioides Romagn. and Agaricus sect. Bivelares (Kauffman) L.A. Parra (syn. Agaricus sect. Duploannulati Wasser, see Kerrigan et al. 2008) and mainly comprised two major clades with a possible third lineage represented by a single species (A. pseudopratensis [Bohus] Wasser). The phylogenetic analysis of Zhao et al. (2011) included five temperate and 10 tropical species that grouped in three different clades: the two major clades recognized by Kerrigan et al. (2005) and a third containing A. microvolvatulus Heinem. and two unidentified species. A. microvolvatulus was represented by a type specimen from Congo and by Thai specimens having identical sequences of the nuc rRNA gene (rDNA) internal transcribed spacers (ITS), but their identification needed morphological confirmation. One of the two unidentified species has been described as the novel A. murinocephalus by Zhao et al. (2013). Four identified species of section Xanthodermatei have been reported from Thailand. Agaricus murinocephalus and A. endoxanthus Berk. & Broome both have been described morphologically from Thailand by Zhao et al. (2013). Agaricus microvolvatulus and A. xanthosarcus Heinem. & Gooss.-Font. both were described initially from Africa and have been reported for Thailand on the sole basis of ITS sequence identity between Thai specimens and African type specimens (Zhao et al. 2011); therefore the identification of the Thai specimens has not been morphologically confirmed. In Pakistan A. endoxanthus is the sole species of section Xanthodermatei that has been reported, having been found in the cities of Sialkot and Murree (Ahmad et al. 1997). Based on the studies of Zhao et al. (2011, 2012, 2013) and on collections made in Thailand and Pakistan over the past 3 y, we selected for further study some samples likely to belong to Xanthodermatei. Our objectives are to

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morphologically and phylogenetically characterize the species to which these samples belong and to investigate their relationships within Xanthodermatei. For this we sequenced the rDNA ITS regions from the new collections to compare them with the other available sequences to update the phylogeny of the section. As a result of this study we redescribe A. microvolvatulus Heinem. and introduce A. bisporiticus sp. nov. and A. fuscopunctatus sp. nov. These three species together with two species with so far unknown relationships belong to a new clade revealed by our phylogenetic analysis. MATERIALS AND METHODS Sampling.—Fifteen samples were examined morphologically, sequenced or both to characterize the three species. Thirteen samples were collected during the past 3 y in tropical areas: one in Pakistan (region of Lahore) and 12 in northern Thailand. These samples have been deposited in BBH (BIOTEC Bangkok Herbarium), LAH (Herbarium of Botany Department of Punjab University), MFLU (Herbarium of School of Science of the Mae Fah Laung University) and SWFC (Herbarium of Southwest Forestry College). The remaining two samples are type specimens of A. microvolvatulus from the Congo. Their detailed origin, the herbarium where they are deposited and GenBank accession numbers of nuc rDNA ITS1+5.8S+ITS2 (ITS barcode) sequences are provided in Materials examined; 11 of the 13 are newly deposited sequences. Forty-four samples are included in the phylogenetic analysis: 13 sequenced samples among the 15 cited above, 25 samples representing species of Xanthodermatei and six samples of Chitonioides or Bivelares representing outgroup species. GenBank accession numbers of 33 samples that have been published (Callac and Guinberteau 2005; Kerrigan et al. 2005, 2008; Parra et al. 2011; Zhao et al. 2011, 2012, 2013; Parra 2013) were included in the phylogenetic analysis. Macro- and micromorphological examination.—Samples were photographed in situ or in the laboratory. Macroscopic characters, such as size, shape, and color of pileus, lamellae and stipe, were observed on fresh specimens. Microscopic observations were performed on fresh or dried material with a compound light microscope following Reid (1984). Sections of specimens were cut and mounted in 5% KOH and dyed with 1% Congo red. Particular attention was given to the structure of the pileipellis and stipitipellis and features of basidiospores, basidia, cystidia, pileus hyphae and stipe hyphae. Measurements of these microscopic characters were obtained based on at least 20 measurements with an ocular micrometer and 1003 oil-immersion objective. The cross reaction test (Scha¨ffer’s) was performed on pileus of fresh specimens by drawing cross lines with aniline and nitric acid respectively. If the point of intersection became red or orange, the reaction was scored as positive, and if no color change was observed at the point of intersection, the reaction was scored negative (Cappelli 1984).

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DNA isolation, PCR and sequencing.—Methods used depended on the laboratory. At the Institut National de la Recherche Agronomique (INRA) DNA was isolated from dry specimens following a CTAB protocol (Saghai- Maroof et al. 1984, Doyle and Doyle 1987) modified as indicated in Zhao et al. (2011). PCR amplification was achieved with primers ITS4 and ITS5 (White et al. 1990) for the ITS1+5.8S+ITS2 regions as indicated in Zhao et al. (2011). Sequencing was performed on ABI Prism genetic analyzers (Applied Biosystems) at Beckman Coulter Genomics, England. Collected basidiomata were vouchered and dried under a fan heater in the laboratory at the University of the Punjab, Pakistan. In preparation for DNA extraction, a small piece of pileus was kept in 2% CTAB buffer. DNA was extracted with modified CTAB method following Gardes and Bruns (1993) and was blended by Q biogen Clean Kit protocol. PCR was carried out following Gardes and Bruns (1993), using fungus-specific forward (ITS1F) and universal backward (ITS4) primers to amplify the rDNA ITS region. PCR conditions were optimized for different basidiomata. The hot-start enzyme JumpStart (Sigma, St Louis, Missouri) was used to catalyse the PCR with 2 min at 94 C, followed by 35 cycles of 30 s at 94 C, 30 s at 53 C, 45 s + 5 s per cycle at 72 C and finishing with 5 min at 72 C. The PCR products were analyzed by agarose gel electrophoresis. PCR products were sequenced at Macrogen, Korea. Sequence alignment and phylogenetic analysis.—After multiple-sequence alignment with T-Coffee 8.99 (Notredame et al. 2000), corrections were made by hand, first because TCoffee did not interpret the heteromorphisms and second to suppress highly variable or ambiguous positions. The maximum likelihood (ML) analysis was performed on the ATGC bioinformatics platform (atgc-montpellier.fr/ phyml/). The phylogenetic tree was constructed with maximum likelihood (ML) implemented in the PhyML 3.0 (Guindon et al. 2010). This method does not allow partitions. The HKY85 substitution model was selected with an estimated proportion of invariable sites of 0.462 and assuming four gamma-distributed rate categories to account for rate heterogeneity across sites. The gamma shape parameter was estimated directly from the data (c 5 0.716). The maximum parsimony (MP) analysis was performed with PAUP* 4.0b10 (Swofford 2004) by heuristic searches with unordered characters, random addition of sequences, gaps treated as missing data or added as coded characters, and tree bisection-reconnection (TBR) branch swapping. The analysis included 100 or 1000 bootstrap replicates for ML or MP respectively. Species-specific ITS markers.— Comparisons were made between the 44 sequences (ITS1+5.8S+ITS2) used for the analyses. The position of a unique nucleotide (nt) in the ITS barcode sequences of a species is indicated as follows: xxxxXxxxx@position where the capital letter represents the characteristic base, and xxxx represents the flanking bases. IUPAC codes as Y does not indicate heteromorphisms or ambiguity but that T or C are found at this position depending on the sequence. Specific markers also can be indicated for a clade; in this case the position in the final

alignment is indicated. It should be noted that comparisons are made with the currently available data for A. sect. Xanthodermatei, but with additional sequences reassessment might be needed.

RESULTS Phylogenetic analyses.—The length of the 44 ITS1, 5.8S, ITS2 sequences varied 649–664 bp (base pairs). The final alignment had 688 characters (702 before corrections). The most likely ML tree is presented (FIG. 1). Alignment and ML tree were deposed in TreeBASE (TB2:S15569). Although section Xanthodermatei was monophyletic, it did not receive strong bootstrap support. The section consists of three clades: Xan I and Xan III were strongly supported (99 and 95 resp.), whereas Xan II was poorly supported. For the MP analysis 27 coded gap characters were added to the 688 characters. A deletion of 7 bp located close to the 39 extremity of the ITS2 region was counted as a single gap. Among a total of 715 characters, 159 were parsimony informative. In the four most parsimonious trees obtained from the analysis, the three sectional clades formed a polytomy; section Xanthodermatei appeared to include three major clades (Xan I, II, III), as was observed in the ML analysis. Moreover in two retained trees clade Xan II was sister to Xan III (FIG. 1), whereas Xan II appeared as a sister to Xan I in the two remaining trees. Some minor differences appeared in clades Xan II or Xan III: for example in the former A. pseudopratensis and A. bisporiticus appeared closely related, but non-sister. As in the ML analysis, Xan I and Xan III were strongly supported but Xan II was not. Molecular characterization of the species and clades.— Each of the five species of clade Xan II possessed at least two unique characters. For the three species described in detail (A. bisporiticus, A. fuscopunctatus, A. microvolvatulus) species-specific ITS markers are indicated below in the taxonomic treatment. Agaricus murinocephalus had the following two species-specific ITS markers: cctt(–)tcag@deletion between 206 and 207 and agggAgatt@595. Kerrigan et al. (2005) indicated that A. pseudopratensis has six speciesspecific ITS markers but after adding related species in our analysis only four remain: gggtaTattgag@110, tcattTcatta@225, and tggccCcttgctTraggt@513-520. Clade Xan I possesses one specific ITS marker, ctcty(a/–)A(w/–)gy(t/–)gttcagc@634. Clade Xan III has two specific ITS markers, tG(k/–)gaggarryr@115 and ggaatc(c/–)tGtyk@210. Clade Xan II likely possesses two species-specific ITS markers, but their exact position in the alignment is somewhat ambiguous. All

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FIG. 1. Maximum likelihood (ML) phylogram of Agaricus section Xanthodermatei based on ITS1+2 sequences of 44 specimens belonging to 28 species of section Xanthodermatei and six species of sections Chitonioides and Bivelares used as outgroups. Topology was globally similar with outgroups at the basis in ML and MP trees. The bootstrap support values above 50% are shown (ML, 100 replicates; MP in boldface, 1000 replicates). The three major clades, Xan I, Xan II and Xan III, are indicated. The three species of clade Xan II described in the present study are A. microvolvatulus and the two novel species A. bisporiticus and A. fuscopunctatus.

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29 sequences (including outgroups) of the species that do not belong to clade Xan II had the following sequence: tmtcagc(YYYB)gytggat, whereas all 15 sequences of the five species belonging to clade Xan II shared the unique nucleotide sequence ATY at the same positions (tatcagc[ATY]gctggat). This unique nucleotide sequence likely results from two mutations that have occurred independently among the four positions YYYB: a deletion possibly at any of the four positions and a transversion (from Y to A) possibly at one of the two first positions. For the ML and MP analyses (FIG. 1) we used the same configuration in all 15 sequences of clade Xan II with the transversion in first position and the deletion in second position (tatcagc[A-TY]gctggat@143-146). To see how this unique nucleotide sequence can affect the final tree we performed a second ML analysis in which the characters at these four positions were deleted from the alignment; the only change in the topology of the resulting tree was that group Xan II appeared as paraphyletic to Xan III with clades Xan I and Xan III as well as the two subclades of Xan II remaining unchanged. A similar result was obtained with the MP analysis when gaps were treated as missing data. Therefore the unique nucleotide sequence ATY supported the monophyly of Xan II. A double homoplasy being unlikely, a common ancestor of the five species of the clade Xan II probably carried the double mutation. TAXONOMY Agaricus bisporiticus Nawaz, Callac, Thongklang & Khalid, sp. nov. FIGS. 2, 3 MycoBank MB808224 Etymology: Having/bearing bisporic (two-spored) basidia.

Macroscopic characters: Pileus 4–8 cm diam and 4 mm thick, conical then convex and finally applanate with center broadly umbonate at first covered with grayish brown to brown fibriles, later becoming squamose, dense at the disk and more scattered toward the margin on a whitish to mouse gray background with age. Surface smooth, dull and dry. Margin incurved when young then straight to recurved and slightly splitting radially. Lamellae free, crowded, ventricose, at first white to light pinkish or light gray to margin on Thai specimens, then light brown, brown and dark brown with age, 3–6 mm broad. Stipe cylindrical and generally curved in the lower half, inconstantly slightly bulbous at the base, 30–70 3 5–12 mm, smooth on surface, white, finally beige. Annulus superous, first thick and appressed to the stipe, later in mature basidiomata pendant or turned toward the stipe apex, narrow, in the upper

surface smooth and in the lower surface smooth with some irregular teeth near the margin, which is cottony-fibrillose. Context firm, pale white, when scratched becoming yellow within 10 s then gray and finally reddish after 10 min. Faint phenol odor. Chemical reactions: In KOH yellowish orange; Scha¨ffer’s negative. Microscopic characters: Basidiospores 4.5–5.9–7.3 3 2.9–3.7–4.7(–4.9) mm Q 5 1.5–1.6–1.9 (n 5 80), ellipsoid with one or two guttules, thick-walled, brown in KOH with dark brown walls, smooth with a prominent apiculus. Basidia 12.4–25 3 5.6–9.1 mm, clavate, hyaline in KOH, sterigmata 1.1–5.7 mm long. Most of basidia were bisporic or trisporic in the type specimen; percentages of bi, tri and tetrasporic basidia varied as follow in three other collections: 28:17:55 (CA910), 31:36:33 (CA929), 37:28:35 (LD2012111). Cheilocystidia 15.2–35.6 3 6–21.4 mm, abundant but sometimes absent in old basidiomata, hyaline and smooth, broadly clavate, pyriform or sphaeropedunculate. Pleurocystidia absent. Pileipellis a cutis constituted by hyaline hyphae 3–11.5 mm diam, as well as brown 3.3–10 mm diam in KOH. Hyphal elements straight, cylindrical or enlarged near the septa, interwoven, lacking clamp connections. Stipitipellis constituted by hyphae 1.9–18 mm diam, hyaline in KOH, smooth, straight, cylindrical. Habitat, distribution, occurrence: Gregarious in gardens or parks, on grass leaf litter, likely a large geographical distribution range including Pakistan and Thailand. Species-specific ITS markers: tttgCtggg@103 (tytg [g/–])Ct[t/–]rgr in the alignment) and taagTctat@ 273 ([h/–)ahgTctat in the alignment). Holotype: PAKISTAN, PUNJAB, Botanical Garden, University of the Punjab, Lahore, at 217 m, gregarious on rich loamy soil of garden, 11 Aug 2011, A.N. Khalid, MCR25 (LAH110811). ITS barcode (GenBank: KJ575608). Additional specimens examined: THAILAND, CHIANG RAI PROV., Mae Fah Luang University, in grass, 9 Jun 2011, P. Callac, S. Karunarathna and S. Rapior, CA910 (MFLU20130431), ITS barcode (GenBank: KJ575609); CHIANG RAI PROV., Mae Fah Luang University, in grass, 16 Jun 2011, P. Callac, S. Karunarathna and S. Rapior, CA929 (MFLU20130430), ITS barcode (GenBank: KJ575610); CHIANG RAI PROV., Mae Fah Luang University, in grass, 20 Jul 2012, J. Chen, LD2012111 (MFLU20120948), ITS barcode (GenBank: KJ575611).

Comments: Agaricus bisporiticus is morphologically well characterized in having at least 50% of basidia being bi- or trisporic and exhibiting reddish discoloration (10 min after bruising). It also is remarkable that all specimens from Pakistan and Thailand have identical sequences without any heteromorphism. Of

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FIG. 2. Microcharacters, A. bisporiticus (LAH110811, holotype). a–e: a. Basidiospores. b. Basidia. c. Lamellae section. d. Cheilocystidia. e. Hyphae from the pileipellis. Agaricus fuscopunctatus (MFLU20120952, holotype). f–j: f. Basidiospores. g. Basidia. h. Lamellae section. i. Cheilocystidia. j. Hyphae from the pileipellis. Agaricus microvolvatulus (MFLU20120910), k–o: k. Basidiospores; l. Basidia; m. Lamellae section; n. Cheilocystidia; o. Hyphae from the pileipellis. Bars: a 5 5 mm; b–o 5 10 mm.

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FIG. 3. Agaricus bisporiticus (a 5 LAH110811 holotype, b 5 LD2012111, c 5 CA929) and A. fuscopunctatus (d 5 MFLU20120952 holotype, e–f 5 CA919). a. Pakistani sporophores in situ. b. Thai sporophores in situ. c. Red brownish discoloration after several minutes on pileus and stipe, which is slightly bulbous at the base. d. Sporophore in situ. e. Scattered grayish-brown squamules on pileus and large umbo. f. Mature and immature coalescent sporophores. Bars: a–f 5 2 cm.

THONGKLANG ET AL.: THREE AGARICUS interest, the closest relative species A. pseudopratensis (FIG. 1) similarly exhibits yellow discoloration followed by wine red discoloration after 10 min. Agaricus fuscopunctatus Thongklang, L.J. Chen, Callac & K.D. Hyde, sp. nov. FIGS. 2, 3 MycoBank MB808225 Etymology: From fuscus ‘‘dark’’ and punctatus ‘‘punctate’’, in reference to the finely spotted squamules on the pileus.

Macroscopic characters: Pileus 4–7 cm diam and 2 mm thick, ovate then convex finally applanate and broadly umbonate, dark brown to dark green olive gray at the disk, covered with concolor dot-like squamules progressively scattering toward the margin on a white background, becoming yellowish after bruising. Margin straight, slightly splitting and striated in dry condition or when mature surface smooth, dull and dry. Lamellae free, crowded, ventricose, at first light pinkish or light gray at the margin then light brown with age, 3–6 mm broad. Stipe cylindrical, often curved, surface smooth and white, almost marginately bulbous with rizomorphs at the base, (40–)68–97 3 6–10 mm, bulb 10–20 mm more or less misshapen. Annulus superous and attached to the upper half of the stipe, narrow but thicker and two-edged at the margin, white with sometimes some dark gray remnants of veil. Context firm, pale white, becoming yellowish when bruised and/or cut. Odor of phenol, ink. Chemical reactions: KOH reaction yellow or bright orange; Scha¨ffer’s reaction negative. Microscopic characters: Basidiospores (3.9–)4.7–5.4– 6.1(–6.3) 3 (2.7–)3–3.5–4(–4.2) mm Q 5 1.54–1.57– 1.61 (n 5 60), ellipsoid with one or two guttules, thick-walled, brown in KOH with dark brown walls, smooth with a prominent apiculus Basidia 13.0–19.7 3 5.1–7.3 mm, clavate, hyaline in KOH, mostly tetrasporic, sterigmata 0.8–3.8 mm long. Cheilocystidia 6.7–38 3 3.8–11.5 mm, hyaline and smooth, broadly clavate to pyriform with cylindrical peduncule. Pleurocystidia absent. Pileipellis a cutis constituted by cylindrical hyphae containing brown vacuolar pigment, smooth, straight, cylindrical, 2.6–8.4 mm diam. Habitat, distribution, occurrence: Scattered in grass as in leaf litter, in parks or forests; known only from Thailand. Species-specific ITS markers: ctagGtggg@19 (cyrrGyggg in the alignment) and tgtaGtaca@287 (trtaGtaca in the alignment). Holotype: THAILAND, CHIANG RAI PROV., Mae Fah Luang University, on litter, 21 Jul 2012, J. Chen, LD2012115 (MFLU20120952). ITS barcode (GenBank: KJ575612). Isotypes: BBH38277, SWFC.

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Additional specimens examined: THAILAND, CHIANG RAI PROV., Meachan, Kewtabyoung, on litter in forest, 16 Jul 2010, N. Thongklang, NTF061 (MFLU20130614), ITS barcode (GenBank: JF514528); CHIANG RAI PROV., Mae Fah Luang University, in grass, 14 Jun 2011, P. Callac, S. Karunarathna and S. Rapior, CA 919 (MFLU20130615), ITS barcode (GenBank: KJ575613).

Comments: It can be difficult to morphologically distinguish Agaricus fuscopunctatus from its sister species A. murinocephalus, although the latter has spores slightly more elongate (6 vs. 5.4 mm) and characteristically exhibits odor or discoloration. However their ITS sequences differ at nine positions. Moreover A. fuscopunctatus as well as A. murinocephalus possess two species-specific ITS markers. Agaricus microvolvatulus Heinem., Cah. Maboke´ 9:6. 1971. Description based on specimens from Thailand. FIGS. 2, 4. Macroscopical characters: Pileus (7–)10–14 cm diam and 7–9 mm (2 mm for NTT084) thick, first parabolic to hemispherical, then convex finally plane or slightly depressed at the center, dark gray to dark brown at the disk, covered with gray to brown dot-like squamules becoming more scattered toward the margin on a white background, turning yellowish when bruised. Margin wavy and straight when mature. Surface smooth, dull and dry. Lamellae free, crowded, ventricose, at first white to light pinkish or light gray at the margin then light brown, finally brown to dark brown, 4–11 mm broad. Stipe cylindrical, hollow, white, smooth, with abrupt bulbous base forming a plinth that can be shared by several sporocarps, 110–120(–188) 3 8–14 mm, bulb reaching 2.5 cm diam, marginate with a thick rounded projecting edge separated from the stipe by a depression 1–2 mm wide. Annulus superous, attached to the upper half of the stipe, upper surface smooth, underside floccose, white with grayish brown remnants of veil forming a colored line on the underside near the insertion on the stipe. Context soft, white, becoming yellowish by bruising or cutting. Odor of phenol or ink. Chemical reactions: KOH reaction yellow or orange; Scha¨ffer’s reaction negative. Microscopic characters: Basidiospores 4.5–5.4–6 (–6.8) 3 (2.6–)2.9–3.3–3.8(–4.4) mm Q 5 1.62–1.64– 1.67 (n 5 100), ellipsoid, thick-walled, brown in KOH with dark brown walls, smooth with a prominent apiculus. Basidia 9–15 3 4–7 mm, clavate, hyaline in KOH, mostly tetrasporic. Cheilocystidia 12.7–22 3 6.7–11.4 mm, hyaline and smooth, broadly clavate with cylindrical peduncule. Pleurocystidia absent. Pileipellis a cutis constituted by cylindrical hyphae containing brown vacuolar pigment, smooth, straight, cylindrical, 2.4–6.9 mm diam. Hyphae at the external surface of the basal volviform bulb with thin, cylindrical, often

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FIG. 4. Agaricus microvolvatulus (a 5 LD2012171, b–c 5 NTT084, d–e 5 LD201271, f 5 LD201272). a. Sporophores in situ. b. Pileus. c. Volviform marginate bulbe at the base of stipe. d. Dark brown dot-like scales on pileus. e. Annulus. f. Two coalescent sporophores at bulbous base. Bars: a–f 5 2 cm.

THONGKLANG ET AL.: THREE AGARICUS contorted terminal elements with attenuated, rounded or slightly capitulate apex 2–4 mm diam. Habitat, distribution, occurrence: Solitary or gregarious in grasslands, parks or forests in North Thailand. Originally described from Congo. Apparently common in northern Thailand. Species-specific ITS markers: tatgGggac@160 (trtgGggay in the alignment), cattTgctg@171 (crbtTgctv in the alignment) and tggaAtgtg@493 (tggaAtrtg in the alignment). Sequences of the five collections are identical excepted one heteromorphism C/T at position 558 in LD201271. Material examined: CONGO, Brazzaville, forest edge, Dec 1966, Grinling61204 (BR, HOLOTYPE); Brazzaville, forest edge, Jan 1967, Grinling70109 (BR, LOCOTYPE), ITS barcode (GenBank: JF514524). THAILAND, CHIANG MAI PROV., Doi Suthep, in forest, 20 Jun 2010, K. Wisitrassameewong, NTT038 (MFLU20120131), ITS barcode (GenBank: KJ575617); CHIANG MAI PROV., Doi Suthep, in forest, 10 Jul 2010, K. Wisitrassameewong, NTT084 (MFLU20120132), ITS barcode (GenBank: KJ575616); CHIANG MAI PROV., Chiang Mai University park, in grassland, 27 Jul 2010, S. Karunarathna and J. Guinberteau, NTS117 (MFLU20100672), ITS barcode (GenBank: KJ575618); CHIANG MAI PROV., Hui nam dang National park, in grassland, 4 Jul 2012, J. Chen, LD201271 (MFLU20120910), ITS barcode (GenBank: KJ575614) and LD201272 (MFLU20120911); CHIANG MAI PROV, Doi suthep National park, in forest, 5 Aug 2012, B. Thongbai (det. J. Chen), LD2012171 (MFLU20120999), ITS barcode (GenBank: KJ575615). Note: we have reexamined collections from Congo (Heinemann 1971). Collections Grinling70109, NTT38, NTT84, and NTS117 have been reported previously by Zhao et al. (2011). Comparison with original collections from Congo: The sequence of the specimen Grinling70109 from Congo is identical to the sequences of four of the five specimens from Thailand. However, we observed two morphological differences. (i) The sporocarp: pileus diameter was larger than 10 cm for the larger specimen of each Thai collection (except NTS117 that was not mature, 7–8 cm), while the specimens from Congo described by Heinemann (1971) had a pileus that did not exceed 5.5 cm diam; such a difference would be significant if it were confirmed with more small African collections. (ii) We re-examined the two type specimens, Ginling61204 and Grinling70109 (Heinemann 1971), and found the following spore dimensions for these collections from Congo: (4.3–)4.5–5–5.5(–5.7) 3 (2.7–)2.8–3.3–3.8(–3.9) mm (n 5 42 + 50). Only the mean spore length of the Thai samples appears higher (5.4 mm); this difference falls within the range of variation that generally is observed in species of

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Agaricus. Again more African samples are necessary to make a statistical comparison between the two continental populations. Comments: This species is macroscopically remarkable by virtue of its marginate bulb with a thick, rounded projecting edge producing a volviform appearance and microscopically by the presence of thin, cylindrical, often contorted terminal elements with attenuated, rounded or slightly capitulate apex 2–4 mm on the external surface of the basal volviform bulb. Moreover the pileus generally exceeds 10 cm diam in the specimens from Thailand and contrasts with the sized type specimens described from Congo (5.5 cm). More samples from Africa are necessary to confirm this morphological difference, possibly reflecting a genetic divergence that would remained cryptic in diagnostic use of the ITS sequences, which are identical in five of the six samples from both continents. DISCUSSION Three tropical species are introduced: the novel species A. bisporiticus from Pakistan and Thailand, the novel species A. fuscopunctatus from Thailand and samples from Thailand of A. microvolvatulus, a species previously described from Africa. The phylogenetic analyses confirmed that these three species belong to Agaricus section Xanthodermatei, in agreement with their characteristic phenol-like odor and yellow discoloration. In addition the section comprised three species recently proposed by Parra et al. (2011), Zhao et al. (2013) and Parra (2013): A. biannulatus, A. murinocephalus and A. moelleroides Guinb. & L.A. Parra respectively. The monophyly of this section was not strongly supported, but more so than in Kerrigan et al. (2005), with ML tree bootstrap values of 66% vs. 54%. The section comprises three clades provisionally named Xan I, Xan II and Xan III. The phylogenetic relationships between Xan II and the two other clades are not yet resolved. Xan I and Xan III both are strongly supported clades in ML and MP analyses. Xan I contains medium to large species generally growing not far from the sea in North America and Europe and which when cut can develop reddish discoloration without first turning yellow. Agaricus biannulatus, recently described from Sardinia, also belongs to this clade as suggested by Parra et al. (2013), although it does not exhibit reddish discoloration at the stipe base and is odorless. Xan III is the core or classical Xanthodermatei group according to Kerrigan et al. (2005) and includes temperate and tropical species. Xan II is a poorly supported clade, but its five members share a unique short nucleotide sequence likely resulting from two mutations and which

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support the monophyly of the clade. Moreover clades Xan I and Xan III have their own specific ITS markers. Whatever its status, monophyletic or paraphyletic, Xan II appears as a new group because it was represented only by A. microvolvatulus in the ML tree of Zhao et al. (2011) or by A. pseudopratensis in the ML tree of Kerrigan et al. (2005) who said that this species could represent a particular lineage. Finally clade Xan II contains five species: the temperate species A. pseudopratensis, a tropical species A. murinocephalus recently described from Thailand (Zhao et al. 2013) and three more tropical species described in the present study. Geographically A. pseudopratensis is reported from Europe and Israel (Wasser 2002). Parra (2013) reports that the range of this species is mainly Mediterranean. The four remaining species of clade Xan II (i.e. A. murinocephalus and the three species described in the present study) are typically tropical and all have been collected in Thailand, whereas A. microvolvatulus originally was described from Congo. The novel species A. bisporiticus also has been collected in Pakistan in the region of Lahore, where it was the first reported species of section Xanthodermatei and where only 11 species of Agaricus have been recognized (Ahmad et al. 1997). Presently there is no record of sample or species of this clade from the Americas although there are species from these continents in both clades Xan I and Xan III. Morphologically the five species of clade II do not form a homogeneous group, although they share a unique short nucleotide sequence. Without any other unifying character other than those characterizing the section, clade II appears to include three or four different lineages: one or two for A. pseudopratensis and A. bisporiticus, a second one for A. fuscopunctatus and A. murinocephalus and a last one for A. microvolvatulus alone. Agaricus pseudopratensis and A. bisporiticus form a poorly supported subclade in clade Xan II. However these two species exhibit yellow or faint yellow discoloration when bruised, which becomes more or less wine red or red-brownish after 10 min. This is an uncommon trait in section Xanthodermatei, which suggests a possible relationship with clade Xan I in which some species exhibit reddish or pink discoloration of the context after cutting. It must be noted that A. murinocephalus also can exhibit a slight reddish brown discoloration after bruising. In other respects the fact that the Pakistani specimen LAH110811 had been identified first as A. romagnesii Wasser is in line with Parra (2013) who noted that A. pseudopratensis often is confused with the species A. bresadolanus (syn. A. romagnesii), although this species belongs to the unrelated A. sect. Spissicaules

(Heinem.) Kerrigan. Agaricus pseudopratensis is a variable species in the ITS sequence, the shape of the sporophore, the aspect and color of the pileus and even the spore size. Agaricus bisporiticus generally has a more slender stature and can be easily identified by examining basidia of which at least the half are bisporic or trisporic. Species producing bisporic basidia generally are considered pseudohomothallic (5 secondarily homothallic) or amphithallic (5 pseudohomothallic and heterothallic). In genus Agaricus bisporic basidia are reported in few taxa, such as A. bisporus var. bisporus, or in certain specimens or in certain conditions in A. subrufescens (Kerrigan and Ross 1987). Moreover in A. bisporus the basidia spore number can vary due to genetic factors (Imbernon et al. 1996). It can be noted that, contrary to what was observed for A. pseudopratensis (Kerrigan et al. 2005), ITS sequences of A. bisporiticus are identical without heteromorphism whatever their origin (Pakistan or Thailand). Agaricus fuscopunctatus and A. murinocephalus are sister species in clade Xan II, which are phylogenetically distinct. However these appear to be morphologically similar and the differences that we noted are not very significant (spore size) or are sensitive to the environment (discoloration) or subjective due to individual interpretation (odor). ITS sequencing therefore is recommended to correctly identify these two species. Agaricus microvolvatulus is well characterized by its volviform bulb and in Thai specimens by the relatively large pileus observed. Temperate species of section Xanthodermatei producing large sporophores (pileus $ 10 cm diam) are phylogenetically related to A. hondensis Murrill in Clade Xan I or to A. xanthodermus Genev. in Clade Xan III. Such species appear to be lacking in Thailand. Consequently, with a pileus reaching 10–14 cm diam in four collections, A. microvolvatulus appears not only as the largest species of clade Xan II but also as the largest species of A. sect. Xanthodermatei in Thailand, in that clade Xan I is not represented and clade Xan III is represented by smaller species, such as A. endoxanthus or A. xanthosarcus. Moreover these large specimens contrast with the smaller pileus, 3.5–5.5 cm diam, reported in the original description of A. microvolvatulus based on two specimens from Congo. With the confirmation of such small pileus diameter in more collections from Africa, the question of African and Asian populations belonging to different taxa would arise despite the fact that samples of both continents have identical ITS sequences. More generally the size of the sporophore is generally not regarded as a relevant taxonomic criterion because it is sensitive to environmental factors, to the competition between sporophores and

THONGKLANG ET AL.: THREE AGARICUS to the number of flushes. However the maximum pileus diameter that the larger sporophore reaches in favorable conditions is a characteristic trait for many species and varies highly among them. Resemblance or apparent convergence between species belonging to different groups complicates their identification. For example, resemblances have been observed between species of clades Xan II and Xan III; Heinemann (1971) said that A. microvolvatulus was probably a close relative of A. volvatulus Heinem. & Gooss because of their similar volviform bulb. In tropical species confusion is possible between Xanthodermatei and species of the tropical clade I of Zhao et al. (2011), which similarly can have phenollike odor and yellow discoloration after bruising (Chen et al. unpubl). Such resemblances possibly reflect variation in the expression of ancestral traits that can be more or less pronounced or hidden, possibly under selection pressure. For instance it would be interesting to know whether traits such as the phenol-like odor or the volviform bulb have a role in defense of the sporophore, in that the stipe base is particularly vulnerable. Because of such morphological or organoleptic resemblances, DNA sequence data are necessary not only for the phylogenetic approach but also for the species identification. In conclusion our phylogenetic analyses revealed that the three species we describe from Pakistan or Thailand belong to a new group in Agaricus sect. Xanthodermatei. Two putatively toxic species of section Xanthodermatei are now recognized in Pakistan and six in Thailand. ACKNOWLEDGMENTS We thank the Royal Golden Jubilee PhD Program-RGJ–I (PHD/0061/2552, 4. S. MF/52/A.1), the French Ministry of Foreign and European Affairs (project AGASIA of the regional program Bio-Asie), the Thailand Research Fund grant (BRG 5580009) under research titled ‘‘Taxonomy, phylogeny and biochemistry of Thai Basidiomycetes’’, for financial support. Institut National de la Recherche Agronomique (INRA), France and School of Science, Mae Fah Luang University and University of the Punjab, Pakistan, is acknowledged for providing facilities and training. We thank the National Natural Science Foundation of China (Project ID: 31000013, 31360014) for financial support. We thank Bencharong Thongbai, Komsit Wisitrassameewong, Samantha Karunarathna and Sylvie Rapior for sample collection.

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